également mis en cause dans l'inhibition du NMD et dans la stabilisation et l'export des ARN viraux non- ou mono-épissés (Nakano et al., 2013).

Par des approches de puces exons, notre laboratoire a publié les premiers profils d'épissage alternatif de patients atteints d'ATLL et de clones cellulaires infectés non transformés obtenus par culture en dilution limite de PBMC de patients (Thénoz et al., 2014). Comparés à leur contrepartie T-CD4+ non infectés, les clones porteurs d'HTLV-1 présentent une diversité transcriptomique très importante avec >2000 exons affectés dans leur épissage pour les clones non-transformés et >23000 pour les clones transformés d'ATLL. De façon intéressante, de nombreux évènements d'épissage sont retrouvés partagés par les clones infectés non transformés et les clones transformés d'ATLL, suggérant qu'ils jouent un rôle fonctionnel dans les processus oncogènes liés à l'infection. Ces épissages alternatifs affectent notamment des transcrits engagés dans les voies de régulation de l'immunité, de l'adhésion cellulaires, et des acteurs de cancers. Par ailleurs, l'analyse par cluster hiérarchisé des évènements d'épissage a permis d'isoler 4000 exons dont le degré d'inclusion dans les ARNm constitue une signature des clones transformés d'ATLL et des clones infectés non-transformés. Enfin, les voies fonctionnelles affectées par les modifications d'épissage apparaissent différentes de celles contrôlées par les gènes dérégulés quantitativement dans leur expression (Thénoz et al., 2014). Ces données suggèrent que les modifications massives de l'épissage alternatif générées par HTLV-1 constituent un nouveau mécanisme de perturbations du transcriptome cellulaire, étudiées jusqu'à présent qu'à travers des différentiels quantitatifs d'expression des transcrits.

Des approches récentes de double hybride ont permis de caractériser l'interaction de Tax avec 35 différents facteurs d'épissage. Notamment, Tax interagit avec U2AF65 et permet de stabiliser le complexe U2AF35/U2AF65, impliqué dans la reconnaissance du 3'SS et du polypirimidine tract. Les auteurs suggèrent que l'interaction de Tax avec U2AF65 permet une reconnaissance de sites d'épissage moins conservés, et ainsi de moduler l'épissage alternatif via cette reconnaissance par le complexe U2AF. L'interaction de U2AF65 avec Tax semble ainsi être un exemple par lequel Tax induit un remodelage du transcriptome cellulaire.

Egalement, les travaux de l'équipe ont pu montrer que l'expression de Tax dans des cellules HEK293T affecte l'épissage alternatif de plus de 900 gènes cellulaires, dont 48% des événements est également retrouvé dans des cellules d'ATLL (Annexe 1) (Ameur et al., 2020;
Vandermeulen et al., 2021). Cet épissage alternatif est indépendant des effets de Tax sur la transcription, et concerne des gènes dont les termes GO correspondent à des voies distinctes de celles régulées en transcription, telle que l'adhésion focale. Plus particulièrement, il a été montré dans les cellules exprimant Tax que le gène CD44 présente une inclusion de l'exon variable v10 qui induit un changement d'adhésion cellulaire. Au niveau moléculaire, Tax interagit avec le facteur d'épissage DDX17 de façon NF-κB-dépendante, et permet de réguler une majorité des exons Tax-dépendants. Dans ce sens, l'analyse de données de ChIP-seq a permis de montrer que l'enrichissement intragénique de p65/RelA est plus proche d'exons alternatifs que constitutifs, et que les exons Tax, et DDX17-dépendants, présentent un enrichissement en sites κB. D'un point de vue mécanistique, l'expression de Tax induit un enrichissement de p65/RelA et de DDX17 proche des exons régulés (Figure 32). Des approches de recrutement par TALE (Transcription-Activator-Like-Effector) ont permis de caractériser que la fixation intragénique de p65/RelA induit le recrutement DDX17 et induit une inclusion de l'exon alternatif v10 de CD44.

<figure><img src="image_34.png" /><figcaption>Figure 32 : Modèle de régulation de l'épissage alternatif par p65/RelA et DDX17.</figcaption></figure>
Pour conclure, Tax induit des dérégulations quantitative et qualitative du transcriptome de la
cellule, au travers de l'activation chronique de la voie NF-κB. Par ailleurs, l'organisation
tridimensionnelle de la chromatine est impliquée dans la régulation de l'expression des gènes
lors d'une activation physiologique de la voie NF-κB. Egalement, l'organisation de la
chromatine joue un rôle dans l'organisation de l'épissage alternatif. Cependant, aucune
information n'est disponible à ce jour permettant de décrire l'impact de Tax sur l'organisation
tridimensionnelle de la chromatine, ni son lien avec la dérégulation quantitative et qualitative
du transcriptome.
Problématique
L'oncoprotéine Tax joue un rôle central dans les altérations transcriptionnelles de la cellule
hôte lors de l'infection par HTLV-1, et dans la leucomogenèse associée à HTLV-1. Notamment,
l'activation chronique de la voie NF-κB par Tax est un élément clé permettant la régulation du
transcriptome des cellules hôte via la fixation des facteurs NF-κB aux promoteurs permettant
l'activation de gènes. Mes travaux (Annexe 1) ont participé dans un premier temps à
démontrer que l'activation de la voie NF-κB Tax-dépendante induit également des altérations
d'épissage alternatif de façon indépendante des régulations transcriptionnelles, qui sont
retrouvées chez des patients infectés par HTLV-1 et chez des patients atteints d'ATL. Sur le plan
fonctionnel, ces dérégulations d'épissage alternatif Tax-dépendantes sont retrouvées dans des
gènes impliqués dans des voies fonctionnelles différentes de celles contrôlées par des gènes
régulés dans leur niveau d'expression par Tax. Ces données suggèrent que l'épissage alternatif
médié par Tax constitue un mécanisme supplémentaire de régulation du transcriptome qui
participe vraisemblablement à la pathogenèse associée à HTLV-1. Plus précisément, nous
avons pu mettre en évidence la fixation intragénique de p65/RelA à proximité des exons
régulés par Tax. Notamment, ces exons régulés par Tax sont majoritairement dépendant de
DDX17. Dans ce sens, nous avons montré que lors de l'activation de la voie NF-κB par Tax, RelA
recrute le facteur DDX17 qui va induire une régulation de l'épissage alternatif de l'exon cible
via son activité hélicase (Annexe 1). Ces données ont ainsi mis en évidence pour la première
fois un rôle fonctionnel de la fixation des facteurs NF-κB au niveau des corps de gènes. Ces
travaux m'ont valu d'être second auteur sur le papier publié dans Nature Communications en
2020. Ces résultats ne seront pas détaillés ici mais l'article est présenté en annexe 1.

En dehors du contexte HTLV-1, plusieurs études ont montré le rôle de l'organisation
tridimensionnelle de la chromatine dans la régulation de la transcription lors de la voie NF-κB.
En effet, l'organisation tridimensionnelle du génome joue un rôle important dans l'expression
des gènes. Il apparaît également à travers de nombreux travaux que des gènes co-régulés par
une voie de signalisation sont retrouvés à proximité les uns des autres, et que leur
rapprochement joue un rôle important dans leur co-régulation (Clowney et al., 2012; Fanucchi
et al., 2013; Fullwood et al., 2009; Karki et al., 2019a; Papantonis et al., 2012). Cependant, ces
données ne renseignent que les processus de régulation quantitative de l'expression des
gènes. Mes travaux ont abordé, à travers une étude intégrative, l'étude des effets de Tax dans
l'altération de l'organisation tridimensionnelle de la chromatine et de leurs implications dans
la co-régulation de l'expression quantitative et qualitative des gènes cellulaires par les facteurs NF-κB.

<figure><img src="image_35.png" /><figcaption>Figure 33 : illustration de la problématique posée.</figcaption></figure>
# Résultats
Résumé :

Les travaux présentés dans l'article qui suit démontrent l'impact de l'oncoprotéine virale Tax dans l'organisation tridimensionnelle du génome. Pour ce faire, des essais de Hi-C, de 4C et de 3C ont été réalisés dans des lignées HEK293T exprimant ou non Tax. Ces expériences nous ont permis de caractériser que Tax induit des changements des interactions de chromatine en renforçant les interactions longue-distance, mais altère également des contacts entre les compartiments A/B et les contacts inter-TAD, sans pour autant affecter la définition des TADs. En comparant ces altérations de contacts de chromatine et nos données de RNA-seq précédemment publiées, nous avons pu mettre en évidence que la majorité des gènes régulés par Tax (au niveau transcriptionnel ou d'épissage alternatif) sont présent dans un réseau de contacts multigéniques à la fois de façon intra- et inter-chromosomique, suggérant que de tels complexes multigéniques puissent permettre la coordination de l'épissage alternatif et de la transcription des gènes co-régules par Tax.

En analysant la nature des gènes présents dans les régions de contacts, nous avons pu constater par analyse d'ontologie un enrichissement de termes impliqués dans les voies de signalisation NF-κB et TNFα ainsi que d'infection virale par HTLV-1. Par ailleurs, l'analyse des motifs présents dans les promoteurs des gènes impliqués dans des contacts longue-distance révèle l'enrichissement de sites κB, suggérant que l'altération des contacts de chromatine puisse coordonner la co-régulation des gènes de la voie NF-κB. Dans ce sens, nous avons identifiés plusieurs contacts entre gènes dérégulés par Tax, à la fois sur le plan transcriptionnel ou de l'épissage alternatif. Plus particulièrement, nous montrons que l'activation de la voie NF-κB par Tax induit un contact entre les gènes NFKBIA et RELA, qui est concomitant à la surexpression de NFKBIA et l'exclusion de l'exon 6 de RELA.

Sur le plan fonctionnel, nous montrons par expression ectopique de la forme RELA alternativement épissée, que cette isoforme tronquée n'induit pas de transactivation des gènes de la voie NF-κB en l'absence de Tax. En revanche, lorsque Tax est exprimée, cette isoforme agit comme modulateur positif ou négatif (selon le gène cible) de l'expression de gènes NF-κB-dépendants, suggérant que l'induction de cette isoforme contribue à définir une signature transcriptionnelle de l'activation de la voie NF-κB par Tax.
Au niveau mécanistique, nous démontrons que le contact entre *NFKBIA* et *RELA* lors de l'expression de Tax s'accompagne d'un enrichissement de p65/RelA au niveau du promoteur de *NFKBIA* et au niveau de l'exon génomique E6 de *RELA*. En utilisant une approche dérivée du système CRISPR/dCas9 capable de perturber le contact entre *NFKBIA* et *RELA* (*dCas9-KRAB*), nous montrons que la perte du contact entre ces deux gènes altère la régulation de l'épissage alternatif de RELA par Tax qui s'accompagne également d'une diminution de la présence de RelA/p65 au promoteur de *NFKBIA* et au niveau de l'exon E6 de *RELA*. L'utilisation d'un second système de CRISPR/dCas9 fusionné à p65/RelA, nous a permis de montrer que le recrutement expérimental de p65/RelA à la chromatine de l'exon 6 de *RELA* induit le contact entre *NFKBIA* et *RELA* et la régulation de l'épissage alternatif de *RELA*.

Ensemble, ces résultats indiquent que p65/RelA joue un rôle central dans l'établissement de complexes multigéniques permettant la coordination des régulations de transcription et d'épissage alternatif des gènes lors de l'activation la voie NF-κB par Tax.

Les tables supplémentaires ainsi que les figures en haute résolutions ont disponibles sur
demande aux auteurs, et jusqu'au 28/06/2023 à l'adresse suivante :

<https://filesender.ens-lyon.fr/?vid=29f818cb-2ef0-7769-cbd5-00007bc90bd7>
Coordinated Regulation of Transcription and Alternative Splicing by 3D
Chromatin Remodeling upon HTLV-1 TAX-induced NF-κB activation

Paul Marie¹, Julien Ladet¹, Matéo Bazire¹, Lamya Ben Ameur¹§, Sanjay Chahar², Nicolas
Fontrodona¹, Tom Sexton², Cyril F. Bourgeois¹, Didier Auboeuf¹, Franck Mortreux¹*

1. Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, Laboratory of Biology and Modelling of the Cell, 46 Allée d'Italie Site Jacques Monod, F-69007, Lyon, France

2. Institute of Genetics and Molecular and Cellular Biology (IGBMC), UMR7104, Centre National de la Recherche Scientifique, U1258, Institut National de la Santé et de la Recherche Médicale, University of Strasbourg, 6704 Illkirch, France

* To whom correspondence should be addressed. Tel: +33 472728663 ; Email: stanck.mortreux@ens-lyon.fr

Present Address: [Lamya Ben Ameur], Vollum Institute, Oregon Health & Science University,
Portland, OR 97239, USA
Summary

The p65/RelA factor of NF-κB plays a pivotal role in coordinating gene expression in response to diverse stimuli, including viral infections. At the chromatin level, p65/RelA governs gene transcription and alternative splicing (AS) through promoter enrichment and genomic exon occupancy, respectively. However, the mechanisms underlying the coordination of these processes remain elusive. In this study, we employed the HTLV-1 TAX viral oncoprotein, a potent activator of NF-κB, to investigate the integrative relationship between 3D chromatin architecture and NF-κB-regulated AS. Our analysis revealed that TAX induces a significant reorganization of the 3D genome, resulting in the formation of multigene complexes that comprise genes co-regulated in transcription and in AS. Notably, we found that the TAX-induced gene-gene contact between the two master genes *NFKBIA* and *RELA* is associated with their differential regulation in gene expression and alternative splicing, respectively. Through dCas9-mediated approaches, we demonstrated that *NFKBIA-RELA* interaction is required for alternative splicing regulation and is caused by an intragenic enrichment of p65/RelA on *RELA*. Via this mechanism, TAX promotes the expression of the splice isoform RELAΔE6 that exhibits specific gene transactivation capacity at the protein level. Our findings shed light on new regulatory mechanisms upon HTLV-1 TAX and underscore the crucial role of p65/RelA in coordinated regulation of NF-κB-responsive genes at both transcriptional and alternative splicing levels in the context of the 3D genome.
INTRODUCTION

The process of gene expression involves multiple steps, including transcription and splicing, that work together to shape the transcriptome and ultimately determine cell behavior. Coordination between transcription and alternative splicing is thus required for proper cell response to stimulus. Cellular pathogens such as viruses can disturb this coordination, leading to both quantitative and qualitative alterations in transcriptome diversity, which can ultimately disturb cellular homeostasis and promote viral replication and persistence. A better understanding of mechanisms involved in coordinating transcription and splicing is thus necessary to comprehend the cellular response to viral infections.

The retrovirus Human T-cell Leukemia Virus type 1 (HTLV-1) primarily infects T lymphocytes, leading to a range of diseases, including adult T-cell leukemia/lymphoma (ATLL) and various inflammatory diseases (1). HTLV-1 replication initially involves cell-to-cell transmission followed by clonal expansion of infected cells which is responsible for the persistence of the virus and associated diseases (2). One of the key viral proteins involved in HTLV-1 infection is the oncoprotein Tax that has been first described as a transcriptional activator interacting with a variety of host cell proteins, including the NF-κB family of transcription factors. By hijacking NF-κB signaling, TAX deeply affects crucial mechanisms involved in immune response, cell survival, and inflammation, and plays critical role in HTLV-1 pathogenesis (3).

At the molecular level, Tax interacts with multiple NF-κB and IκB proteins, including p65/RelA (4), p50 (5), c-Rel (4), p100 (6-8), p105 (9), IκBα (10,11), and NEMO (10,12). Tax activates the NF-κB pathway by promoting the degradation of the inhibitory protein IκBα, which controls the oscillating dynamics of NF-κB signaling (13,14). Tax induces a constitutive nuclear translocation of p65/RelA, resulting in chronic activation of target genes. Through post-translational modifications (15), TAX is able to form nuclear bodies enriched in NF-κB p65/RelA and splicing factors SFRS2/SC35 that have been proposed to participate in Tax-mediated activation of gene expression via NF-κB pathway (16,17). In this setting, we have recently demonstrated that when TAX is expressed, p65/RelA not only impacts promoters but also enriches gene bodies and induces alternative splicing changes, thereby broadening the influence of NF-κB on
transcriptome diversity both quantitatively and qualitatively (18). The transcription factor p65/RelA interacts with various splicing factors (18-22), among which DDX17 that regulates splicing of GC-rich exons when recruited by p65/RelA in close proximity to the genomic target exon (18). It is noteworthy that Tax-induced alternative splicing primarily impacts genes that are not modified at the gene expression level (18,23), yet experimental tethering of p65/RelA to its genomic target exon is sufficient to locally affect alternative splicing, indicating that p65/RelA acts on splicing regardless of its activity on the target gene promoter (18). Given the tightly controlled coordination of NF-κB-regulated genes to mediate cellular functions and responses to stimuli, these findings raise questions whether these two distinct activities of p65/RelA are coordinated.

The transcriptional coordination of NF-κB-responsive genes is regulated through a complex interplay of mechanisms that includes the oscillating dynamics of NF-κB signaling (13,14,24), the differential DNA affinity of homo- or heterodimers formed by NF-κB subunits (25,26), the recognition of κB sites and flanking sequences (26-28), as well as specific co-factors and post-translational modifications of NF-κB (29,30). More recently, the 3D conformation of chromatin has emerged as a critical determinant of NF-κB-mediated gene expression regulation (31). NF-κB p65/RelA is able to promote DNA looping through interactions with various transcription and chromatin remodeling factors (32,33). NF-κB signaling upon TNF-α has been associated with the formation of specialized transcription factories, also called NF-κB factories, where responsive-coding and -miRNA genes are transcribed in a coordinated manner (34). Such long-range interactions of co-transcribed genes can occur in a hierarchical relationship that contributes to the temporal coordinated regulation of responsive genes (35). The question arises whether these mechanisms could play a role in coordinating transcription and alternative splicing events promoted by p65/RelA upon TAX expression.

Alternative splicing is a co-transcriptional process regulated by the combination of various factors, such as RNA binding proteins, elongation rate of transcription, and chromatin remodeling factors (36,37). Additional studies have provided evidence for the involvement of 3D chromatin conformation in alternative splicing regulation (33-35,38-41). Using chromosome conformation capture (3C)-based approaches such as Hi-C, active chromatin has
been observed highly concentrated in the nuclear center and around nuclear speckles (NS), which are dynamic subnuclear structures enriched in splicing factors and other RNA processing proteins (42). NS are enriched in GC-rich genomic sequences in their close vicinity (43). In particular, it appears that exon-intron units with high-GC content are preferentially localized in the nuclear center in association with NS, compared to their low-GC counterparts (44,45). Importantly, high- and low-GC exon-intron units display different splicing patterns, and their experimental shifting from one genomic location to another affects both their transcription and splicing regulation, indicating that the nuclear location of the gene determines its regulation in alternative splicing (45). In line with this, the formation of multigene complexes via trans-chromosomal contacts in foci enriched in splicing factor RBM20 has been shown to function as a splicing factory, suggesting that nuclear condensates may act as hubs regulating both transcription and alternative splicing of cell-signaling responsive genes (46).

Here, we provide molecular evidence that the viral oncogene HTLV-1 TAX induces drastic changes in the 3D conformation of the cell genome that are accompanied by differential gene expression and alternative splicing modifications. In particular, we show that a trans-chromosomal interaction involving *NFKBIA* and *RELA* genes is required for TAX-induced skipping of exon E6 of *RELA*, leading to the expression of an isoform of p65/RelA with a unique gene expression activation pattern. Engineering-based approaches via dCas9 demonstrate that *NFKBIA-RELA* contacts are caused by the DNA binding of p65/RelA at the close vicinity of the genomic *RELA* E6, without affecting target gene expression. These results provide insight into the interplay between HTLV-1, NF-κB signaling, and 3D genome conformation in reprogramming cellular function, demonstrating how the formation of a p65/RelA dependent multigene complex coordinates NF-κB-related transcription and alternative splicing events.
MATERIAL & METHODS

Cell culture and transfections

The human embryonic kidney (HEK) 293T-LTR-GFP cell contains an integrated GFP reporter gene under the control of the viral HTLV-1 long terminal repeat (LTR). HEK293T-LTR-GFP were cultured in DMEM + 4.5 g/L D-Glucose + Pyruvate + GlutaMAX™, supplemented with 10 % heat-inactivated FBS and 1 % Penicillin/Streptomycin. The transfection efficiency of cells with Tax, Tax-M22, and dCas9-T2a-GFP constructs was assessed by measuring GFP fluorescence with EVOS® FL Imaging System. RNA guides for dCas9 (sgRNA) (Supplementary Table 1) were designed using UCSC 'CRISPR targets' tracks and sequences with overall highest prediction scores and lowest off-targets. The sgRNAs were cloned into the BsmBI sites of the CRIZI plasmid (generously gifted by Philippe Mangeot, CIRI, Lyon, France). For dCas9-p65 cloning, WT-p65 was amplified from HEK-293T-LTR-GFP cDNA and cloned into the BamHI and XbaI sites of dCas9-empty-GFP plasmid (generously gifted by Reini F. Luco, Institut Curie, Orsay, France). The expression vectors pRELA-WT and pRELAΔE6 were produced using sequences amplified from HEK293T cDNA and the V5 sequence was added in 5'. Sequences were then cloned in pXJ41 plasmid using NotI and BamHI restriction sites. All sequences were verified by Sanger sequencing.

Transfections were performed using pSG5M-Tax-WT, pSG5M-Tax-M22, pLV hU6-sgRNA hUbC-
dCas9-KRAB-T2a-GFP (addgene plasmid #71237), sgRNA-CRIZI, pBabe-Puro-IKBalpha-wt
(Addgene plasmid #15290), dCas9-p65, dCas9-empty using Jeprime (Polyplus Transfection)
following manufacturer's instructions, and cells were harvested 48h post transfection.

HTLV-1-chronically infected lymphocytes C91PL and non-infected Jurkat (both kindly gifted by Renaud Mahieux CIRI, Lyon, France) were cultivated in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 25 mM HEPES, 10 % heat-inactivated FBS and 1 % Penicillin/Streptomycin.

RNA-seq analysis
RNA-seq data have been previously published (18) and deposited on NCBI GEO under the
accession number GSE123752. Briefly, poly-A transcripts were extracted at 48h post-
transfection from 293T-LTR-GFP cells transfected with pSG5M-Tax or pSG5M empty vectors.
RNA-seq libraries were prepared from biological triplicates and sequenced using the illumina
HiSeq 2500. Paired-end pairs of reads were then analyzed using the pipeline FaRLine and the
FasterDB database (47). HTSeq-count (v0.7.2)(48) and DESeq2 (v1.10.1) (49) were used
calculating gene expression level and for computing differential gene expression levels,
respectively. The significance thresholds were set to 5% for ΔPSI (differential percentage of
spliced-in sequence) (p < 0.01, Fisher's exact test) and 0.6 for log2-gene expression changes
(p<0.05, Wald test using Benjamini and Hochberg method), respectively. Relative genomic
distance analysis between alternative splicing events (ASE) and differential gene expression
was carried out using reldist from Bedtools. Control set of ASE was generated using the shuffle
function of Bedtools.

Hi-C assays and data processing

The Hi-C experiment and library preparation were conducted following the *in situ* method described by Rao and colleagues with slight modifications (50). Briefly, HindIII enzyme was used to digest cells overnight at 37°C with 500U, followed by biotin filling and proximity ligation for 4 hours at 18°C with 2000U T4 DNA Ligase. After reverse-crosslinking, DNA was purified and sheared to ~300 bp fragments using Covaris S220 sonicator. The biotin-containing fragments were then immobilized on MyOne Streptavidin T1 beads. The NEXTflex adaptors were ligated, and the fragments were PCR amplified for 8 cycles using the KAPA HiFi Library Amplification Kit. Double-size selection was performed using AMPure XP beads to isolate fragments between 300 and 800bp. Sequencing of Hi-C libraries was performed on Illumina Nextseq 550 with 150 bp single-end reads (Genomeast, IGBMC, Illkirch, France). The raw reads were independently mapped to the hg19 reference genome using the HiC-Pro pipeline (version v3.1.0), and the output statistics, including the total number of sequenced reads and the number of valid interactions after filtering, are presented in Sup Fig 1.

The Hic explorer pipeline (v3.7.2) was used to process the Hi-C data and plot the matrices (51).
The matrices were first normalized to each other (hicNormalize), then corrected
(hicCorrectMatrix) for removing bins with low numbers of reads and balanced using ICE (52) at
resolutions of 10k, 50k, 100k, and 500k bp. Corrected matrices were plotted (hicPlotMatrix) on a log scale at a resolution of 500k. The differential matrix was obtained using a log2ratio comparison between TAX and CTL cells on corrected matrices at the 500k resolution (hicCompareMatrices). The ratio between short-range and long-range contacts was computed using hicPlotSVL with the threshold distance set to 1e6 nucleotides.

The inference of A/B compartments and changes in compartmentation between TAX and CTL matrices were analyzed using the pipeline dcHiC with matrices at a resolution of 100k (53). The full genome compartment strength was calculated as (AA + BB) / (AB + BA) from the log2 ratio of observed versus expected contacts sorted by their eigen vector values (hicCompartmentalization). A saddle plot was generated with cooltools (https://github.com/open2c/cooltools) using cis-contacts from matrices at a resolution of 50k. The eight subcompartments were assigned by the pipeline CALDER with matrices at a resolution of 10k and the binning variable set to 50k (54). The Deeptools' PlotEnrichment (55) function was then used to validate CALDER predictions regarding their subcompartment enrichment levels in histone marks H3K27ac, H3K4me3, and H3K9me3 publicly available for HEK293T cells in ENCODE (ENCFF706GDY, ENCFF219COP, ENCFF800LFN, ENCFF063WXN, ENCFF626VTP, and ENCFF295FIO). Comparison of both compartment and subcompartment orientations between TAX and CTL conditions was carried out using Bedtools (56). Sankey diagram was built in R using the networkD3 package. Genomic regions corresponding to both subcompartment transition and inter-chromosomal changes upon TAX were obtained using bedtools intersect function with coordinates of TAXvsCTL log2ratio contact matrix computed at 50kbp and those of the subcompartment changes grouped as follows: A.1 (A.1.1 + A.1.2), A.2 (A.1.1 + A.1.2), B.1 (B.1.1 + B.1.2), and B.2 (B.2.1 + B.2.1). Gene ontology analysis and motif enrichment in promoters involved in 3D chromatin contacts in TAX expressing cells was performed with ShinyGO 0.76.3.

The identification of topologically associating domains (TADs) was carried out using the HicFindTADs function of the hicexplorer software with a matrix resolution of 50k. A minimum and maximum window length of 150k and 500k, respectively, were set for the analysis, and a q-value threshold of false discovery rate (FDR) was set to 0.05. The putative boundary and surrounding bins were differentiated by setting a minimum differential threshold (delta) to 0.01. The similarity of TAD boundaries between the control (CTL) and TAX-expressing cells was
assessed by using the Bedtools' Jaccard statistic with a tolerance window of 50 kbp. Intra-TAD
and inter-TAD interactions were analyzed by merging the TAD domains obtained from both
CTL and TAX conditions using the hiCMergeDomains function of Hicexplorer. The CTCF binding
sites (ENCODE ENCFF895AOX) were used to delineate TAD boundaries, considering that
boundaries within 20Kb of each other were merged as one. The merged TAD boundaries were
then utilized to calculate the intra-TAD and inter-TAD differential contacts between TAX and
CTL matrices (hicDifferentialTAD). Significant intra-TAD and inter-TAD interactions were
defined thanks to the Wilcoxon rank-sum test with a p-value threshold set to 0.05. Bedtools
intersect function allowed assigning gene deregulations to differential chromatin interactions
between TAX vs CTL (log2(obs/exp) at 50 k). Network analysis of ASE, DGE, and chromatin
contacts was performed with Cytoscape (3.9.1).

Western Blot

Cells were washed twice with 1 X PBS, harvested and incubated with lysis buffer (10 mM Tris
HCl pH 8, 50 mM NaCl, 5 mM EDTA, 1 % NP-40, 1 % SDS, 1 mM DTT, supplemented with
protease inhibitors and 10 mM NaF) for 30 min on ice. Total proteins were sonicated on a
Bioruptor Plus (Diagenode) (position high, 5 x 30'/30' cycles), and cell debris were pelleted and
discarded. 20 µg of total proteins were used for SDS-PAGE electrophoresis on NuPAGE™ 4–
12% Bis-Tris Protein Gels and transferred on nitrocellulose membranes using the Trans-Blot®
TurboTM Blotting System. Membranes were saturated in 1 X TBS-Tween containing 5% non-
fat milk for 1 h and were incubated overnight at 4°C with primary antibodies diluted in 1 X TBS-
Tween containing 5% non-fat milk. The primary antibodies used were the following: RELA (sc-
109, Santa Cruz), Tax (1A3, Covalab), Vinculin (ab129002, abcam), IkBα (L35A5 Cell Signaling
Technology). Membranes were washed three times in 1X TBS-Tween and incubated with HRP-
conjugated secondary antibodies for 1 h before washing for three times. HRP substrate
(Immobilon Forte (Millipore)) was deposed onto the membrane before reading
chemiluminescence on a Chemidoc (BioRad).

RNA extraction, PCR, and real-time quantitative PCR

Total RNAs were extracted using TRIzol (Invitrogen). 2,5 µg of total RNA was treated with dsDNase (Thermo Scientific) and retro-transcribed using Maxima First Strand cDNA Synthesis (Thermo Scientific) according to manufacturer's instructions. PCRs were performed with
GoTaq polymerase (Promega, Madison, WI, USA) using 7.5 ng cDNA as template, were run on agarose gel electrophoresis and read using Geldoc (BioRad). Quantitative PCR were performed with SYBR® Premix Ex Taq TM II (Tli RNaseH Plus) on LightCycler 480 II using 7.5 ng of cDNAs. Melting curves were checked for non-specific product absence. Relative expression levels were calculated using the ΔΔCt method on technical duplicates or triplicates and normalized to GAPDH expression. Alternative exons inclusion rates were calculated using ΔΔCt method and were normalized to a constitutive exon. Oligonucleotide sequences used are listed in Supplementary table 1.

## Chromatin immunoprecipitation

2x10⁷ cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched by addition of Glycin to a final concentration of 0.125 M. Cells were harvested and incubated 30 min at 4°C in lysis buffer (1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140 mM NaCl, 50 mM HEPES-KOH pH 7.5, 1 mM EDTA, supplemented with protease inhibitor and phosphatase inhibitor). Nuclei were pelleted and resuspended in shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 mM EDTA, 0.1% SDS, supplemented with protease inhibitor and phosphatase inhibitor). Chromatin was sheared to an average fragments size of 100-500 bp using Covaris S220 (Peak Power: 140; Duty Factor: 5; Cycles/burst: 200, 20 min). Immunoprecipitations were performed using 25 µg of chromatin diluted in dilution buffer (1% Triton X-100, 0.01% SDS, 150 mM NaCl, 10 mM Tris HCl pH 8, 1 mM EDTA, supplemented with protease inhibitor and phosphatase inhibitor), and incubated with 5 µg of anti-RELA (#36369, Active Motif), anti HA (H3663, Sigma-Aldrich) or control Rabbit or Mouse IgG (10500C and 10400C respectively, ThermoFisher) overnight at 4 °C under rotation. chromatin-antibodies complexes were recovered by incubation with 30 µl of Dynabeads® Protein A/G 1:1 mix (ThermoFisher) for 2 h at 4°C under rotation. Complexes were washed for 5 min at 4°C under rotation with the following buffers : Wash 1 (1% Triton X-100, 0.1% NaDOC, 150 mM NaCl, 10 mM Tris-HCl pH 8), Wash 2 (1% NP-40, 1% NaDOC, 150 mM KCl, 10 mM Tris-HCl pH 8), Wash 3 (0.5% Triton X-100, 0.1% NaDOC, 500 mM NaCl, 10 mM Tris-HCl pH 8), Wash 4 (0.5% NP-40, 0.5% NaDOC, 250 mM LiCl, 20 mM Tris-HCl pH 8, 1 mM EDTA), Wash 5 (0.1% NP-40, 150 mM NaCl, 20 mM Tris-HCl pH 8, 1 mM EDTA) and twice with Tris-EDTA buffer (10 mM Tris pH 8.0, 1 mM EDTA). The immunoprecipitated chromatin was eluted in elution buffer (1% SDS, 200 mM NaCl, 100 mM NaHCO3) and crosslink was reversed
by adding 100 µg proteinase K and incubating overnight at 65°C. Immunoprecipitated
chromatin was purified by phenol-chloroform extraction, and quantitative PCR was performed
using SYBR® Premix Ex Taq (Tli RNaseH Plus) (Takara) on a LightCycler 480 II (Roche, Mannheim,
Germany), using manufacturer's recommended thermocycling conditions. Values were
expressed as fold enrichment relative to the signal obtained for the control IgG
immunoprecipitation. Primers used for quantitative ChIP experiments are available in
Supplementary Table 1.

Chromosome Conformation Capture (3C)

1 x 107 cells were cross-linked with 1 % formaldehyde (ThermoFisher) for 10 min at RT. Formaldehyde cross-linking was stopped by addition of Glycine for 5 min to a final concentration of 0.125 M. Cells were washed twice with cold PBS and harvested. Cells pellets were lysed for 20 min on ice using lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, 0.5 % NP-40, 1 % Triton X-100, 5 mM EDTA and supplemented with protease inhibitor cocktail). Nuclei were harvested by centrifugation and washed in 1,2 X restriction buffer (depending on the restriction enzyme used) and resuspended in 500 µl of the same buffer. For nuclear membrane permeabilization, SDS was added to a final concentration of 0.3 % followed by incubation at 37°C for 1 h under agitation (800 rpm). SDS was neutralized by addition of Triton X-100 to a final concentration of 2.5 % at 37 °C for 1h h at 900 rpm. Dlgestion was realized by addition of 50 U Dpnll or Apol at 37 °C for 3h at 900 rpm followed by another 50 U and overnight incubation at 37 °C at 900 rpm. Digestion efficiency was assessed on a reverse cross-linked and purified aliquot by agarose gel electrophoresis (data not shown). Restriction enzyme was inactivated by incubation at 65 °C for 20 min at 900 rpm. Digested chromatin was proximity-ligated by diluting restriction reaction in 7 ml of 1X ligation buffer. Ligation was performed by addition of 50 U T4 DNA ligase and 3h incubation at 16 °C at 750 rpm followed by another 50 U and overnight incubation at 16 °C at 750 rpm. Ligation efficiency was assessed on a reverse cross-linked and purified aliquot by agarose gel electrophoresis (data not shown). Cross-link was revered by addition of 30 µg proteinase K and overnight incubation at 65 °C at 900 rpm. 3C templates were purified by phenol/chloroform extraction and ethanol precipitation.

3C-PCR were performed using semi nested primers (Supplementary Table 1) and two rounds
of PCR. for the first round, 50 ng of 3C template was used and amplified using Phusion Green
HSII High-Fidelity PCR Master Mix and a final primer concentration of 0.5 µM using the following program : 3 min of denaturation at 98°C, followed by 20x (98°C for 10 s, primer-specific annealing temperature for 30 s, 72 °C for 45 s) and 10 min final extension at 72 °C. the first-round of 3C-PCR was analyzed by gel agarose electrophoresis to check the absence of any significant amplification band before performing second-round 3C-PCR. Second-round 3C-PCR was performed using the same primer than the first round for RELA viewpoint, and a different primer closer to the restriction site for NFKBIA viewpoint. 1 µl of the first round PCR was amplified using Phusion Green HSII High-Fidelity PCR Master Mix and a final primer concentration of 0.5 µM using the following program: 3 min of denaturation at 98°C, followed by 25x (98°C for 10 s, primer-specific annealing temperature for 30 s, 72 °C for 45 s) and 10 min final extension at 72 °C. Optimal annealing temperatures were determined using Thermofisher Phusion tm calculator (www.thermofisher.com/tm calculator). 3C-PCR products were analyzed by gel agarose electrophoresis.

Circular Chromosome Conformation Capture (4C-seq) and data processing

For Circular Chromosome Conformation Capture, the same protocol as for 3C was performed. 50 µg of 3C template was digested by 50 U second restriction enzyme (Csp6I, ThermoFisher) in 1X Buffer B (ThermoFisher) for 2 h at 37 °C at 500 rpm. Digestion efficiency was assessed on a purified aliquot by agarose gel electrophoresis (data not shown). Restriction enzyme was inactivated by incubation at 65 °C for 20 min at 900 rpm. The second ligation was performed under diluted conditions by addition of 50 U T4 DNA ligase and 2 h incubation at 16 °C at 750 rpm followed by another 50 U and 2 h incubation at 16 °C at 750 rpm. Ligation efficiency was assessed on a reverse cross-linked and purified aliquot by agarose gel electrophoresis (data not shown). 4C templates were purified by phenol/chloroform extraction and ethanol precipitation followed by an additional JetSeq™ Clean purification.

4C sequencing primers (Supplementary Table 1) were designed as previously described (57) and tested on a small portion of 4C template with increasing templates amounts to determine linear range, efficiency, minimal primer dimer amplification, and optimal annealing temperature of each primer pairs. 4C libraries were prepared in two steps of PCR. The first step consisted in an amplification of ligation fragments specific of each viewpoint using 100 to 400 ng template depending on primer linearity. Expand™ Long Template PCR System (Roche) was
used with the following thermocycling program: 2 min of denaturation at 94°C, followed by 16x (94°C for 15 s, primer-specific annealing temperature for 1 min, 68°C for 3 min) and 7 min final extension at 68 °C. All first step PCR primers are listed in Supplementary Table 1. Multiple PCR reactions were performed, pooled together and purified using JetSeq™ Clean purification according to manufacturer's instructions. The second PCR step (used to add illumina adapters, indexes and sequencing sequences) was performed on 5 µl of purified first step PCR products using Expand™ Long Template PCR System (Roche) with the following thermocycling program: 2 min of denaturation at 94°C, followed by 20x (94°C for 15 s, 60 °C for 1 min, 68°C for 3 min) and 5 min final extension at 68 °C. All second step PCR primers are listed in Supplementary Table 1. 4C templates were purified using JetSeq™ Clean purification according to manufacturer's instructions. Libraries quantity and purity were determined on a 2200 TaPEstation (Agilent) with a D500 ScreenTape (Agilent). Sequencing of 4C libraries was performed on Illumina Nextseq 550 with 150 bp single-end reads (Genomeast, IGBMC, Illkirch, France).

Computational analysis of 4C-seq data was performed for each replicate and viewpoint using the fourSig pipeline (58) with the following parameters: window.size=5, iterations=1000, fdr=0.001, and fdr(prob=0.01. For each viewpoint, triplicate outputs were normalized to each other using multiBamsummary and Bamcompare from deepTools (55). Differential chromatin contacts between CTL and TAX conditions were determined using the DESeq2 R package (FDR=0.05). Figures were generated using pygenometrack (version 3.6) (59).
# RESULTS

## TAX reshapes the 3D genome.

To evaluate the impact of TAX on 3D chromatin organization, we performed an unbiased chromatin interaction mapping using Hi-C analysis of HEK293T cells with and without TAX expression. Hi-C data were analyzed using the HiC-pro pipeline (60), which ensured high-quality experiments as evidenced by high mapping rates (Sup Fig 1). We identified over 160 million interactions, including over 20 million trans-chromosomal contacts, in both control (CTL) and TAX-expressing cells (Sup Table 2). Consistent with previous studies on chromosome territories (61,62), the Hi-C contact maps demonstrated higher frequencies of cis- versus trans-chromosomal interactions (Figure 1A). While the Hi-C maps from CTL and TAX-expressing cells showed similar shapes (Figure 1A and Sup Fig 2), we noticed a decrease in short-range (<1 Mb) and an increase in long-range (>1 Mb) interaction frequencies upon Tax expression (Sup Fig 3 A-B). These changes were particularly evident in differential heatmaps (Figure 1A, right panel).

Principal component analysis (PCA) of the Hi-C contact matrices allowed us to assess the effect of TAX on genomic compartments A and B, which correspond to euchromatin and heterochromatin regions, respectively (Figure 1B). The eigen vectors calculated from CTL and TAX samples were highly correlated (Pearson test, R=0.948, p-value <0.0001) (Sup Fig 3 C), indicating no significant changes in genomic locations of A and B compartments between the two experimental conditions. Homotypic compartment interactions (AA and BB) were predominant in CTL cells (Figure 1C). In comparison, TAX-expressing cells presented increased heterotypic compartment interactions and decreased AA contacts (Figure 1C), indicating that TAX weakens chromatin compartmentalization. Comparative analysis of compartment distribution between CTL and TAX-expressing cells revealed 13% compartment switching (A to B and B to A, Figure 1D), suggesting that the viral oncoprotein structurally affects both large heterochromatin and euchromatin regions.

Trans-chromosomal interactions have been linked to the definition of sub-compartment domains that associated with distinct epigenetic regulations (50). Subcompartments A1 have been identified as highly active chromatin regions that are closely localized to nuclear speckles
(63,64), while B1 and B2 are typically enriched in repressive marks, such as H3K27me3 and H3K9me3 (50,54). We used the CALDER algorithm (54) to infer eight subcompartments in both CTL and TAX-expressing cells (Figure 1E and Sup Fig 3 E-F). Using publicly available ChIP-seq datasets for HEK 293T cells, we confirmed that the predicted subcompartments A.1.1 and A.1.2 in control cells were markedly enriched in active marks H3K27ac and H3K4me3, while compartments B.2.1 and B.2.2 corresponded to H3K9me3 embedded regions (Sup Fig 3 E). We then compared the subcompartment annotations between the two experimental conditions by considering 4 large subcompartments: A.1 (A.1.1 + A.1.2), A.2 (A.1.1 + A.1.2), B.1 (B.1.1 + B.1.2), and B.2 (B.2.1 + B.2.1). Subcompartment transitions were observed in TAX expressing cells, as demonstrated in Figure 1E and Sup Fig 3F. These transitions involved shifts from active to inactive subcompartments, with some coinciding with differential trans-chromosomal interactions (Figure 1F). Overall, these data indicated that TAX promotes a profound reorganization of the 3D genome that likely follows from dynamic chromatin regulation.

We next investigated the effects of TAX on topologically associating domains (TADs). To this end, we employed the HiC-explorer pipeline to assess the TAD-separation score, which measures the degree of separation between the left and right regions in each Hi-C matrix. We set the minimum distance between two boundaries to 50 kilobase pairs (kbp) and used publicly available CTCF binding sites for determining TAD boundaries (ENCFF895A0X) (Sup Fig 3G). Our analysis revealed 3539 TADs shared by control cells and TAX-expressing cells, indicating that TAD boundaries were largely unmodified between samples (Figure 1G and Sup Fig 3G). Nevertheless, we observed differential chromatin contacts at both intra- and inter-TAD levels, accounting for 40.3% and 23.4% of TADs, respectively (Figure 1H). Notably, we found a statistically significant increase in inter-TAD contacts in TAX-expressing cells (one-way ANOVA, p=0.004), indicating that TAX functions as a potent modifier of host 3D chromatin architecture.

**TAX-induced 3D chromatin contacts associate with changes in both gene expression and alternative splicing.**

We next sought to assess the functional significance of TAX-induced 3D chromatin remodeling. Gene expression profiles including alternative splicing events of CTL and TAX(+) cells have been previously published (18). Using stringent thresholds to analyze differential gene expression
(DGE) (glm-adjusted p-value < 0.05, Log2-fold change ≥ 0.6) and alternative splicing events (ASE) (p < 0.01 adjusted Fisher test, ΔPSI ≥ 0.05), we identified 523 genes with altered whole gene expression and 1,104 genes producing alternatively spliced transcripts upon TAX expression (Figure 2A). A total of 1345 alternative splicing events consisted of 1020 skipped exon (SE), 15 alternative 5' splice site (A5SS), 62 alternative 3' splice site (A3SS), 244 multi-exon skipping (MES), and 4 mutually exclusive exons (MXE) (Figure 2B). In line with previously published data (18,23), a minority of TAX-regulated genes (5%, 23/523) were affected at both DGE and ASE levels (Figure 2A), suggesting that TAX-induced transcription and alternative splicing are two distinct and complementary mechanisms that profoundly modify the host transcriptome upon HTLV-1 infection. We examined the relative distribution of TAX-regulated genes across the genome and found that genes producing alternatively spliced transcripts tended to be located closer to differentially expressed genes than expected by chance, suggesting that these two independent mechanisms could be locally interrelated (Figure 2C). The coordinates of TAX-regulated genes (DGE + ASE) significantly overlapped with differential topologically associating domains (TADs) (Figure 2D), leading us to examine whether TAX-induced gene regulations could colocalize with differential 3D chromatin interactions. To this end, we cross-compared the differential Hi-C contact matrix with genome coordinates of TAX-regulated genes and found that the majority of TAX-regulated genes appear to be intertwined in a highly interconnected network of 3D chromatin contacts that occur at both cis-chromosomal and trans-chromosomal levels (Figure 2E and Sup Fig 4). Using differential Hi-C matrix binned to 30 kbp, we identified 73% deregulated genes (including both DGE and ASE, 1173/1604) distributed in 1451 differential 3D chromatin interactions (Log2FC ≥ 0.5). Of those, 237 chromatin contacts involved genes affected in expression, consistent with previously described transcriptional condensates clustering multiple genes co-regulated in transcription (34,35,39). Additionally, 1047 chromatin contacts involved genes undergoing alternative splicing, of which 731 were spatially gathered with genes differentially expressed, suggesting that multigene complexes might coordinate both transcription and alternative splicing of co-regulated genes.

To further improve this Hi-C prediction, we conducted 4C assays to investigate physical interactions between genes expressing alternative spliced isoforms and distant genes that were concomitantly deregulated at the gene expression level by TAX. We selected three
distinct viewpoints in genes *CCNL1*, *SEC31B*, and *RFX2*, which were identified above as TAX-regulated targets for alternative splicing of exons E4, E9, and E18, respectively (Figure 2F-H) (18). Using these viewpoints in triplicate 4C assays, we found that upon TAX expression, *CCNL1*, *SEC31B*, and *RFX2* were connected more frequently with distal TAX-regulated genes *PTX3*, *ABCC2*, and *FUT3*, respectively (see Figure 2F-H). Overall, these results provide compelling evidence that TAX triggers long-range chromatin interactions that involve genes undergoing differential expression and/or alternative splicing regulation.

**TAX-induced NF-κB activation promotes gene-gene contacts and coordinated gene regulation between *NFKBIA* and *RELA*.**

To gain insight into the molecular mechanism underlying gene-gene contacts, we next aimed to assess whether specific gene subsets were enriched in chromatin interactions generated by TAX. Gene ontology analysis of TAX-regulated genes (including both DGE and ASE) identified in 3D chromatin contacts pointed to a significant enrichment in TNF-α and NF-κB signaling pathways, while remaining terms were related to oncoviral infection including that of HTLV-1 (Figure 3A). In line with this, analysis of transcription factor motifs in promoter sequences involved in chromatin contacts revealed a significant enrichment in NF-κB motifs (Sup Fig 5A). These results were reminiscent of previous studies reporting the formation of multigene complexes in which interacting genes are especially activated by NF-κB upon TNF-α stimulation (34,35). Accordingly, we sought to assess by chromosome conformation capture (3C) whether such NF-κB multigene complexes could pertain into TAX expressing cells. To this end, we developed a 3C assay and an inverse PCR (IPCR) assay (with no crosslinked chromatin) used as negative control. Validating our approach and ruling out false positive products derived from ligation of free DNA fragments, the 3C analysis, but not IPCR assay, allowed to detect a previously described TNF-α-related gene-gene interaction between *SAMD4A* and *GCH1* genes (distant of ~0.8 Mbp on chromosome 14) (Figure 3B) (34). Similar results were obtained in cells expressing TAX, indicating that TAX mimics TNF-α-activated *cis*-contacts (Figure 3B). In both experimental conditions, the *SAMD4A-GCH1* interaction coincided with a significant increase in *SAMD4A* expression (Figure 3B, right panel), thereby proposing that, as described for TNF-α, TAX promotes the formation of multigene complexes in which genes are coregulated in transcription.
As a logical progression, we next examined whether *trans*-chromosomal contacts previously
evidenced upon TNF-α between chromosomes 11 and 14 (34,35) may involve TAX-
deregulated genes. Our attention was drawn to RELA (chr11:65421067-65430443) and NFKBIA
(chr14:35870716-35873960), which were the only genes regulated by TAX found in both
chromosomes and because of their critical roles in NF-κB signaling, as well as in viral
persistence and ATL development (65). Both NFKBIA and RELA, which respectively encode the
inhibitor IκBα and the effector p65/RelA of NF-κB signaling, have been identified among the
most critical genes encoding TAX interactome and frequently affected by activating somatic
mutations in ATLL (65,66). RNA-seq data showed that NFKBIA was significantly upregulated
upon TAX expression, while RELA displayed no significant changes in gene expression but was
regulated at the alternative splicing level by the exon skipping of exon 6 (Sup Table 3). We
confirmed these RNA-seq predictions by RT-qPCR assays in HEK cells expressing TAX as well as
in HTLV-1-transformed C91PL cells (Figure 3C and Sup Fig 5C). Of note, the protein level of
p65/RelA in HEK cells appeared unaffected by TAX, whereas that of IκBα was downregulated,
which was consistent with its proteasomal degradation described upon TAX expression (Sup
Fig 5B) (67,68). In contrast to the wild-type TAX, the ectopic expression of TAXM22, a mutant
deficient for NF-κB activation, failed to influence both NFKBIA expression and RELA exon
skipping (Figure 3C), indicating that both transcriptomic events affecting NFKBIA and RELA
upon TAX expression relied on NF-κB activation.

To assess the impact of exon E6 skipping on protein translation, we used the GenBank
sequence NM_021975 as reference for the full transcript of RELA. An open-reading frame
prediction using Expasy tools revealed that, in comparison to the wild-type isoform that
encodes 551 amino acids, the mRNA isoform lacking exon E6 - referred to as RelAΔE6 - encodes
a truncated protein isoform of p65/RelA that consists of 153 amino acids (predicted at 44 kDa)
(Supplemental Figure 5D). This truncated isoform shares the rel homology domain with its wild-
type counterpart but lacks other domains that are involved in DNA binding and protein-protein
interactions. Ectopic expression of RelAΔE6 demonstrated that this p65/RelA isoform does not
possess transactivating effects in the absence of TAX when compared to its wild-type
counterparts (Figure 3D and Sup Fig 5E-F). In contrast, upon Tax expression, RelAΔE6 acts as a
repressive regulator for a subset of NF-κB-responsive genes, including *A20* and *BCL3*, while having no significant effects on *NFKB2* expression (Figure 3D and Sup Fig 5F). Additionally, RelAΔE6 displays an enhanced transactivating effect on the expression of other genes, such as IL8 (Figure 3D). These data indicate that the RelAΔE6 isoform induced by TAX is capable of fine-tuning gene expression related to NF-κB.

We next sought to test whether the concomitant deregulation of *NFKBIA* and *RELA* corresponded to increased *trans*-chromosomal contacts between chromosomes 11 and 14 upon TAX expression. To this end, we developed semi-nested 3C assays using both promoters of *RELA* and *NFKBIA* as anchors (Figure 3E). This approach revealed a single PCR signal attributed to *RELA-NFKBIA* contacts in TAX expressing cells, but not in CTL cells (Figure 3F). In comparison, there was no change in PCR signals deriving from an intragenic fragment of *RELA* used as loading control, as well as from an intra-*GAPDH* chromatin loop used as 3C control (34). These data demonstrated that TAX induced chromatin contacts between *NFKBIA* and *RELA* genes localized on chromosomes 14 and 11, respectively.

To determine whether the formation of a TAX-induced multigene complex involving *NFKBIA* and *RELA* participates in regulating the alternative splicing of *RELA* transcripts, we aimed to examine the *RELA* E6 skipping event in cells that are impaired for *NFKBIA-RELA* interaction. To this end, we used the dCas9-KRAB system (69), already identified as an effective steric barrier of chromatin contacts (70), to disrupt the contact between *NFKBIA* and *RELA* genes. Four distinct guide RNAs were designed to tether the dCas9-KRAB construct to the *RELA*-interacting genomic region of *NFKBIA* (Figure 3E). Quantitative ChIP (qChIP) experiments confirmed the specific tethering of dCas9-KRAB onto the *NFKBIA* target when compared to a gRNA-free experiment (Figure 3G). Under these conditions, the effect of TAX on the *RELA-NFKBIA* interaction was impaired (Figure 3H). Tethering the dCas9-KRAB on *NFKBIA* promoter did not interfere with the expression level of *RELA* but it significantly reduced the effect of TAX on *RELA* E6 exon skipping (Figure 3I). Importantly, we ascertained that the overexpression of ectopic IκBα had no impact on this negative effect of dCas9-KRAB (Sup Fig 5F-G), indicating that the loss of TAX-induced skipping of *RELA* E6 through dCas9-KRAB tethering to *NFKBIA* is due to disrupted contacts between *NFKBIA* and *RELA* rather than a reduced cellular abundance of IκBα. Collectively, these data define *trans*-chromosomal contacts between chromosomes 11
and 14 as an underlying mechanism for the concomitant TAX-driven regulation of *NFKBIA* expression and alternative splicing of *RELA* transcripts.

**Intragenic p65/RELA occupancy of RELA leads to RELA-NFKBIA contacts and alternative splicing of RELA E6.**

In light of our previous findings that p65/RelA occupancy of genomic exons can influence gene expression through alternative splicing (18), we next conducted qChIP assays to assess the chromatin distribution of p65/RelA across *RELA* and *NFKBIA* in cells with or without TAX expression. As shown in Figure 4A, TAX induced an average 2-fold enrichment of p65/RelA binding on the *NFKBIA* promoter, whereas it had no significant effects on *RELA* promoter occupancy. These data reflected the effects of TAX on the expression levels of *NFKBIA* and *RELA* genes (Figure 3C). In addition, we observed a significant increase in p65/RelA occupancy at the genomic exon E6 of *RELA* of TAX-expressing cells compared to control cells, suggesting that exon E6 belongs to exon subsets that are regulated by p65/RELA (Figure 4A). Moreover, disrupting *NFKBIA-RELA* contacts with the dCas9-KRAB in TAX-expressing cells led to a marked reduction in p65/RelA recruitment at the *NFKBIA* promoter, along with a significant decrease in p65/RelA occupancy of genomic exon E6, while there was no significant impact on p65/RelA occupancy of the *RELA* promoter. These findings suggest a direct relationship between p65/RelA, alternative splicing, and gene-gene contact regulation.

We previously demonstrated that experimental chromatin enrichment of p65/RELA in proximity to its genomic exon target can effectively lead to alternative splicing modification (18). To further investigate the role of p65/RelA in alternative splicing and 3D chromatin contacts, we designed dCas9 constructs fused or not to p65/RelA to assess the effect of p65/RelA locally tethered to the genomic *RELA* E6. We validated the specific tethering of the dCas9 constructs to this region using qChIP assays, compared to a dCas9-empty construct (Figure 4C). At the RNA level, this dCas9-mediated local p65/RelA enrichment resulted in exon skipping of E6 (Figure 4D), confirming that *RELA* E6 is an NF-κB responsive exon. Additionally, semi-nested 3C analysis of *NFKBIA-RELA* interactions revealed that E6-tethered dCas9-p65/RelA was sufficient to promote gene-gene interactions between *NFKBIA* and *RELA* (Figure 4E), thus identifying p65/RelA as an effector of multigene complex formation where NF-κB
Résultats

Coordinated Regulation of Transcription and Alternative Splicing by 3D Chromatin Remodeling upon HTLV-1 TAX-induced NF-κB activation

responsive genes are coordinated in their regulation at both transcriptional and alternative
splicing levels.
DISCUSSION

The 3D genome conformation is crucial for gene regulation, and understanding how viruses
manipulate it can reveal fundamental principles of gene regulation and cell behavior. Here, we
present evidence that the HTLV-1 viral oncoprotein TAX remodels the 3D chromatin
architecture of its host cells. Using Hi-C and 4C analyses, we found that TAX-induced changes
in long range chromatin interaction frequencies, compartmentalization, and inter-TAD
interactions coincided with differential gene expression and alternative splicing. We
demonstrated that gene-gene interactions led to coordinated splicing and transcriptional
events upon NF-κB activation. Our data show that TAX-induced NF-κB activation promotes
contacts between NFKBIA and RELA genes, which coincides with the skipping of exon E6 of
RELA and upregulation of NFKBIA expression. Disrupting the NFKBIA-RELA interaction impacted
RELA E6 exclusion at the RNA level, while targeted chromatin recruitment of dCas9-p65/RelA
triggered NFKBIA-RELA interaction and RELA E6 skipping. Our findings suggest that p65/RelA
plays a role in assembling multigene complexes, coordinating transcription and alternative
splicing regulation of NF-κB-responsive genes. This highlights a new mechanism coordinating
transcriptional and post-transcriptional changes through the NF-κB pathway and shows how
HTLV-1 TAX impacts molecular mechanisms linking 3D genome conformation to transcriptome
diversity.

Numerous cellular signaling pathways are known to influence alternative splicing (71,72), in addition to their effects on gene transcription. However, the specific mechanisms responsible for fine-tuning cellular responses to stimuli are not yet fully understood. In this context, studies examining the dynamics of 3D genome conformation have revealed the importance of multigene complex formation in gene coregulation upon cell signaling (34,35,40), where long-range interactions between cotranscribed genes help to organize transcription within nuclear condensates by establishing hierarchical relationships between gene loops. In light of our findings, we propose a model in which TAX-induced NF-κB activation leads to a persistent accumulation of p65/RelA at promoters and enhancers, forming nuclear condensates that assemble NF-κB-responsive genes for coordinated transcription, in line with previous reports (34,35,40). Simultaneously, based on the same physicochemical properties, genes with p65/RelA bound to genomic exons might be attracted to NF-κB factories without any
consequence on their transcriptional activity, due to the absence of NF-κB factors at their promoters. However, these multigene complexes could create a suitable environment for facilitating p65/RelA-dependent alternative splicing regulation.

Accordingly, the exon target specificity and the resulting splicing outcomes may then depend on the p65/RelA interactome, in which the presence of various RNA helicases like DDX17, DDX5, DHX9, DDX3X, G3BP1, DHX15, DDX1, and DDX21 is notable, as well as other splicing regulators including SF3B130 (19) and FUS (22,73). Interestingly, these factors have been recently linked to splicing regulatory subnetworks associated with genes located in the center of nucleus and composed of GC-rich intron-exon units (45). This observation is consistent with the features of splicing events initiated by TAX, which predominantly affect GC-rich exons (18). Furthermore, as an additional layer, splicing-associated chromatin signatures (SACS) have been shown to potentially recruit distinct splicing regulators to pre-mRNA of genes within the same functional pathway (74,75). Given its capacity to recruit diverse chromatin remodelers and splicing factors, p65/RelA emerges as a strong candidate for facilitating SACS. Since histone modifications influence both local and global 3D genome organization, p65/RelA could act as a key initiator of intricate and interconnected mechanisms that govern gene movements and splicing decisions upon NF-κB activation. Future studies should examine these points, focusing on characterizing the p65/RelA interactome and its role in alternative splicing regulation in the context of 3D genome.

Although the long-term effects of TAX-induced NF-κB activation have been widely studied, it remains unclear whether this activation leads to unique expression patterns of NF-κB-responsive genes that are specific to TAX. Our study reveals that TAX-induced NF-κB activation leads to gene-gene contact-dependent regulation of alternative splicing for exon E6 of RELA in both HEK and HTLV-1 transformed cells, resulting in the expression of a spliced isoform lacking E6. This isoform encodes a shorter p65/RelA protein of approximately 44 kDa (p65/RelAΔE6), which, unlike its wild-type counterpart, does not independently alter the expression levels of NF-κB-responsive genes. However, when TAX is expressed, ectopic expression of p65/RelAΔE6 appears to repress A20 and BCL3 while upregulating IL8 (Figure 3D and Sup Fig 5F). These findings suggest that NFKBIA-RELA contacts upon TAX-induced NF-κB activation allow the
generation of a RELA spliced isoform that might contribute to refining TAX activation-specific gene expression profiles.

Recent studies have reported that other oncogenic viruses, including human papillomavirus (HPV) (76), Epstein-Barr virus (EBV) (77), and hepatitis B virus (HBV)(78), reshape the 3D structure of their host cell genome, potentially contributing to complex mechanisms underlying gene perturbations that lead to cancer development. Regarding HTLV-1, its integration has been demonstrated to alter the 3D genome structure by forming loops between the proviral CTCF binding site and host genome regions located in *cis* (79). It has been proposed that the integration of an HTLV-1 provirus into a genomic region that typically occupies a transcriptionally repressive compartment in the nucleus, such as lamina-associated domains (LADs), could favor the survival of HTLV-1-infected cellular clones *in vivo* (80). However, some gene promoters associated with the nuclear lamina have been shown to escape transcription repression (81), suggesting a more complex and dynamic regulation in peripheral region of the nucleus. Given our findings that TAX can promote changes in long-range chromatin interactions affecting both *cis*- and *trans*-chromosomal regions, future investigations should examine how bursts of TAX expression influence the magnitude of proviral-genome contacts and their consequences on the clonal selection of infected cellular clones and the development of HTLV-1-associated diseases.

In summary, our study demonstrates that the HTLV-1 viral oncoprotein TAX remodels the 3D chromatin architecture of its host cell, influencing gene-gene interactions and coordinating transcription and alternative splicing regulation upon NF-κB activation. This sheds new light on the orchestration of the interplay between 3D genome conformation and transcriptome diversity, building upon earlier works from our group and others that demonstrated the role of intragenic chromatin loops in regulating transcription and alternative splicing (41,82-84). Our findings raise the question whether similar mechanisms could pertain to other cellular signaling pathways, such as TGFβ that has been linked to TGFβ factories and splicing regulation through a SMAD3:HNRNPE1 axis (34,85). Further investigations are needed to fully understand the complex relationship between cellular signaling pathways, chromatin architecture, coordinated transcriptional and alternative splicing regulation, and the impact of HTLV-1.
DATA AVAILABILITY

The raw data for Hi-C, 4C and RNA-seq have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number XXXXX, XXXX, and XXXX, respectively.
SUPPLEMENTARY DATA

Supplementary Data are available at NAR online.

**Supplementary Figure 1:** Hi-C data quality control analysis by HiC-Pro pipeline for TAX(+) and CTL conditions. (A-B) Quality control of read alignment and pairing for the CTL and TAX conditions, respectively. During this step, low-quality alignment, singleton, and multiple hits are removed. (C-D) represent the read pair filtering step, where read pairs are assigned to a restriction fragment and invalid pairs such as dangling-ends and self-circles are tracked but excluded from downstream analysis.

**Supplementary Figure 2:** Hi-C heatmaps and compartment analysis at 500kbp resolution. The figure shows Hi-C contact maps for each chromosome, visualized in logarithmic scale. Each heatmap represents the contact frequencies between genomic loci at a resolution of 500kbp. Below each heatmap, the eigen vector value of the corresponding chromosomal segment is presented. This value characterizes the compartmentalization of the genome into A (positive) and B (negative) compartments, which correspond to open and closed chromatin, respectively. The data were preprocessed and normalized using ICE algorithm. The visualization was generated using hicexplorer software.

**Supplementary Figure 3:** TAX alters long range chromosomal interaction and the compartmentalization of the genome. (A) Contact probability was calculated using hicexplorer as a function of genomic distance for interactions within individual chromosome arms across the whole genome. Lower panel indicates the log2FC of differential contact frequency between TAX and CTL condition. (B) The ratio of short-range (≤1 Mb) versus long-range (≥1 Mb) interactions (SVL) was calculated for each chromosome arm across the whole genome using hicexplorer. The SVL is significantly lower in Tax-expressing cells than in CTL cells (****P < 0.0001). (C) cis-eigen values are not affected genome wide by the expression of TAX Pearson test, R=0.948, p-value <0.0001). (D) Compartmentalization strength in CTL and TAX expressing cells. The global strength for compartmentalization is computed genome-wide as (AA + BB) / (AB + BA) after rearranging the bins of obs/exp based on their corresponding eigen values. (E) Correlation of 8 subcompartments identified by CALDER in CTL cells and histone mark enrichment for H3K27ac, H3K4me3, H3K9me3 obtained from publicly available Chip-seq data in HEK cells. (F) Sankey plot showing subcompartments switching upon TAX expression. (G) Stacked Hi-C data showing Hi-C heatmap matrix of CTL and TAX, eigen vector values indicating compartments A (red) and B
(bleu), insulation score of CTL (grey) and TAX expressing cells (orange) calculated using CTCF binding sites (below) obtained from public data.

**Supplementary Figure 4: Network analysis of TAX-regulated genes (DGE and ASE) involved in 3D chromatin contacts.** Overall, 1369 genes were identified to be involved in 3D chromatin contacts in TAX expressing cells, forming a main network consisting of 1247 TAX-regulated genes that are connected through 2671 chromatin contacts. Genes that belong to the same chromosome are color-coded uniformly. The shape of nodes representing genes indicates their modification upon TAX expression. The edges between the nodes (chromatin contacts) are indicated by different shapes, representing whether the interactions involved two genes deregulated both in transcription (dashed line), in alternative splicing (backward slash line), or one gene in transcription and the other at the alternative splicing level. Red edges indicate differential contact frequency between TAX expressing cells and CTL cells (abs(Log2FC)>0.5).

**Supplementary Figure 5: DNA binding of p65/RelA to the genomic exon E6 of RELA leads to concomitant RELA-NFKBIA interactions and exon skipping of RELA E6.** (A) DNA motifs enrichment analysis in the promoter regions of genes involved in 3D chromatin contacts in TAX-expressing cells using ShinyGO. (B) Quantitative RT-PCR analysis of gene expression levels of NFKBIA and RELA, as well as E6 exon skipping of RELA in Jurkat cells and HTLV-1 transformed T-cells C91PL. (C) Western blot analysis of p65/RelA, IκBα, and TAX in cells expressing TAX, TAX M22, and control cells. Vinculin was used as a loading control. (D) Prediction of the protein encoded by the splicing isoform of RELA skipped for exon E6 using Expasy-based tools. (E) Western blot analysis of RelA-WT and RelAΔE6 constructs using V5 antibodies. HEK293T cells transiently transfected with 200ng and 400ng of expression vectors for p65/RelA-WT (pRelA-WT) and p65/RelAΔE6 (pRelAΔE6) or empty vectors. (F) Effect of ectopic expression of p65/RelA-WT and the splicing isoform p65/RelAΔE6 on gene expression levels of NFKB2 and BCL3. HEK293T cells were transiently transfected with 400ng of expression vectors as described in Figure 3D. (G) Western blot analysis of p65/RelA, IκBα, and TAX in control cells and TAX-expressing cells transfected with pdCas9-KRAB along with the expression or not of guide RNAs and the vector pNFKBIA encoding IκBα. Vinculin was used as a loading control. (H) The ectopic expression of IκBα does not affect the effect of TAX, and that of RELA E6-tethered dCas9-KRAB, on exon skipping of RELA E6 (compared to Figure 3C). Quantitative RT-PCR analysis was carried out as described in Figure 3C.
ACKNOWLEDGEMENTS

We gratefully acknowledge support from Laurent Modolo at the LBMC for bio-
informatic advice, and from the PSMN (Pôle Scientifique de Modélisation Numérique)
of the ENS de Lyon for computing resources.

FUNDING

This work was supported by the Ligue Contre le Cancer (Comité de du Rhône), the
Fondation ARC (ARC PJA20151203399), and the Agence Nationale pour la Recherche
(program INFLASPLICE). P.M. was supported by a bursary from the French Ministry of
Higher Education and Science, the Fondation de France, and ANR INFLASPLICE; J.L.,
M.B., and L.B.A. by the Ligue Contre le Cancer; S.C. by XXXXX; N.F by ENS-Lyon; and
T.S., C.F.B, D.A., and F.M. by INSERM.

CONFLICT OF INTEREST

The authors declare no competing interests.
## FIGURES LEGENDS

**Figure 1. Tax reshapes the 3D chromatin conformation of the genome.** (A) Heatmap showing the normalized Hi-C interaction frequency map of chromosomes in control cells (CTL) and cells expressing the Tax protein (TAX). The upper panel depicts all chromosome matrices with diagonal line representing the intra-chromosomal contacts, while the off-diagonal regions show the inter-chromosomal interactions. The lower panel zooms at 500 kb resolution across entire chromosome 11. The right panel represents the differential heatmap between TAX and CTL conditions, with the log2 ratio indicated by the color scale. (B) compartment A and B in both TAX and CTL conditions. Compartment A and B are respectively shown in red and blue. The panel demonstrates all possible switch between compartment A and B (bleu square). The x-axis shows the genomic coordinates, and the y-axis represents the normalized compartment score. (C) Saddle plot of Hi-C data showing the homotypic (A-A and B-B) and heterotypic (A-B and B-A) cis-interaction frequencies. The right panel shows the differential saddle plot between TAX and CTL in log2 ratio contacts. (D) Percentage of switching compartments in cells expressing Tax (TAX) compared to control cells (CTL). (E) Sankey plot showing the proportion of genomic segments that switch between each subcompartment in TAX and CTL conditions. (F) Circos plot showing trans-chromosomal contacts identified by Hi-C data involved in the switching of subcompartments from control cells to TAX-expressing cells. (G) Venn diagram showing the overlap of TADs boundaries identified in control cells and cells expressing Tax. (H) Boxplots of the observed over expected (obs/exp) interaction frequencies for intra-TAD and inter-TAD interactions in control cells and TAX expressing cells. The obs/exp ratios are expressed as logarithmic value.

**Figure 2. TAX-induced 3D chromatin contacts associate with changes in both gene expression and alternative splicing.** (A) Venn diagram illustrating the genes affected by TAX expression in alternative splicing events (ASE) and those that are differentially expressed (DGE). The gene expression threshold is set at log2fc = 0.6 with a p-value of <0.05 (glm test), and the significant threshold for Δpsi is set at 5% with a p-value of <0.01 (Fisher test). (B) Number of different types of ASE that occur upon TAX expression. (C) Bedtools analysis (reldist) of the relative distance of gene submitted to alternative splicing modification to genes differentially expressed upon TAX. Control set was generated using shuffle function of bedtools that randomly permutes the genomic locations among the genome hg19. (D) Venn diagram displaying the number of TAX-regulated genes (DGE + ASE) that are located in differential TADs (Figure 1H). A hypergeometric statistical test was used to determine the significance of the overlap. (E) Cytoscape view of the network of genes that are connected to each other via 3D chromatin contacts. Genes that belong to the same chromosome are color-coded uniformly. The shape of nodes representing genes indicates their modification upon TAX expression. The edges between the
nodes (chromatin contacts) are indicated by different shapes, representing whether the interactions involved two genes deregulated both in transcription (dashed line), in alternative splicing (backward slash line), or one gene in transcription and the other at the alternative splicing level. The complete image is shown in Supplementary Figure 4. (F-H) chromatin contacts evidenced in triplicated 4C analysis of CCNL1 (E), SEC31B (F), and RFX2 (G), which were used as anchors (VP stands for viewpoint). Gene expression levels are shown for CTL (blue) and TAX(+) (red) conditions. The differentially expressed genes (DGE) and alternative splicing events (ASE) are specified above annotated genes (hg19), and TAX vs CTL values are indicated. The right panels show a zoom of the highlighted region (blue). The standard deviation across the 3 replicates of 4C assays is displayed in grey, and asterisks indicate significant changes in 4C signal (p-value <0.05, Fisher test).

**Figure 3. TAX-mediated changes in the 3D genome involved contacts of NF-κB responsive genes in both gene expression and alternative splicing.** (A) Gene Ontology (GO) analysis (KEGG pathways) of TAX-regulated genes involved in 3D chromatin contacts. The histogram indicates the significant enrichment of certain gene sets in the TAX-regulated genes (DGE+ASE). (B) Chromosome Conformation Capture (3C) analysis of SAMD4A and GCH1 upon either 24h TNFα exposure or TAX expression is depicted. The 3C templates were prepared using HindIII, and PCR was conducted using equal weights of DNA. The resulting gels were subjected to gel electrophoresis and SYBR green staining. The presence of a band indicates a high contact frequency between the respective primer targets. Internal SAMD4A primers were used as loading control amplimers. GAPDH primers were used for amplifying an intra-GAPDH loop as previously described (34). IPCR (inverse PCR) was conducted as 3C experiments with no chromatin crosslinking steps and was used to rule out signal providing from intra-looping of HindIII-undigested fragments. The 3C products were analyzed on agarose gels stained by SYBR green. The right panel shows the relative gene expression levels induced upon TNFα exposure and TAX expression. (C) Effects of TAX-WT and mutant TAX-M22, deficient for NF-κB activation, on gene expression levels of NFKBIA and RELA and on skipping of RELA exon E6. (D) Effect of ectopic expression of RelA-WT and the splicing isoform RelAΔE6 on gene expression levels of A20 and IL8. HEK cells were transiently transfected with 400 ng of expression vectors for p65/RelA-WT (pRelA-WT) and p65/ (pRelAΔE6) (or empty vector as control) along with TAX expression vector or its empty vector control. Total RNA was extracted from the cells at 48 hours post-transfection, and RTqPCR was performed as described in the Methods section. (E) Design of semi-nested 3C experiments targeting NFKBIA and RELA. Black arrows represent primers used for semi-nested 3C PCR, and grey arrows represent RELA primers used for loading control. (F) Semi-nested 3C analysis of chromatin contacts between RELA and NFKBIA in CTL and TAX-expressing cells. The resulting 3C products were analyzed in an agarose gel stained with Sybr-green. GAPDH primers were used for amplifying an intra-GAPDH loop as in panel B (G) Validation by qChip of dCas9-
KRAB tethering using four gRNAs (see Methods) on the *NFKBIA* promoter. The percentage input is presented for cells expressing TAX and those not expressing TAX. (H) Effect of dCas9-KRAB tethered to the *NFKBIA* promoter on *NFKBIA-RELA* contacts. Semi-nested 3C analysis of contacts between *RELA* and *NFKBIA* was conducted in CTL and TAX-expressing cells transected with the dCas9-KRAB expression vector along with the vector expressing the four gRNAs or its empty counterpart. The resulting 3C products were analyzed in an agarose gel stained with Sybr-green, as described in Panel F. (I) Functional impact of dCas9-KRAB-mediated inhibition of *NFKBIA-RELA* contacts on gene expression levels of *RELA* and exon skipping of exon *RELA* E6. For all panels, asterisks correspond to statistical significance calculated using Fisher's exact test (p-value threshold: *0.05, **0.01, ***0.001, ****0.0001).

**Figure 4.** The recruitment of p65/RelA to the gene body of RELA leads to an increase in exon skipping of RELA E6 concomitantly to an enrichment of chromatin contacts between RELA and NFKBIA (A) Quantitative ChIP analysis of p65/RelA occupancy of NFKBIA promoter, RELA promoter, and genomic exon E6 of RELA in cells with and without TAX expression, and with or without disruption of NFKBIA-RELA contacts by dCas9-KRAB. Histograms show the mean fold enrichment relative to input DNA, with error bars indicating SEM. (B) Design of semi-nested 3C experiment for analyzing NFKBIA-RELA contacts in cells expressing dCas9-p65/RelA tethered to the genomic exon E6 of RELA using 4 distinct gRNAs. (C) Validation of dCas9-p65/RelA tethering to the genomic exon RELA E6 by qChIP against the HA tag and p65/RelA. HEK cells were transfected with the expression vector of HA-dCas9-p65/RelA or the empty vector expressing HA-dCas9-empty (with no p65/RelA fused). (D) Effect of genomic RELA E6-tethered dCas9-empty and dCas9-p65/RelA on exon skipping of RELA E6. RT-qPCR analysis was performed using RNA extracted from cells expressing HA-dCas9-empty or HA-dCas9-p65/RelA and with or without TAX expression. (E) Effect of tethered dCas9-empty and dCas9-p65/RelA into genomic exon E6 on gene-gene contacts between NFKBIA and RELA. Semi-nested 3C experiments were carried out in cells expressing dCas9-p65/RelA tethered to the genomic exon E6, as illustrated in panel B. Cells with and without TAX expression were used as controls. For all panels, asterisk corresponds to statistical significance assessed using the Fisher test (*p* < 0.05).
**Supplementary Tables :**

All supplementary tables are available at the following link until 2023/06/12 :

<https://filesender.ens-lyon.fr/?vid=57848aa5-0566-2588-33a9-000008d25e86>

<caption>**Supplementary Table 1: list of oligonucleotides**</caption>

<caption>**Supplementary Table 2: comprehensive Overview of Interaction Data**</caption>

**Supplementary Table 3: Whole gene expression and alternative splicing changes upon Tax expression.** Whole gene expression threshold was set to log2(FC)=0.6, p<0.05 (glm and Wald test). For differential splicing, deltaPsi threshold was set to 0.5 p<0.01 (Fisher test). These data have been already published in Ameur LB, *et al*. in Nature Communications 2020.
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# Discussion générale et perspectives
Dans une première partie de mes travaux, j’ai contribué à mettre en évidence les effets de l’oncogène viral Tax du HTLV-1 sur les mécanismes de régulation de l’épissage alternatif. Nous avons montré que Tax pouvait altérer les profils d’épissage des transcrits cellulaires et aboutir à la synthèse d’isoformes retrouvées *in vivo* dans des cellules infectées non-transformées et les ATLL (Thénoz et al., 2014) (Annexe 1). Nous avons notamment identifié un nouveau mécanisme de contrôle de l’épissage alternatif qui s’opère lors de l’activation de la voie NF-κB à travers l’enrichissement intragénique du facteur NF-κB p65/RelA qui, par ces capacités à interagir avec les facteurs d’épissage, permet de définir des exons cibles propres à l’activation de la signalisation NF-κB. En particulier, via le recrutement de DDX17, nous avons montré que p65/RelA est capable de contrôler l’inclusion d’un grand nombre d’exons cassette (58%) régulés par Tax. L’enrichissement local de p65/RelA à lui seul par des essais de TALE est suffisant pour induire le recrutement du facteur d’épissage et influencer en conséquence le choix d’épissage de l’exon cible (Annexe 1). De façon intéressante, ce mécanisme est indépendant de la présence de Tax car il est retrouvé lors d’une activation de la voie NF-κB par le PMA, suggérant que Tax exacerbe un mécanisme physiologique qui permet de fournir des cibles de régulation d’épissage à travers p65/RELA lors de la signalisation NF-κB. Enfin, les régulations d’épissage par p65/RelA sont indépendantes de ses fonctions au promoteur du gène, posant la question des mécanismes de coordination des fonctions de régulation transcriptionnelle et d’épissage de p65/RelA à l’échelle du génome au cours de l’activation chronique de la voie NF-κB par Tax.

La seconde partie de mon travail a abordé cette question à travers une étude intégrative des régulations de l'expression des gènes et de la conformation tridimensionnelle de la chromatine lors de l'expression de Tax. Nos résultats montrent que Tax induit un remaniement des interactions chromatiniennes à longue distance et trans-chromosomiques. Les bordures de TADs semblent largement conservées lorsque Tax est exprimé, mais de nombreuses interactions intra-TADs, inter-TADs et également entre les compartiments A et B sont altérées. De façon intéressante, ces modifications des contacts chromatiniens à l'échelle tridimensionnelle coïncident avec des régions génomiques comportant des gènes dont les niveaux de transcription ou d'épissage alternatif sont affectés par Tax.
D'un point de vue moléculaire, nos données révèlent que l'activation de la voie NF-κB par Tax induit un rapprochement spatial entre les gènes *NFKBIA* et *RELA* qui dépend de l'enrichissement de p65/RelA dans la région génomique de l'exon E6 de *RELA* et qui induit une exclusion de l'exon 6 au niveau de l'ARNm de *RELA*. L'utilisation de constructions dCas9 nous ont permis de valider la relation de cause à effet entre l'enrichissement de p65/RelA intragénique, la formation des contacts chromatiniens entre les deux gènes et la régulation de l'épissage alternatif. Ces données révèlent un nouveau mécanisme de coordination des régulations transcriptionnelles et co-transcriptionnelles de la voie NF-κB à travers la formation de complexes multigéniques dans un contexte de modification de la conformation tridimensionnelle du génome par Tax.

<figure><img src="image_56.png" /><figcaption>Figure 34 : modèle de la coordination de la régulation transcriptionnelle et co-transcriptionnelle de la voie NF-κB par Tax.</figcaption></figure>
# Spécificité de l'activation de la voie NF-κB Tax-dépendante

## Intégration de plusieurs voies de signalisations par les stimuli d'activation de NF-κB

Nous avons montré que l'activation de la voie NF-κB par Tax et la présence de p65/RelA à la chromatine sont nécessaire au rapprochement de RELA et NFKBIA et à la régulation de l'épissage alternatif de l'exon RELA E6. De façon intéressante, l'exclusion de l'exon E6 de RELA n'est pas retrouvée lorsque la voie est activée par le TNFα (Figure 35) mais nous pouvons l'induire lors de l'exposition des cellules au PMA. Nous n'avons pas à ce jour de données permettant de corréler ces régulations à des contacts entre les deux gènes. Cela peut être dû au fait que Tax n'active pas seulement la voie NF-κB, mais module de nombreuses voies qui peuvent également influencer des gènes co-régulés par la voie NF-κB, tels que certains gènes contrôlés par la voie MAPK contrôlée également par Tax. Il a été observé que les complexes multigéniques observés lors de l'activation de la voie NF-κB comportent un certain chevauchement avec les complexes multigéniques générés lors d'une activation de la voie TGF-β (Papantonis et al., 2012). Les auteurs avancent l'hypothèse selon laquelle des usines à transcription pourraient permettre l'intégration de multiples signaux présentant des chevauchements dans leurs gènes cibles. Ainsi, Tax pourrait influencer le fonctionnement de ces usines à transcription et détourner l'activation de gènes contrôlés par plusieurs voies à la fois. Nous avons notamment identifié que les gènes formant des contacts et régulés dans leur expression, quantitativement ou qualitativement, par Tax présentent un enrichissement en motifs κB mais également bHLH et E2F (voir partie résultat, figure supplémentaire 5.A). Il serait ainsi intéressant de comprendre par des essais de ChIP et ChIP-seq si ces facteurs sont présents au niveau des gènes modulés et s'ils participent à la mise en place de contacts de chromatine et à la régulation de la transcription et de l'épissage alternatif par p65/RelA.
<figure><img src="image_57.png" /><figcaption>Figure 35 : Effets de la PMA et du TNFa sur l'exclusion de l'exon 6 de RELA.</figcaption></figure>

Coopération de p65/RelA avec des cofacteurs

Les enrichissements de motifs décrits ci-dessus pourraient signer la participation d'autres facteurs dans la formation de contacts entre les gènes induits par Tax. Des boucles de chromatine ont en effet déjà été observées au sein d'un même gène entre les sites AP-1/c-Jun et les sites κB, montrant que p65/RelA est capable de coopérer avec d'autres facteurs pour mettre en place de tels contacts (Chen and Chang, 2015; Zhao et al., 2011). Tax pourrait ainsi altérer ce type de mécanisme en modifiant l'expression des cofacteurs de p65/RelA ou via des interactions directes, et ainsi modifier, positivement ou négativement, certains contacts générés par p65/RelA lors d'une activation physiologique. Ces observations vont dans le sens des données de la littérature montrant que seulement un tiers des sites de fixations de p65/RelA à la chromatine sont communs quel que soit le stimulus d'activation de la voie NF-κB, montrant donc une très grande spécificité de la distribution de p65/RelA en fonction des stimuli (Borghini et al., 2018). C'est ce que nous observons ici pour la régulation de RELA E6, qui semble stimulus-spécifique. Tax semble ainsi induire une répartition spécifique de RelA à la chromatine et donc une activation singulière de la voie NF-κB.

Nos précédents travaux ont pu montrer que l'hélicase à ARN DDX17 est recrutée par p65/RelA à la chromatine (Annexe 1). L'activité hélicase de DDX17 permet de réguler au niveau de l'ARN l'inclusion de l'exon à proximité de la fixation intragénique de p65/RelA. Nous avons évalué que près de 58% des événements d'épissage alternatifs générés par Tax sont sensibles à
l'extinction de DDX17 et de son orthologue DDX5 par siRNA. L'interactôme de p65/RelA est en réalité riche en facteurs d'épissage, incluant d'autre hélicase à ARN telles que DDX3X, DDX1, DDX21, ou DHX15 (Feldman et al., 2007; Li et al., 2014). Ces hélicases à domaine DEAD-box sont par ailleurs des acteurs majeurs dans la formation de condensats transcriptionnels (Hondele et al., 2019). Il serait donc intéressant de réaliser des essais de marquage de proximité in vivo des protéines interagissant avec p65/RelA à la chromatine, comme par exemple en utilisant des technologies telles que Bio-ID ou APEX, techniques reposant sur une protéine de fusion induisant une biotinylation de proximité, suivie d'une purification et d'une identification protéomique des protéines biotinylées (Qin et al., 2021). Ces technologies permettraient de déterminer les interactions de p65/RelA en l'absence ou en présence de l'activation de la voie par Tax, et ainsi d'identifier des facteurs autres que DDX17 pouvant relier p65/RelA aux contrôle des interactions gène-gène et de l'épissage alternatif.

Outre les facteurs d'épissage, les marques d'histone peuvent aussi permettre le recrutement de facteurs d'épissage, ou influencer la vitesse d'élongation de l'ARNPII via un remodelage de la chromatine, et dans les deux cas moduler l'épissage des gènes (voir introduction partie 2.b.iii). Ainsi, outre DDX17, la fixation de p65/RelA à proximité d'exons régulés pourrait induire des modifications des marques d'histones permettant d'influencer l'épissage alternatif selon un modèle de recrutement ou un modèle de cinétique. Dans ce sens, le facteur de transcription p65/RelA interagit avec de nombreux facteurs de remodelage de la chromatine tels que des HDACs ou des HAT comme CBP/p300 (Zhong et al., 1998). Pour aborder cette question, il serait intéressant de générer des données de Chip-seq de différentes marques d'histones lors de l'expression de Tax, et notamment des marques déjà identifiés dans la régulation de l'épissage alternatif comme par exemple H3K27Ac, H3K36me3, ou H3K4me3 (Luco et al., 2010).

Outre p65/RelA, les facteurs NF-κB agissent en dimères à la chromatine, et la composition des dimères permet de moduler leur fixation au niveau des sites κB et la transactivation transcriptionnelle des gènes (Saccani et al., 2003). Il est fort probable que ces effets de composition de dimères NF-κB puissent exister lors de la reconnaissance des sites κB intragéniques proches des exons cibles. Cependant, les études par ChIP-seq de la distribution de NF-κB à la chromatine ne ciblent pour la plupart que le facteur p65/RelA. Il serait intéressant
de pouvoir évaluer par ChIP-seq la distribution des autres membres NF-κB lors de l'activation par Tax, et d'aborder par ChIA-PET l'engagement de ces facteurs dans la formation de boucles de chromatine pour mieux comprendre le rôle de chaque dimère dans les mécanismes qui relient le remodelage de l'organisation tridimensionnelle du génome et l'épissage alternatif lors de l'expression de Tax.

## Epissage alternatif et structures subnucléaires

### Localisation nucléaire et épissage alternatif

Des données récentes montrent que le mode de régulation de l'épissage alternatif est dépendant de la position radiale des gènes dans le noyau (central vs périphérique) et notamment de leur proximité avec les nuclear speckles (NS) (Barutcu et al., 2022; Tammer et al., 2022). Les NS situées au centre du noyau sont associées à une régulation des exons riches en GC. De façon intéressante, les exons régulés par Tax sont également des exons riches en GC (Annexe 1). Dans ce contexte, nous proposons un modèle dynamique qui concernerait la régulation de gènes localisés au centre du noyau qui pourraient modifier leurs interactions avec d'autres gènes par la formation de complexe multigéniques lors de l'activation de la voie NF-κB par Tax.

D'un point de vue mécanistique, notre modèle propose que lors de l'activation de la voie NF-κB par Tax, l'import nucléaire de p65/RelA permet un enrichissement massif des promoteurs comme des régions intragéniques cibles de p65/RelA. Comme précédemment décrit (Bertero et al., 2019; Papantonis et al., 2012), les complexes p65/RelA à la chromatine participent à la formation de condensat multigénique, vraisemblablement par des processus de transition de phase (LLPS), qui favorise la génération d'un microenvironnement riche en facteurs de transcription et d'épissage, comme déjà démontré pour les usines à transcription (Caudron-Herger et al., 2015; Melnik et al., 2011). Dans ces structures, seuls les gènes porteurs de p65/RelA au niveau de leur promoteur seraient régulés au plan transcriptionnel. En revanche, les gènes porteurs de p65/RelA à proximité des exons subiraient des régulations co-transcriptionnelles propres aux facteurs qui partagent des interactions directes ou indirectes avec p65/RelA, tels que des modifications des histones par le recrutement d'HDAC et ou HAT (comme CBP/p300) et/ou le recrutement de régulateurs de l'épissage tels que DDX17. Ce
mécanisme permettrait vraisemblablement de contrôler à travers l'enrichissement d'un unique effecteur de la voie, p65/RelA, un programme transcriptionnel et post-transcriptionnel approprié à la réponse cellulaire.

Reste à préciser les fonctions exactes de Tax dans ce mécanisme. Néanmoins, nos expériences de ChIP dirigées contre Tax ne permettent pas aujourd'hui d'affirmer la présence de Tax à la chromatine avec p65/RelA. Un tel complexe à la chromatine permettrait de recruter de nombreux facteurs transcriptionnels avec lesquels interagit Tax à la chromatine, et ainsi proposer que Tax puisse affecter la composition et les activités des transcriptions factories spécialisées pour ces facteurs. Il serait donc intéressant de pouvoir obtenir des données de ChIP-seq de la distribution de Tax et de p65 à la chromatine, et encore plus intéressant de réaliser des essais de ChIA-PET en immunoprécipitant Tax ou RelA afin d'obtenir un paysage des interactions longues distances dépendant directement de RelA et/ou de Tax, et relier ces dernières aux dérégulation en transcription et en épissage alternatif. Dans ce but, nous avons récemment développé une construction Avitag-Tax (tag cible de biotinylation) afin de pouvoir générer une protéine immunoprécipitable avec une grande spécificité et ainsi pouvoir générer des données de ChIP et ChIP-seq ou également de CHIA-PET de Tax.

## Tax speckles, nuclear speckles et usines à transcription

Dans le noyau, Tax est capable de former des corps nucléaires denses appelés Tax speckles (Bex et al., 1997; Semmes and Jeang, 1996). Ces structures sont enrichies en facteurs d'épissage tels que Sc35 et Sm, en facteurs NF-κB, en ARNPII active, et en ARN naissants à leur périphérie. Ces structures partagent donc des composantes avec les nuclear speckles (NS) habituellement marqués par Sc35, compartiments jouant un rôle important dans l'épissage. Mais la présence d'ARNPII active et de transcrits néo-synthétisés à la périphérie des Tax speckles pourrait suggérer que ces structures partagent également des fonctions avec les usines à transcription (Papantonis and Cook, 2013). Nous ne savons cependant pas si les Tax speckles sont des structures propres à Tax ou s'il s'agit de nuclear speckles, ou bien de usines à transcription, infiltrées et remodelées structurellement et fonctionnellement par Tax. Si c'était le cas, un tel mécanisme permettrait vraisemblablement de coordonner une régulation à la fois de la transcription et de l'épissage alternatif de certains gènes cibles pour générer un
profil d'expression d'isoformes transcriptionnelles propices à la prolifération des cellules infectées et à l'oncogenèse liée à HTLV-1.

Il a été récemment observé que l'ARNPII est capable de transiter entre condensats d'initiation (enrichis en Mediator) et les nuclear speckles, participant ainsi à la régulation de l'épissage des transcrits naissants (Guo et al., 2019). Les structures subnucléaires dédiées à la transcription, et à l'épissage semblent ainsi intimement liées. Une des hypothèses serait que Tax pourrait transiter entre ces condensats de régulation de la transcription et de l'épissage. Nous avons récemment observé que RelA co-localise avec DDX17 dans les corps nucléaires Tax (Figure 36). La présence de DDX17 dans ces structures suggère que les modifications d'épissage régulées par le complexe p65/RelA::DDX17 lors de l'activation de la voie NF-κB par Tax pourrait avoir lieu au sein de ces structures qui pourraient alors être considérées comme des « Tax factories » ou des « NF-κB factories » infiltrées et détournées par Tax.

Il serait intéressant d'étudier cette hypothèse en réalisant des essais de RNA-FISH sur des transcrits altérés par RelA (comme NFKBIA et RELA) combinés à des essais d'immunofluorescences de l'ARNPII (sous sa forme active S2P/S5P), afin de voir leur co-localisation. Il serait également possible d'envisager de pouvoir purifier de façon native ces structures subnucléaires, afin d'en déterminer la composition protéique par des approches de protéomique (Melnik et al., 2011).
Tax est-elle impliquée dans les processus de transition de phase liquide-liquide (LLPS)

La formation d'usines à transcription et de nuclear speckles est en partie expliquée par la capacité de facteurs à former une séparation de phase liquide-liquide (LLPS) pour générer un condensat nucléaire. Parmi les caractéristiques de la formation de LLPS, les propriétés physico-chimiques et notamment la composition des protéines joue un rôle important (Martin and Holehouse, 2020). Notamment, la présence de régions de désordre intrinsèque (IDR) dans les séquences protéiques est en particulier un élément prédictif de formation de nombreuses interactions multivalentes non spécifiques de faible force pouvant mener à la formation de condensats par LLPS. Une étude récente indique que Tax porte une IDR, ce qui permet par ailleurs d'expliquer les difficultés rencontrées dans les analyses cristallographiques de la structure de Tax (Boxus et al., 2008; Lyngdoh et al., 2019). Il serait donc intéressant d'étudier les capacités de Tax à former de la LLPS *in vitro* et d'évaluer si ses partenaires, tels que les hélicases de la famille DEAD-box particulièrement engagées dans la formation de condensat transcriptionnels, pourraient contribuer à former ces corps nucléaires Tax (Hondele et al., 2019). La co-localisation entre Tax et DDX17 dans les condensats Tax semblent soutenir cette hypothèse (Figure 36). Il faudra rechercher par RNA-FISH si ces structures Tax::p65/RelA::DDX17 peuvent participer à regrouper des gènes co-régulés transcriptionnellement et post-transcriptionnellement. Dans cette voie, il a récemment été observé que le facteur de transcription viral d'EBV EBNA2 est capable d'induire de la LLPS avec des composants du splicéosome pour réguler l'épissage alternatif cellulaire (Peng et al., 2021).
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Figure 36 : Tax co-localise avec RelA et DDX17 dans des structures discrètes subnucléaires. Des cellules HEK293T ont été transfectées transitoirement par des plasmides exprimant His-Tax (B) ou par un plasmide contrôle (A). 48 heures après transfection, les cellules ont été fixées, perméabilisées et marquées par des anticorps anti RelA, DDX17 ou His. Les cellules ont été montées dans du milieu de montage contenant du 4,6-diamidino-2-phenylindole. Les cellules ont été imagées sur un ZEISS LSM 980 avec Airyscan 2. Les images sont représentatives de la population cellulaire après transfection.

La réorganisation tridimensionnelle du génome par Tax dans les processus mutagènes.

La formation de complexe multigéniques par Tax et l'activation de la voie NF-κB pourraient-elles être impliquées dans l'incidence élevée de mutations somatiques observées dans les ATLL ? En effet, il a été démontré que l'organisation tridimensionnelle du génome est impliquée dans la cooccurrence de mutations entre des régions impliquées dans des contacts chromatiniens (Shi et al., 2016). De façon similaire, NFKBIA et RELA sont fréquemment retrouvés mutés dans des cellules d'ATLL (Kataoka et al., 2015). Le renforcement des contacts longue-distance vs courte distance par Tax pourrait refléter un relâchement de la chromatine qui permettrait sa diffusion plus facile dans le noyau, permettant ainsi à des régions chromatiniennes distantes d'entrer en contact. Une chromatine plus ouverte serait donc plus sensible à subir des mutations, car plus exposée aux espèces réactives de l'oxygène et aux agents mutagènes. Dans ce sens, il a été montré que les mutations apparaissant dans des régions de chromatine ouvertes sont associées à de nombreux cancers (Kikutake and Suyama,
2022). Il serait ainsi intéressant de pouvoir obtenir des données de Hi-C de cellules d'ATLL afin de comprendre si les régions fréquemment mutées dans les ATLL coïncident avec des fréquences de contacts plus importantes pour ces régions chromosomiques, en particulier si les gènes répondeurs à NF-κB mutés fréquemment dans les ATLL partagent les mêmes complexes multigéniques.

## Limites du modèle utilisé

Les travaux présentés ont été réalisées dans des cellules 293T, cellules n'étant pas la cible de l'infection par HTLV-1. Cependant, il existe un recouvrement entre les évènements d'épissage observés dans ce modèle et des cellules d'ATLL. En effet, 48% des évènements d'épissage alternatif observés dans les 293T exprimant Tax sont retrouvés *in vivo* dans des cellules d'ATLL. De plus, les régulations transcriptionnelles de RELA et NFKBIA observées dans les 293T exprimant Tax sont également retrouvées dans les cellules C91PL, infectées par HTLV-1. Bien que le paysage transcriptionnel et tridimensionnel des 293T puisse différer des lymphocytes T cibles du HTLV-1, et donc que la nature des gènes affectés de manière tridimensionnelle et (co)transcriptionnelle par Tax puissent différer, les effets de Tax seront les même. De plus, ces travaux se concentrent uniquement sur Tax, et ne prennent pas en compte l'expression de l'ensemble des protéines virales, qui pourraient influencer les effets de Tax à la fois sur le transcriptome et sur l'organisation du génome. Le modèle utilisé ici est cependant adéquat afin d'étudier l'effet de Tax seul, de façon simplifiée (facilité de manipulation des cellules 293T pour les méthodes employées), afin de disséquer un mécanisme général de Tax dans une cellule. Il serait cependant intéressant de comprendre quels sont les effets de Tax dans des lymphocytes T ou même dans des lignées HTLV-1 dans la réorganisation du génome.

## L'intégration de HTLV-1 dans le génome altère l'organisation tridimensionnelle du génome

Les modifications de la structure tridimensionnelle du génome hôte lors de l'infection par HTLV-1 ont déjà été suggérées à travers les fonctions d'un site CTCF (vCTCF) dans la séquence provirale (Satou et al., 2016). Des essais de 4C ciblant vCTCF ont montrés que le provirus forme des contacts avec les régions flanquantes et des régions distantes (>300kb) du site d'intégration. La mutation de vCTCF abouti à l'inhibition de la formation des boucles de
chromatine ainsi qu'à une dérégulation des gènes à proximité des régions en contact. Il a été
proposé que l'insertion du provirus permettrait de former des néo-TAD et/ou de modifier les
TADs présents dans la cellule, et d'interférer avec l'expression des gènes cellulaires.
Cependant, cette étude n'apporte pas de données concernant des contacts très longue
distance (à plusieurs Mb) ou inter-chromosomiques, qui permettraient d'avoir une vision plus
large des contacts et dérégulations engendrées par le provirus. Plus récemment, il a été
identifié que la région pX comporte une séquence enhancer pouvant également être mise en
jeu dans la formation de boucles de la chromatine et l'altération de l'expression des gènes
cellulaires (Matsuo et al., 2022). Il serait important de comprendre le rôle de Tax dans la
dynamique des contacts générés par le provirus HTLV-1, et d'en étudier les conséquences
fonctionnelles, à la fois en transcription et en épissage alternatif, comme dans la progression
de l'infection via l'expansion clonale des cellules infectées. Les analyses récentes de la
distribution des sites d'intégration suggèrent que la présence du provirus dans les régions
proches de la lamine seraient enrichies dans les clones cellulaires infectés retrouvés in vivo
(Melamed et al., 2022). Les auteurs proposent que ce type de localisation en périphérie du
noyau permettrait une extinction de l'expression d'HTLV-1 et un meilleur échappement des
cellules infectées à la pression du système immunitaire. La localisation du provirus dans
l'espace nucléaire participerait ainsi à la sélection clonale des cellules infectées in vivo. Il serait
intéressant de réaliser des analyses Hi-C dans des cellules infectées par HTLV-1 ou encore dans
des cellules d'ATLL afin d'obtenir une vision d'ensemble des dérégulations de l'organisation du
génome par HTLV-1. Il serait également important de mesurer l'impact de l'expression de Tax
sur la localisation du provirus dans l'espace nucléaire et de ses contacts cis- et trans-
chromosomique. Il serait pour cela envisageable de réaliser des essais de 4C en ciblant le
génome proviral dans des cellules issues de patients et cellules cultivées ex-vivo pour
permettre la réactivation de l'expression virale. La comparaison des données de ce type
d'approche combinée aux analyses transcriptomiques (expression de gène et épissage)
réalisées chez les individus asymptomatiques et les patients atteints de TSP/HAM et d'ATLL,
permettrait de mieux comprendre le rôle de cette nouvelle composante fonctionnelle d'HTLV-
1 sur la dynamique des interactions tridimensionnelle du génome dans le développement des
pathologies associées au virus.
Conclusion

Pour conclure, mes travaux de thèse ont permis de montrer que l'activation de la voie NF-κB par l'oncogène Tax de HTLV-1 induit une réorganisation du génome qui induit la formation de complexes multigéniques composés de gènes régulés par p65/RelA dans leur niveau transcriptionnel mais également dans leur épissage alternatif. Ces données démontrent pour la première fois que la formation de ces complexes multigéniques permet à p65/RelA de coordonner à la fois la régulation de la transcription et de l'épissage alternatif des transcrits de façon NF-κB-dépendante. Ces données redéfinissent la fonction de p65/RelA, initialement défini comme un facteur de transcription, comme un facteur régulant à la fois de manière quantitative et qualitative le transcriptome. Son action n'est pas restreinte aux régions flanquantes de sa fixation au niveau d'un promoteur ou d'un corps de gènes mais elle affecte la régulation d'autres gènes à distance (en cis ou en trans-chromosomique) via des modifications de la conformation tridimensionnelle du génome. Ces données révèlent un mécanisme supplémentaire dans la complexité de régulation des gènes associés à l'activation de la voie NF-κB qui pourrait concerner d'autres voies de signalisation de manière plus générale. Enfin, ces travaux permettent d'apporter un regard nouveau sur les mécanismes moléculaires impliqués dans les phénomènes d'inflammation cellulaire et d'oncogenèse, reliés à HTLV-1 ou à d'autres situations pathologiques associées à l'activation chronique de la voie NF-κB.
# Annexes
Annexe 1
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ARTICLE

https://doi.org/10.1038/s41467-020-16853-x

OPEN

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# Intragenic recruitment of NF-κB drives splicing modifications upon activation by the oncogene Tax of HTLV-1

Lamya Ben Ameur<fnref n="1" />, Paul Marie<fnref n="1" />,⁸, Morgan Thenoz<fnref n="1" />,²,⁸, Guillaume Giraud<fnref n="1" />,⁸, Emmanuel Combe<fnref n="1" />, Jean-Baptiste Claude<fnref n="1" />, Sebastien Lemaire<fnref n="1" />, Nicolas Fontrodona<fnref n="1" />, Hélène Polveche³, Marine Bastien<fnref n="1" />,⁴, Antoine Gessain⁵, Eric Wattel⁶,⁷, Cyril F. Bourgeois<fnref n="1" />, Didier Auboeuf<fnref n="1" />✉ & Franck Mortreux<fnref n="1" />✉

Chronic NF-κB activation in inflammation and cancer has long been linked to persistent activation of NF-κB-responsive gene promoters. However, NF-κB factors also massively bind to gene bodies. Here, we demonstrate that recruitment of the NF-κB factor RELA to intragenic regions regulates alternative splicing upon NF-κB activation by the viral oncogene Tax of HTLV-1. Integrative analyses of RNA splicing and chromatin occupancy, combined with chromatin tethering assays, demonstrate that DNA-bound RELA interacts with and recruits the splicing regulator DDX17, in an NF-κB activation-dependent manner. This leads to alternative splicing of target exons due to the RNA helicase activity of DDX17. Similar results were obtained upon Tax-independent NF-κB activation, indicating that Tax likely exacerbates a physiological process where RELA provides splice target specificity. Collectively, our results demonstrate a physical and direct involvement of NF-κB in alternative splicing regulation, which significantly revisits our knowledge of HTLV-1 pathogenesis and other NF-κB-related diseases.

<footnote n="1">¹Laboratory of Biology and Modelling of the Cell, Univ Lyon, ENS de Lyon, Univ Claude Bernard, CNRS UMR 5239, INSERM U1210, 46 Allée d'Italie Site Jacques Monod, 69007 Lyon, France. ²Department of Pediatrics and Medical Genetics, Faculty of Medicine and Health Sciences, 9000 Gent, Belgium. ³CECS, I-Stem, Corbeil-Essonnes 91100, France. ⁴School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, Liverpool, UK. ⁵Unité d'Epidémiologie et Physiopathologie des Virus Oncogènes, Institut Pasteur, Paris, France. ⁶Université Lyon 1, CNRS UMR5239, Oncovirologie et Biothérapies, Faculté de Médecine Lyon Sud, ENS - HCL, Pierre Bénite, France. ⁷Université Lyon 1, Service d'Hématologie, Pavillon Marcel Bérard, Centre Hospitalier Lyon-Sud, Pierre Bénite, France. ⁸These authors contributed equally: Paul Marie, Morgan Thenoz, Guillaume Giraud. ✉email: didier.auboeuf@inserm.fr; franck.mortreux@ens-lyon.fr</footnote>
The human T-cell leukemia virus (HTLV-1) is the etiologic agent of numerous diseases, including adult T-cell leukemia/lymphoma (ATLL)¹, an aggressive CD4⁺ T-cell malignancy, and various inflammatory diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)². Changes at the gene expression level participate in the persistent clonal expansion of HTLV-infected CD4⁺ and CD8⁺ T-cells, leading ultimately to HTLV-1-associated diseases³. We recently reported that alternative splicing events help to discriminate between ATLL cells, untransformed infected cells, and their uninfected counterparts derived from patients⁴. Alternative splicing of pre-messenger RNAs is a co-transcriptional processing step that controls diversity in both the transcriptome and the proteome and that governs cell fate. Its regulation relies on a complex and still incompletely understood interplay between splicing factors, chromatin regulators, and transcription factors⁵,⁶. For instance, we still do not fully understand the molecular mechanisms of HTLV-1-induced splicing modifications, or whether these effects rely on interplay between transcription and splicing.

Tax is an HTLV-1-encoded protein that regulates viral and cellular gene transcription. Tax also alters host signaling pathways that sustain cell proliferation, leading ultimately to cell immortalization⁷. The nuclear factor-κB (NF-κB) signaling pathway is the most critical target of Tax for cell transformation⁸. The NF-κB transcription factors (RELA, p50, c-Rel, RelB, and p52) govern immune functions, cell differentiation, and proliferation⁹. NF-κB activation involves the degradation of IκB, which sequesters NF-κB factors in the cytoplasmic compartment; this leads to NF-κB nuclear translocation and binding of NF-κB dimers (of which RELA:p50 is the most abundant) to their target promoters¹⁰,¹¹. Tax induces IKK phosphorylation and IκB degradation, leading to persistent nuclear translocation of NF-κB¹²,¹³. In addition, Tax interacts with nuclear NF-κB factors and enhances their effects on transcription¹⁴,¹⁵.

Interestingly, genome-wide analyses of NF-κB distribution have unveiled that the vast majority of RELA peaks is outside promoter regions and can be localized in introns and exons¹⁶⁻¹⁹. Some of those promoter-distant RELA-binding sites correspond to cis-regulatory transcriptional elements²⁰,²¹ but globally, there is a weak correlation between the binding of RELA to genes and regulation of their steady-state expression¹⁷,¹⁸. These data suggest that NF-κB could have other functions than its initially described function as a transcription factor.

Here, we show that NF-κB activation accounts for alternative splicing modifications generated upon Tax expression. These effects rely on a tight physical and functional interplay between RELA and the DDX17 splicing factor, which dynamically occurs upon NF-κB activation. Our results reveal that RELA DNA binding in the vicinity of genomic exons regulates alternative splicing through DDX17 recruitment, which then modulates exon inclusion via its RNA helicase activity.

## Results

**Tax regulates splicing regardless of its transcriptional effects.**

We performed RNA-seq analyses on 293T-LTR-GFP cells transiently transfected with a Tax expression vector. We then identified Tax-induced changes in gene expression level and in alternative splicing and annotated them as previously described²²,²³ (Supplementary Data 1 and 2). Notably, the ectopic expression of Tax affected the splicing and gene expression levels of 939 and 523 genes, respectively (Fig. 1a). A total of 1108 alternative splicing events was predicted, including 710 exon skipping events (Fig. 1b). A small percentage of genes (3.5%, 33/939) was altered at both their expression and splicing levels,

indicating that Tax largely affects alternative splicing independently of its transcriptional activity. We validate a subset of splicing events by RT-PCR (Fig. 1c). To test whether Tax-induced splicing modifications occurred in vivo, we took advantage of a publicly available RNA-seq dataset (EGAS00001001296²⁴) from three healthy control donors, three asymptomatic carriers (AC), and 55 ATLL patients (Supplementary Data 1). Although lowly expressed, Tax mRNA was detected in the vast majority of infected samples (Supplementary Fig. 1). It is important to note that ATL can sporadically express Tax in bursts²⁵,²⁶; thereby explaining, at least in part, their Tax-like features even though Tax is poorly detected in ATL samples²⁴. Based on this, we examined the percent spliced-in (PSI) values of exons expressed in samples from donors, carriers, and patients with ATLL (Supplementary Data 1). Overall, 542 (48%) Tax-induced splicing modifications were detected at least once in 55 ATL samples (Supplementary Data 1). This low frequency was anticipated due to the high fluctuations between individuals of Tax expression levels, proviral loads, and infected cell clonality, as well as somatic alterations (e.g., gene mutations, deletions, and duplications), which accumulate in ATLL cells²⁴. Some Tax-related splicing events were recurrently detected in asymptomatic and ATLL samples (Fig. 1d). In addition, using exon-specific RT-PCR assays, we confirmed that Tax promotes splicing events that have been previously described in HTLV-1-infected individuals, including in the AASS, CASK, RFX2, and CD44 genes⁴,²⁷ (Fig. 1c). Altogether, these results uncovered a large number of splicing modifications that occurred upon Tax expression, which to a degree coincide with alternative splicing events observed in HTLV-1 patients.

**DDX17 interacts with RELA upon Tax-induced NF-κB activation.** As Tax is a well-known trans-acting transcription regulator, we first analyzed whether Tax could affect gene expression levels of splicing factors. However, no significant change was measured for 227 genes encoding splicing regulators (Supplementary Data 1, Fig. 2a), thereby suggesting a direct role of Tax in alternative splicing regulatory mechanisms. To tackle this question, we focused on the auxiliary component of the spliceosome DDX17, which has been previously identified, but not validated, in a recent mass spectrometry screen for putative protein partners of Tax²⁸.

We therefore aimed to validate the interaction between Tax and DDX17. As shown in Fig. 2b, Tax co-immunoprecipitated with the two endogenous isoforms of DDX17, namely p72 and p82. Reciprocal IP confirmed this interaction (Fig. 2c). Due to the involvement of NF-κB signaling in Tax-positive cells (Fig. 1d¹¹), we examined whether DDX17 interacts with a Tax mutated form, namely M22 (G137A, L138S), which is defective for IKK and NF-κB activation²⁹⁻³². Despite similar expression levels and immunoprecipitation efficiencies of Tax and M22 (Fig. 2d), we failed to detect any interaction between M22 and DDX17 (Fig. 2b,c), suggesting that NF-κB is required for recruiting DDX17. In this setting, RELA co-immunoprecipitated with DDX17 and Tax, but not with M22 (Fig. 2b, c). Moreover, DDX17 was co-immunoprecipitated with RELA in a Tax-dependent manner (Fig. 2e). This interaction did not require RNA as the DDX17: RELA complex remained detected when cell extracts were pretreated with RNAse A (Fig. 2f).

As DDX17:RELA complexes were observed neither in control cells (that do not expressed Tax) nor in M22-expressing cells, this suggested that NF-κB activation is necessary for the binding of DDX17 to RELA. This hypothesis was confirmed by exposing M22-expressing cells to TNFα, a potent NF-κB activator that allowed retrieving DDX17:RELA complexes (Fig. 2g). Altogether,
<figure><img src="image_61.png" /><figcaption>**Fig. 1** Tax induces alternative splicing modifications independently of its transcriptional effects. **a** Genes regulated at the steady-state expression level and at the splicing level upon Tax expression in HEK cells. The significance thresholds were typically set to 10% for ΔPSI (differential percentage of spliced-in sequence) and 0.6 for log2-gene expression changes (*p* < 0.05, Fisher's exact test), respectively. **b** Different alternative splicing events induced by Tax: alternative acceptor (acc), alternative donor (don), exon skipping (ES), multi-exon skipping (MES), and multi-exclusive exon skipping (MEx). **c** Validation of alternative splicing predictions by RT-PCR (using 35 cycles). The exon number is indicated in red. CD44 full variants (Ev*) were assessed using primers C13 and C12A (Supplementary Fig. 5). Representative image from three independent experiments is shown. **d** Exon-based hierarchical clustering. Kruskal-Wallis ANOVA (*p* < 0.05) was carried out with Mev4.0 software (http://www.tm4.org/) using the PSI values of exons that share similar regulations upon Tax and in clinical samples (EGAS00001001296). Only the most significant exon regulations are presented. Source data are provided as a Source Data file.</figcaption></figure>

these results revealed that Tax dynamically orchestrates interactions between the transcription factor RELA and the splicing regulator DDX17 by activating the NF-κB signaling pathway (Fig. 2h).

**Tax-mediated effects on splicing depend on DDX5/17.** To estimate the role of DDX17 in Tax-regulated splicing events, RNA-sequencing was performed using 293T-LTR-GFP cells expressing or not Tax and depleted or not for DDX17 and its paralog DDX5, which cross-regulate and complement each other<sup>22,33,34</sup>. Tax had no effect on the expression of DDX5 and DDX17 (Figs. 2a and 3a) and RELA protein level was not significantly changed upon both Tax expression and DDX5/17 silencing (Fig. 3b).

Overall, 58.5% (648/1108) of Tax-regulated exons were affected by DDX5/17 knockdown, a significantly higher proportion than expected by chance (Fig. 3c, Supplementary Fig. 2a). Of particular significance, 423 Tax-induced splicing events were completely dependent on the presence of DDX5/17 (Supplementary Data 3). For example, DDX5/17 silencing completely abolished the

Tax-mediated effect on splicing of SEC31B, CASK, MYCBP2, CCNL1, ROBO1, and ADD3 transcripts (Fig. 3d). Tax, as well as siRNA-mediated DDX5/17 depletion, had no marked effects on gene expression levels of those genes (Supplementary Fig. 2c, d). Of note, splicing specific RT-PCR assays permitted to validate the effect of DDX5/17 on Tax-dependent splicing changes for CD44, ADD3, and EIF4A2 transcripts, even though their predicted differential inclusion fell below the arbitrary computational threshold (Fig. 3d and Supplementary Fig. 2b). This suggested that the contribution of DDX5/17 to Tax-mediated alternative splicing regulation might be under-estimated.

As NF-κB activation modified the interactions between DDX17, RELA, and Tax (Fig. 2), we next examined the interplay between NF-κB activation and DDX17-mediated splicing regulation. As shown in Fig. 3d, M22 did not have any effect on DDX5/17-sensitive splicing events, arguing that Tax splicing targets are regulated by RNA helicases DDX5/17 in an NF-κB-dependent manner. This was further confirmed using siRNA-mediated depletion of RELA that abolished DDX17-dependent splicing regulations by Tax (Fig. 3d).
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**Fig. 2** Physical interactions between Tax, RELA, and DDX17 in an NF-κB dependent manner. **a** Mean average plot (n = 3, *p* < 0.05) of cellular gene expressions upon Tax. Each gene is plotted according to its expression level (log10(BaseMean) from DESeq2 analysis) and to fold change (log2-FC) upon Tax. Red dots show significant gene expression changes in HEK cells (log2-FC > 0.6, *p* < 0.05, Fisher's exact test). Black dots highlight genes encoding splicing factors. DDX5 and DDX17 are indicated. **b** Immunoprecipitation assays (IP) were carried out in HEK cells using isotype IgG or anti-DDX17 (b, g), anti-REL A (e, g), and anti-Tax (c, g) antibodies, followed by immunoblotting (IB) with indicated antibodies. **c** Western blot analysis of Tax and M22 expression 48-h post-transfection. **d** RNA-free IP assays. **e** TNFα exposure of M22-expressing cells promotes RELA-DDX17 interactions. **f** Model of NF-κB-dependent interplay between Tax, RELA, and DDX17. For **b–g**, a representative image from three independent experiments is shown. Source data are provided as a Source Data file.

**Functional insight of Tax and DDX17-dependent spliced genes.** Gene ontology (GO) analysis of quantitatively altered genes revealed several signaling pathways that are well described in Tax-expressing cells, including NF-κB, TNF, and MAPK signaling (Supplementary Fig. 3C)¹¹,³⁵. In contrast, genes modified at the splicing level belong to membrane-related regulatory processes including focal adhesion and ABC transporters (Supplementary Fig. 3C). In particular, the term *Focal Adhesion* was

shared between Tax splicing targets identified in infected samples (RNA-seq datasets, Supplementary Data 1) and those regulated by DDX17 (Supplementary Data 3) (Supplementary Fig. 3C). In addition, using an exon ontology (EO) approach that we recently developed to estimate enrichment in protein features encoded by exons³⁶, we found that Tax- and DDX17-regulated exons encoded for regions involved in functionally validated post-translational modifications (PTM), protein structure and
<figure><img src="image_70.png" /><figcaption>**Fig. 3** DDX5/17 regulates Tax splicing targets in an NF-κB-dependent manner. **a** Western blot analysis of DDX5 and DDX17 expression in HEK cells expressing or not Tax and depleted or not of DDX5 and DDX17 by siRNA. **b** Western blot analysis of RELA and β-actin upon Tax expression and siRNA-DDX5/17 delivery. **c** Splicing events modified upon the depletion of DDX5/17 in Tax-positive HEK cells. The significant threshold was set to ≥2 in comparisons between TaxvsCTL and TaxsiDDX5/17vsCTL. For **b** and **c**, a representative image from three independent experiments is shown. **d** Validation of alternative splicing predictions of a set of Tax- and DDX5/17-regulated exons. TaxM22 and siRNA-mediated RELA depletion were used in order to assess the dependency of splicing events on NF-κB activation. Histograms represent the results of exon-specific quantitative RT-PCR measurements computed as a relative exon inclusion (alternatively spliced exon vs constitutive exon reflecting the total gene expression level). All genes but MYCBP2 were unmodified at the whole transcript level upon Tax expression (Supplementary Fig. 2c). Data are presented as the mean ± SEM values from biological replicates. Each black square represents a biological replicate. Statistical significance was determined with two-way ANOVA followed by Fisher's LSD test (\*\*p < 0.01, \*\*\*p < 0.0001). Exact *p*-values for Tax vs CTL: 0.0068 for SEC31B; 0.0084 for CASK; <0.0001 for MYCBP2; <0.0001 for CCNL1; 0.0063 for ROBO1; <0.0001 for ADD3. Source data are provided as a Source Data file.</figcaption></figure>

binding functions (Supplementary Data 4 and Supplementary Fig. 3C). These data are reminiscent of PTM related to protein regions encoded by variable CD44 exons that also control the cell-adhesion properties of CD44 (Supplementary Fig. 3D). Accordingly, we observed that Tax-expressing HEK cells displayed switched cell-adhesion properties from hyaluronate- to type IV collagen-coated surfaces, which is in accordance with the substrate affinity of the CD44 v10 isoform³⁷ (Supplementary Fig. 3E).

**RELΑ recruits DDX17 at the vicinity of genomic exons.** The results described above prompted us to focus on *CD44* as gene model to further examine the mechanisms underlying RELA and DDX17 splicing regulations upon Tax. *CD44* is composed of 10 constitutive exons and 10 variable exons. The constitutive exons 1–5 and 15–20 encode the standard *CD44* transcripts, while *CD44* variants (*CD44v*) are produced by extensive splicing leading to alternative inclusion of variable exons 5a-14 also named v1-v10 (Fig. 4b)³⁸. As shown in Fig. 4a, the exon v10 inclusion rate is markedly influenced by Tax in a DDX5/17- and NF-κB activation-dependent manner. The importance of NF-κB in this process was further confirmed as the inactivation of NF-κB via the ectopic expression of the *IκBα* super repressor (*IκBSR*) abolished the effects of Tax on *CD44* v10 inclusion (Supplementary Fig. 4A). Remarkably, in line with previous work suggesting that NF-κB directly regulates the *CD44* promoter, we also noticed a slight reduction in *CD44* expression in Tax-expressing cells knocked down for RELA (Supplementary Fig. 4B), thereby defining *CD44*

regulations as an appropriate situation to address whether or not RELA-dependent transcription and splicing are two interrelated processes.

Using quantitative ChIP (qChIP) analyses, we first observed that Tax expression led to recruit RELA not only to the *CD44* promoter, but also to a genomic region spanning the alternative exon v10, but not a downstream constitutive exon (E16) (Fig. 4b, c, left panel). To assess whether RELA occupancies at the v10 exon and *CD44* promoter are interrelated, a stable cell line was generated in which the κB site localized at -218 bp from the transcription start site (TSS) was deleted using a CRISPR-Cas9 approach. Positive clones (*CD44ΔκB*) were screened and sequenced to confirm the 40 bp deletion in the promoter region (Fig. 4b). As expected, Tax expression failed to promote RELA binding at the promoter in CD44ΔκB cells (Fig. 4c, right panel), coinciding with a decreased *CD44* expression comparable to that observed upon RELA depletion (Supplementary Fig. 4B, C). Nevertheless, Tax still promoted RELA binding at the v10 region. Importantly, Tax expression induced v10 inclusion at a similar level in both CD44ΔκB and parental cells (Fig. 4d). These results suggested that Tax-mediated effect on exon v10 splicing could depend on RELA binding in the vicinity of the alternative v10 exon. Supporting this hypothesis, the analysis of publicly available RELA ChIP-seq datasets revealed that intragenic RELA peaks are significantly closer to alternative exons than to constitutive exons (Supplementary Fig. 4D). More particularly, alternative exons regulated by Tax, including those sensitive to DDX5/17 depletion, were identified in genomic regions enriched in NF-κB-binding sites compared to alternative exons non-regulated by Tax (Fig. 4e).
<figure><img src="image_71.png" /><figcaption>**Fig. 4** Intragenic RELA-binding sites associate with alternative splicing events. **a** Alternative splicing modifications of the CD44 exon v10 upon in HEK cells expressing or not Tax and knocked down or not for RELA or DDX5/17 expressions. TaxM22 and siRNA-mediated RELA depletion were used to assess the dependency of splicing events after NF-κB activation. Histograms represent the results of exon-specific quantitative RT-PCR measurements computed as a relative exon inclusion (alternatively spliced exon vs constitutive exon reflecting the total gene expression level). **b** Schematic representation of the human CD44 gene. Black and white boxes represent constitutive and alternative exons, respectively, as previously annotated (50). The orange box represents the kB site localized at ~218 bp from the TSS and the 40 bp fragment deleted by CRISPR/Cas9 in CD44ΔkB HEK cells. **c** qChIP analysis of RELA occupancy across the promoter, the exon v10, and the constitutive exon E16 of CD44. RELA enrichment is expressed as the fold-increase in signal relative to the background signal obtained using a control IgG. **d** Relative exon inclusion of CD44 exon v10 was quantified by qRT-PCR in parental cells and its CD44ΔkB counterparts. **e** Distribution of alternative exons that are regulated or not by Tax and DDX5/17 in RELA-enriched intragenic regions. The analysis was restricted to alternative exons expressed in HEK cells and regulated or not by Tax. Boxes extend from the 25th to 75th percentiles, the mid line represents the median and the whiskers indicate the maximum and the minimum values. **f** Bootstrapped distribution of median distance between intragenic RELA peaks and either Tax-regulated exons (red line, 1079 bp) or randomly chosen exons (10⁵ repetitions) (blue). **p**-values were determined by sample t-test. **g** Consensus de novo motif for RELA-binding sites <1 kb of Tax-regulated exons. Data are presented as the mean ± SEM values from biological replicates. Each black square represents a biological replicate. Statistical significance was determined with two-way ANOVA followed by Fisher's LSD test (*p* < 0.05, **p** < 0.01, ***p** < 0.001) (a, c, and d) and two-tailed Wilcoxon test (e, ****p* < 0.0001). Exact **p**-values for Tax vs CTL: a <0.0001, c parental, promoter: 0.0248 and V10: 0.0005; CD44ΔkB, V10: 0.021; d parental: 0.0028; CD44ΔkB: 0.0054. Source data are provided as a Source Data file.</figcaption></figure>

Accordingly, we observed that RELA-binding sites are often found in the vicinity of Tax-regulated exons (Fig. 4f). Using the MEME-ChIP suite as motif discovery algorithm³⁹, we uncovered that RELA-binding sites located within the closest range (<1 kb) of Tax-regulated exons coincided with the typical NF-κB consensus motif (Fig. 4g). Furthermore, this subset of Tax-regulated exons displayed weak 3' and 5' splice sites together with significant low minimum free energy (MFE) value (Supplementary Fig. 4E) and high GC-content (Supplementary Fig. 4F) when compared to all human exons. This emphasizes the high potential of these splice sites to form stable secondary RNA structures, a typical feature of exons regulated by RNA helicases DDX5/17³³.

Taken together, these data define a signature of splicing target specificity for RELA, and they suggest that RELA and DDX17 might control together the inclusion of a subset of Tax-regulated exons. We therefore investigated the genomic occupancy of some

target exons by RELA and DDX17 by qChIP analysis of cells expressing or not Tax. For all tested genes (CD44, SEC31B, CASK, and MYCBP2), both RELA and DDX17 bound specifically the regulated alternative exon in a Tax-dependent manner, compared to a downstream constitutive exon (Fig. 5a and Supplementary 4g). Remarkably, the knockdown of RELA expression by siRNA affected DDX17 chromatin occupancy on Tax-regulated exons (Fig. 5a), despite its slight positive impact on DDX17 expression (Supplementary Fig. 2d). We further validated the RELA/DDX17-dependent alternative splicing events in C91PL and ATL2 cells compared to the non-infected MOLT4 cells (Fig. 5c). The magnitude of splicing regulation was higher in ATL2 than in C91PL cells, and appeared to positively correlate to a higher expression level of both DDX17 and RELA, and a high level of constitutive NF-κB activation, as reflected by IL8 expression (Fig. 5b). As formerly observed in HEK cells expressing Tax, these
<figure><img src="image_72.png" /><figcaption>**Fig. 5 RELA locally recruits DDX17 at the genomic target exons, leading to splicing regulation.** a Relative occupancy of DDX17 at Tax-regulated genomic exons in cells that did or did not express Tax and knocked down or not with RELA-specific siRNA. b Relative gene expression levels of IL8, DDX17, and RELA in ATL2, C91PL, or HEK cells transiently transfected by pSG5M-Tax as compared uninfected MOLT4 cells. Tax mRNAs levels are expressed in arbitrary units (a.u.). c Alternative splicing modifications in the HTLV-1-infected cell lines ATL2 and C91PL as compared to those in the uninfected cell line MOLT4. Relative exon inclusion was measured as described in Fig. 3. d Relative RELA and DDX17 occupancies of regulated exons in ATL2 cells as compared to MOLT4 cells. Each occupancy of regulated exon by RELA and DDX17 is represented as a fold of that measured at its neighboring constitutive exon. Source data are provided as a Source Data file. Data are presented as the mean ± SEM values from biological replicates. Each black square represents a biological replicate. Statistical significance was determined with two-way ANOVA followed by Fisher's LSD test (a, d) and two-tailed unpaired t-test (b, c) (*p* < 0.05, **p** < 0.01, ***p** < 0.001, ****p** < 0.0001). Exact *p*-values for Tax vs CTL (a): 0.0312 (CD44), 0.0337 (SEC31B), 0.0002 (CASK), 0.0171 (MYCBP2). Exact *p*-values for ATL2- and C91PL vs MOLT4 (b): 0.0001, 0.0004, 0.0003 and <0.0001, 0.0365, 0.0007 corresponding to IL8, DDX17, and RELA, respectively. For Tax expression, *p* = 0.0016 HEK + Tax vs ATL2. Exact *p*-values for ATL2- and C91PL vs MOLT4 (c): 0.0002, 0.0008, 0.0153, 0.0105 and 0.019, 0.0013, <0.0001, 0.11 for CD44, SEC31B, CASK, and MYCBP2 respectively. Exact *p*-values (d) for ATL2 vs MOLT4: qChIP-RELA < 0.0001 (CD44), 0.0003 (SEC31B), 0.0004 (CASK), 0.0029 (MYCBP2); qChIP-DDX17 0.0004 (CD44), 0.0211 (SEC31B), 0.0207 (CASK), 0.0184 (MYCBP2). Source data are provided as a Source Data file.</figcaption></figure>

splicing events in HTLV-1-infected cells coincided with a high and significant increase in chromatin occupancy of both RELA and DDX17 at the vicinity of regulated exons (Fig. 5d). These data reveal that the RELA:DDX17 axis in the splicing regulatory network pertains to both HEK293T and CD4⁺-infected cells expressing Tax.

**Causal relationship linking RELA and DDX17 to splicing regulation.** To more confidently assess the causative relationship linking RELA and DDX17 to alternative splicing, we experimentally tethered DDX17 or RELA to the CD44 v10 exon locus using modified TALE (Transcription-Activator-Like-Effector)⁴⁰. For this, we designed a TALE domain that recognizes specifically an exonic 20 bp DNA sequence located 12 bp upstream from the 5' splice site (SS) of exon v10. This TALE domain was fused to either RELA or DDX17 proteins. We also used an additional construct consisting in the same TALE fused to GFP to rule out

non-specific effects resulting from the DNA binding of the TALE. After transient transfection of each TALE construct into 293T-LTR-GFP cells (Fig. 6a), we monitored the relative effects on the recruitment of endogenous RELA and DDX17 as well as on exon v10 splicing; all results were normalized and expressed as relative effects compared to the TALE-GFP (Fig. 6a-c). As expected, and validating our approach, TALE-RELA tethering to the exon v10 led to a significant chromatin recruitment of RELA to its target site, and not to the downstream exon E16 used as control (Fig. 6a, left panel). Further, a significant and specific DDX17 enrichment was observed at exon v10 after expression of TALE-RELA as compared to TALE-GFP (Fig. 6a, left panel), indicating that tethering RELA to exon v10 induced local recruitment of the endogenous DDX17 protein. At the RNA level, this TALE-RELA-mediated recruitment of DDX17 coincided with a significant increase in the exon v10 inclusion rate (Fig. 6a, right panel).
<figure><img src="image_73.png" /><figcaption>**Fig. 6** Chromatin relationship between RELA and DDX17. **a–c** Chromatin and splicing regulation upon TALE-mediated tethering of RELA and DDX17. The TALE domain was designed to bind the v10 exon of CD44 and fused to either GFP (**a–c**), RELA (**a**), DDX17, (**b**), or a helicase-deficient mutant DDX17_K142R (**c**). The effect of TALEs on RELA and DDX17 chromatin enrichment (left panels) and on the relative v10 exon inclusion (right panels) was monitored in HEK cells by qChIP and qRT-PCR, respectively. Results were normalized to measures obtained in TALE-GFP assays. (**d**) Relative exon inclusion rate of exon v10 of CD44 in HEK cells expressing or not the Tax mutant M22 and the TALE-DDX17 construct. **e** Relative exon inclusion rate of exon v10 of CD44 in HEK cells exposed to TNFα and PMA. **f** Relative exon inclusion rate of exon v10 of CD44 in HEK cells transiently transfected with increasing amounts of RELA expression vector (200 and 500 ng). **g** Model of NF-κB-dependent regulation of alternative splicing. Upon NF-κB activation, DNA-bound RELA proteins act as chromatin anchors for DDX17, which then provides splicing target specificity due to its RNA helicase activity. Data are presented as the mean ± SEM values from biological replicates. Each black square represents a biological replicate. Statistical significance was determined with two-tailed unpaired t-test (qChIP in **a–c**) and one-way ANOVA followed by Fisher's LSD test (relative exon inclusion (REI) in **a–f**) (*p* < 0.05, **p** < 0.01, ***p* < 0.001, ****p < 0.0001). In **a–c**, exact *p*-values for TALE-RELA vs TALE-GFP on V10: 0.049 (qChIP RELA), 0.0079 (qChIP DDX17), 0.0276 (REI). Exact *p*-values for TALE-DDX17 vs TALE-GFP on V10: 0.0139 (qChIP DDX17), 0.0276 (REI). Exact *p*-values for TALE-DDX17_K142R vs TALE-GFP on V10: 0.0312 (qChIP DDX17). In **d–f**, exact *p*-values are <0.0001 (M22 + TALE-DDX17 (**d**)), 0.0125 (TNFα (**e**)), <0.0001 (PMA (**e**)), 0.0369 (0.2 µg (**f**)), 0.0012 (0.5 µg (**f**)). Source data are provided as a Source Data file.</figcaption></figure>

We next investigated whether DDX17 tethering could result in similar effects. Quantitative ChIP analysis demonstrated that DDX17 was properly tethered to exon v10 when fused to the designed TALE, but TALE-DDX17 had no effect on RELA

recruitment (Fig. 6b, left panel). This was expected since the formation of RELA:DDX17 complexes only occurs upon NF-κB activation (Fig. 2). Nevertheless, TALE-DDX17-expressing cells exhibited a reproducible and significant increase in v10 inclusion
(Fig. 6b, right panel), indicating that chromatin-bound DDX17 alone (e.g., without RELA) can modulate splicing efficiency. Of note, the levels of v10 exon inclusion induced by TALE-RELA and TALE-DDX17 were comparable to that measured in cells transiently transfected with a Tax expression vector (Fig. 4d and Supplementary Fig. 4A). Although less quantitative, a nested RT-PCR assay clearly confirmed these results (Supplementary Fig. 5b). Strikingly, however, the mutant TALE-DDX17_K142R (a DDX17 helicase mutant$^{33,41-43}$) failed to increase the levels of exon v10 inclusion, despite a clear chromatin enrichment of the DDX17 mutant (Fig. 6c and Supplementary Fig. 5b). Collectively, these results demonstrate that RELA binding in the vicinity of genomic exons recruits the RNA helicase DDX17, which positively regulates the inclusion rate of the target exon due to its RNA helicase activity. Of note, this causal relationship between RELA, DDX17, and splicing regulation was observed irrespectively of the expression of Tax. Indeed, TALE-DDX17 rescued the inclusion of exon v10 in cells expressing the NF-κB defective mutant M22 (Fig. 6d). In addition, Tax-independent activation of NF-κB in HEK cells (using either TNFα or Phorbol 12-myristate 13-acetate (PMA)) mimicked the effects of Tax on splicing regulation (Fig. 6e). More importantly, ectopic RELA expression had similar effects, in a dose-dependent manner (Fig. 6f). Further, we observed a dynamic induction of RELA:DDX17 interactions after RELA expression or PMA activation (Supplementary Fig. 5c), but not after TNFα activation, likely due to a lower level of RELA nuclear translocation in this condition (Supplementary Fig. 5d). Collectively, these results suggest that the mechanistic role of Tax is to promote DDX17-dependent splicing regulation by promoting constitutive activation of NF-κB pathway.

Discussion

Since the finding of splicing dysregulations in HTLV-1-infected individuals$^{4,24,27,44}$, deciphering how HTLV-1 interferes with the splicing regulatory network has become a new challenging issue for improving our knowledge of HTLV-1 infection and its associated diseases. Here, we provide the molecular evidence that upon Tax-induced NF-κB activation, RELA directly regulates splicing by binding to gene bodies at the vicinity of GC-rich exons and by locally recruiting the splicing factor DDX17, which regulates splicing via its RNA helicase activity.

Our results demonstrate that Tax deeply impacts alternative
splicing independently from its effects on transcription. In
addition, Tax-regulated exons were found in transcripts enriched
in functional pathways that are distinct from those enriched by
Tax transcriptional targets, suggesting that splicing reprogram-
ming may constitute an additional layer of regulation by which
HTLV-1 modifies the host cell phenotype. Supporting this, we
also showed that the Tax-induced splicing variant CD44 v10,
which was previously identified in circulating blood of HAM/TSP
patients²⁷ and confirmed here ex vivo in infected CD4⁺ T-cell
clones, contributes to modulating cell-adhesion affinity in vitro.
GO analyses of Tax splicing targets also identified the GO terms
phosphatidylinositol signaling system and inositol phosphate
metabolism, two processes that are particularly connected to NF-
κB signaling and that have critical roles in oncogenesis and dis-
ease progression of malignant diseases, including ATLL$^{45,46}$.
Furthermore, exon ontology analysis predicted that critical
changes at the protein level would affect the experimentally
validated protein structure and post-translational modifications,
which together are likely to affect the connectivity network
between proteins, and subsequently contribute to modifying cell
phenotypes. This suggests that, beside its transcriptional effects,
splicing regulatory functions of Tax might contribute to its
oncogenic properties. Indeed, a large number of Tax-regulated

exons could be observed in ATLL samples, which rarely express
Tax but typically exhibit NF-κB addiction for survival and
proliferation<sup>24,26,47</sup>.

At the molecular level, we showed that increased chromatin
occupancy of RELA upon Tax expression is not restricted to
promoter regions but also occurs in the vicinity of exons that are
regulated at the splicing level (Figs. 4 and 5). Exons regulated by
Tax, especially those localized within 1 kb of intragenic RELA-
binding sites, are characterized by a high GC-content, a typical
feature of exons regulated by the DDX5 and DDX17 RNA heli-
cases<sup>33</sup> (Supplementary Fig. 4E, F). Accordingly, we found that a
majority of Tax-regulated exons depend on the expression of
these proteins (Fig. 3c). A local chromatin recruitment of DDX17
and RELA was validated on several Tax-regulated exons (Figs. 4
and 5). More importantly, we identified a confident causal rela-
tionship between exon tethering of RELA, local recruitment to
chromatin of DDX17, and subsequent splicing regulation via
DDX17 RNA helicase activity (Fig. 6). This catalytic activity of
DDX17 was strictly required for its splicing regulatory functions
(Fig. 6), as previously reported<sup>33</sup>. Indeed, the RNA helicase
activities of DDX5 and DDX17 have been implicated in resolving
RNA structures, facilitating the recognition of the 5' splice site
(which can be embedded in secondary structures), and exposing
RNA-binding motifs to additional splicing regulators<sup>3,34,42,48-50</sup>.
However, even though some RNA-binding specificity has been
reported for DDX17<sup>51,52</sup>, these RNA helicases are devoid of a
proper RNA-binding domain, and their activity in splicing likely
depends on additional factors that are able to provide target
specificity. Here, we suggest that RELA is a DDX17 recruiter,
acting as a chromatin anchor for DDX17 in the vicinity of exons
dynamically selected upon NF-κB activation. While performing
complementary experiments, we notified that siRNA-DDX5/17
affected the RELA chromatin occupancy of both exons and
promoters regulated by Tax (Supplementary Fig. 6). This is
reminiscent of recent report indicating that DDX5/17 depletion
affects interactions between the transcription factor REST and
DNA, defining DDX5/17 as REST transcriptional coregulators<sup>22</sup>.
Altogether, these data indicate that the RELA-DDX17 chromatin
interplay relies on complex mechanisms that deserve future
investigations.

The target specificity of NF-κB factors remains a complex
question. It has been estimated that ~30–50% of genomic RELA-
binding sites do not harbor a typical NF-κB site, and only a
minority of RELA-binding events have been associated with
transcriptional change$^{16–19}$, thereby indicating that neither hav-
ing a consensus site nor significant NF-κB occupancy are suffi-
cient criteria for defining RELA’s target specificity. Here, we
identified a typical NF-κB consensus motif at RELA-binding loci
that are close to alternatively spliced exons but we also uncovered
that weak splice sites, low MFE, and significant GC-content bias
of exons likely contribute to RELA’s target specificity. Because
low MFE and high GC-content confer a high propensity to form
stable RNA secondary structures, the recognition and the selec-
tion of such GC-rich exons with weak splice sites by the splicing
machinery typically depend on the RNA helicases DDX5/17$^{33}$.
Based on these observations, we propose a model of RELA-
induced splicing target specificity (Fig. 6g), whereby, upon NF-κB
activation, RELA binds to intragenic-binding consensus motifs
and locally recruits DDX17. When the RELA:DDX17 complex is
located in close proximity of GC-rich exons flanked by weak
splice sites, DDX17 can increase their inclusion rate by
unwinding GC-rich secondary structures of the nascent RNA
transcript, and potentially also by unmasking binding motifs for
additional splicing regulators. Of note, although Tax was identi-
fied in both RELA- and DDX17-containing complexes, we
demonstrated that Tax-independent NF-κB activation is sufficient
for promoting interactions between RELA and DDX17 and the corresponding splicing regulation. Without ruling out possible additional effects of Tax on the RELA:DDX17 complex, such as favoring dimer formation (as already proposed for NF-κB dimers¹⁵), these data indicate that Tax exacerbates a dynamic and physiologic process involving RELA in splicing target specificity.

In conclusion, our results provide conceptual advances for understanding how cell signaling pathways may drive target specificity in splicing by dynamically recruiting cognate transcription factors at the vicinity of target exons that act as chromatin anchor for splicing regulators. In the context of NF-κB signaling, such a mechanism likely has a significant impact on cell fate determination and disease development associated with HTLV-1 infection and other situations linked to chronic NF-κB activation, such as human inflammatory diseases and cancer.

## Methods

**Cell culture and transfections.** Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll separation of whole blood from HTLV-1-infected individuals in the context of a Biomedical Research Program approved by the Committee for the Protection of Persons, Ile-de-France II, Paris (2012-10-04 SC). All individuals gave informed consent. PBMCs were cloned by limiting dilution in RPMI 1640 supplemented with penicillin and streptomycin, sodium pyruvate, non-essential amino acids, 2-mercaptoethanol, 10% filtered human AB serum, 100 U/ml recombinant IL-2 (Chiron Corporation), and 75 µM HTLV-1 integrase inhibitor L-731,988. Clones were phenotyped by flow cytometry using antibodies against CD4 (Dako-Cytomation) and isotype-matched controls on a FACScan system using CellQuest software (Becton Dickinson). HTLV-1-positive clones were assessed by PCR. The human embryonic kidney 293T-LTR-GFP cells⁵³, which contain an integrated GFP reporter gene under the control of the Tax-responsive HTLV-1 LTR, were cultured in DMEM + glutamax medium supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. This cell line was used to measure transcription efficiency in Tax and TaxM22 conditions. In standard transfection experiments, siRNAs (Supplementary Data 5) and/or expression vectors (pSG5M empty, pSG5M-Tax-WT, pSG5M-M22) were mixed with JetPrime (Polyplus Transfection) following the manufacturer's instructions, and cells were harvested 48 h after transfection. TNFα exposure consisted in treating cells with 10 ng/ml of TNFα for 24 h. HTLV-1-chronically infected lymphocytes ATL2 (kind gift from Masao Matsuoka (Kyoto University, Kyoto, Japan) and Roberto Accolla (Università degli Studi dell'Insubria, Varese, Italia)), C91PL (kind gift from Cynthia Pise-Maison (National Cancer Institute, NIH, Bethesda, MD)) and Renaud Mahieux (Center for Research in Infectious Diseases, Lyon, France) and non-infected MOLT4 cells (CRL-1582, ATCC) were grown in RPMI 1640 medium (Gibco, Life Technologies) supplemented with 10% heat-inactivated fetal calf serum, 20 IU/ml penicillin, 20 µg/ml streptomycin, and 25 mM HEPES.

**Cell-adhesion assays.** Culture plates were prepared by coating with 40 µg/ml hyaluronic acid from human umbilical cord (Sigma) and 25 µg/ml type IV collagen from human placenta (Sigma) overnight at 4 °C. Non-specific binding sites were blocked for 1 h with PBS containing 1 mg/ml heat-denatured BSA. After three washes with 1× PBS, 5 × 10⁴ cells transiently transfected with pSG5M-Tax vector or its empty control were added at 48-h post-transfection. Cell adhesion was allowed to proceed for 20 min at room temperature. Non-adherent cells were removed with three washes with 1× PBS, and adherent cells were quantified. All experiments were done in triplicate.

**Cell fractionation.** HEK cells were resuspended in A buffer complemented with DTT, PhosSTOP⁻ and protease inhibitor cocktail (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 1× PhosSTOP⁻, 1× protease inhibitor cocktail) then incubated on ice for 10 min. The lysates were then centrifuged at 4 °C for 3 min at 1000×g. Supernatants were next resuspended in A⁺ buffer complemented with DTT, PhosSTOP⁻ and protease inhibitor cocktail (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 0.2% IGEPAL® CA-630, 0.5 mM DTT, 1X PhosSTOP⁻, 1× protease inhibitor cocktail) then incubated on ice for 2 min. Lysates were then centrifuged at 4 °C for 1 min at 1000×g and supernatant were kept as cytoplasmic extract. Pellets were extensively washed with A⁺ buffer and centrifuged, then resuspended in Lysis Buffer complemented with DTT, PhosSTOP⁻ and protease inhibitor cocktail (50 mM Tris-HCl pH 8.0, 400 mM NaCl, 5 mM EDTA, 0.2% SDS, 1% IGEPAL® CA-630, 1 mM DTT, 1× PhosSTOP⁻, 1× cComplete⁻ EDTA free protease inhibitor cocktail (Roche)) and incubated 30 min one ice before sonication using Diagenode Bioruptor™ Plus (6 cycles 30°/30°, high power). Nuclei lysates were centrifuged at 4 °C for 10 min at 16,000×g and the supernatant were kept as nuclear extracts.

**Western blot.** Cells were washed twice with 1× PBS and total proteins were directly extracted in RIPA buffer (50 mM Tris-HCl pH 7.4, 50 mM NaCl, 2 mM EDTA, 0.1% SDS, 1× PhosSTOP⁻, 1× cComplete⁻ EDTA free protease inhibitor cocktail (Roche)). A total of 20 µg of whole-cell proteins were separated on a NuPAGE™ 4–12% Bis-Tris Protein Gels and transferred on a nitrocellulose membrane using Trans-Blot® Turbo™ Blotting System. Membranes were saturated with 5% milk and incubated overnight at 4 °C with the primary antibodies against RELA (sc-109, Santa Cruz, 1:1000), Tax (1A3, Covalab, 1:500), DDX17 (ab24601, Abcam, 1:2000), DDX5 (ab10261, Abcam, 1:2000), actin (sc-1616, Santa Cruz, 1:1000), GAPDH (sc-32233, Santa Cruz, 1:10,000), H3 (ab1791, Abcam, 1:10,000), V5 (AB3792, Millipore, 1:1000), and α-Tubulin (sc-32293, Santa Cruz, 1:2000). After three washes with 1× TBS-Tween, membranes were incubated 1 h at room temperature with the secondary antibodies conjugated with the HRP enzyme and washed three times as above. Finally, the HRP substrate (GE Healthcare or Immobilon Forte (Millipore)) was applied to the membrane for 5 min, and the chemiluminescence was read on Chemidoc (BioRad).

**Co-immunoprecipitation.** Cells were harvested in IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 10% glycerol). Extracts were incubated overnight with 5 µg of antibodies recognizing RELA (C20 sc-372, Santa Cruz), Tax (1A3, Covalab), and DDX17 (ProteinTech) in the presence of 30 µl Dynabeads® Protein A/G (ThermoFisher). Isotype IgG rabbit (Invitrogen) or mouse (Santa Cruz) was also used as negative control. The immunoprecipitated complexes were washed three times with IP lysis buffer.

**Chromatin immunoprecipitation.** A total of 10⁷ cells were crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched by addition of 0.125 M glycine. Nuclei were isolated by sonication using a Covaris S220 (2 min, Peak Power: 75; Duty Factor: 2; Cycles/burst: 200), pelleted by centrifugation at 1000×g for 5 min at 4 °C, washed once with FL buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% NP-40) and resuspended in 1 ml shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 mM EDTA, 0.1% SDS). Chromatin was sheared in order to obtain fragments ranging from 200 to 800 bp using Covaris S220 (20 min, Peak Power: 140; Duty Factor: 5; Cycles/burst: 200). Chromatin was next immunoprecipitated overnight at 4 °C with 5 µg of antibodies, of anti-RELA (C20 sc-372, Santa Cruz), anti-DDX17 (19910-1-AP, ProteinTech), or anti-V5 (AB3792, Millipore), and 30 µl Dynabeads® Protein A/G (ThermoFisher) were added. Complexes were washed with 5 different buffers: Wash 1 (1% Triton, 0.1% NaDOC, 150 mM NaCl, 10 mM Tris-HCl pH 8), Wash 2 (1% NP-40, 1% NaDOC, 150 mM KCl, 10 mM Tris-HCl pH 8), Wash 3 (0.5% Triton, 0.1% NaDOC, 500 mM NaCl, 10 mM Tris-HCl pH 8), Wash 4 (0.5% NP-40, 0.5% NaDOC, 250 mM LiCl, 20 mM Tris-HCl pH 8, 1 mM EDTA), and Wash 5 (0.1% NP-40, 150 mM NaCl, 20 mM Tris-HCl pH 8, 1 mM EDTA). The immunoprecipitated chromatin was purified by phenol-chloroform extraction, and quantitative PCR was performed using Rotor-Gene 3000 cycler (Corbett) or LightCycler 480 II (Roche, Mannheim, Germany). Values were expressed relative to the signal obtained for the immunoprecipitation with control IgG. Primers used for ChIP experiments were designed for exon/intron junction (Supplementary Data 5). For TALE ChIP experiments, DDX17 and RelA enrichment were normalized to the signal observed with V5 antibody corresponding to TALE recruitment. The TALE-GFP condition was used as control and set to 1. Note that in TALE assays qChIP experiments and exon-specific RT-qPCR were carried out at 24-h post-transfection.

**RNA extraction, PCR, and real-time quantitative PCR.** Total RNAs were extracted using TRIzol (Invitrogen). RNAs (2.5 µg) were retro-transcribed with Maxima First Strand cDNA Synthesis Kit after treatment with dsDNase (Thermo Scientific) following the manufacturer's instructions. PCRs were performed using 7.5 ng of cDNAs with GoTaq polymerase (Promega, Madison, WI, USA). PCR products were separated by ethidium bromide-labeled agarose gel electrophoresis. Band intensity was quantified using the ImageLab software (BioRad). Quantitative PCR was then performed using 5 ng of cDNAs with SYBR® Premix Ex Taq TM II (Tli RNaseH Plus) on LightCycler 480 II. Relative levels of the target sequence were normalized to the 18 S or GAPDH gene expression (ΔCt), and controls were set to 1(ΔΔCt). The inclusion rate of alternative exons was calculated as 2⁻²⁻ΔΔCt (included exon)/2⁻²⁻ΔΔCt (constitutive exon). Oligonucleotide sequences used are listed in Supplementary Data 5.

**RNA-seq and bio-informatic analysis.** RNA-seq analyses were performed with poly-A transcripts extracted from 293T-LTR-GFP cells transfected with pSG5M-Tax or pSG5M empty vectors and knocked down or not for DDX5-17. RNA-seq libraries were generated at Aros Applied Biotechnology (Aarhus, Denmark) using Stranded mRNA Sample Prep kit (Illumina) and sequenced using illumina HiSeq 2500 technology. Each sample had in average 6 × 10⁷ of paired-end pairs of reads. RNA-seq data were analyzed using FaLine, a computational program dedicated to analyzing alternative splicing with FasterDB database²,³,⁵⁴. The gene expression level in each sample was calculated with HTSeq-count (v0.7.2)⁵⁵, and differential expression between conditions was computed with DESeq2 (v1.10.1) (abs(log2-FoldChange) ≥ 0.4, *p* ≤ 0.05)⁵⁶. Tax expression in the RNA-seq dataset EGAS00001001296 was examined by Kallisto⁵⁷ using the nucleotide sequence of Tax/Rex sequence (coordinates 6951-8078) from NC_001436.1 as a reference.
Ontology analysis were performed using DAVID software for gene ontology and Exon Ontology v1.5.0³⁶.

In silico screening of NF-κB-responsive elements in the CD44 promoter sequence was carried out using the PROMO database (based on TRANSFAC v8.3)⁵⁸. The MEME-ChIP suite was used to discover the regulatory motifs in the NF-κB ChIP-seq data³⁹.

To predict splice site strengths, scores were computed using MaxEntScan⁵⁹ for the sequence covering both sides of the splicing site (using 3 bases into the exon and 6 bases into the intron for 5' splice sites, and 20 bases into the intron and 3 bases into the exon for 3' splice sites). MaxEntScan uses Maximum Entropy Models (MEMs) to compute log-odds ratios. The minimum free energy was computed from exon-intron junction sequences using RNAfold from the ViennaRNA package (v 2.4.1; http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). Analyzed sequences include 25 nucleotides within the intron and 25 nucleotides within the exon. The GC-content was calculated for exons defined in FasterDB⁵⁴.

The distribution of RELA peaks across alternative and constitutive exons, and the average distance between RELA peaks and Tax exon targets was measured using ChiP-seq datasets from GEO⁶⁰, ENCODE⁶¹, and CISTROME⁶² databases: from GEO GSE63736, GSM1239484, GSM486271, GSM486293, GSM486298, GSM486318, GSM847876, GSM847877, GSM2394419, GSM2394421, GSM2394423, from ENCODE ENCFF002CPA, ENCFF002CQB, ENCFF002CQJ, ENCFF002CQN, ENCFF580QGA, and from CISTROME 53597, 5388, 5389, 4940, 36310, 36316, 4971. For another GEO dataset, GSM2628088, reads were mapped to the hg19 build of the human genome with Bowtie²⁶³ and RELA peaks were identified with Macs²⁶⁴. Alternative and Constitutive spliced exons were obtained from FasterDB³⁴. To focus on intragenic RELA peaks, we used the bedtools⁶⁵ intersect command to remove all intergenic RELA peaks and all RELA peaks localized on first exon (or at least at less than 500 nt distance) for each gene. A Perl script was specifically created to measure the distance between RELA peaks and Tax-regulated exons. Briefly, RELA peaks and exons are provided as BED files, and the script reports for each exon the distance in nucleotides of the nearest RELA peak. Closest peak distances from the 710 Tax-regulated exon-cassettes were compared to closest peak distances from 710 exons chosen by chance (10⁵ runs). Using bootstrap analysis, the random samples were compared with the set of Tax-regulated exons, and the normal distribution of these counts was used in a sample *t*-test to assess the significance of the RELA-binding sites enrichment at the vicinity of Tax-regulated exons. *p* = 2e-4 was considered significant.

**TALE design and construct.** The TALE constructs were obtained from ThermoFisher Scientific. TALEs were constructed using the Golden Gate Assembly method⁴⁰. The RVDs HD, NI, NG, and NN were chosen to specifically recognize the nucleotides C, A, T, and G, respectively. The TALE-targeting CD44 v10 sequence was 5'-TCCAACTCTAATGTCAATC-3'. This TALE construct was fused to a V5 sequence and a SV40 NLS at its 5' end and cloned in the *NotI*-HindIII fragment of the pXJ41 backbone plasmid. DDX17-WT and DDX17-K142R cDNA were obtained by PCR from pcDNA3-HA-DDX17 and pcDNA3-HA-DDX17-K142R and were cloned in the *HindIII-BglII* fragment in the MCS downstream to the TALE sequence.

**CRISPR design and construct.** The sequence-specific sgRNA for site-specific interference of genomic targets were designed using CRISPRseek R package, and sequences were selected to minimize off-target effect⁶⁶. Two complementary oligonucleotides were annealed and cloned into BbsI site of pSPCas9(BB)-2A-Puro (PX459) V2.0 (Addgene plasmid #62988)⁶⁷ for co-expression with Cas9 using 5U of T4 DNA ligase, T4 DNA ligase buffer (1x) (Roche). 293T-LTR-GFP cells were transfected with the mix of equimolar ratio of PX459-sgRNA1 and PX459-sgRNA2 (Supplementary Data 5). At 24-h post-transfection, the medium was changed, 1 µg/ml puromycin was added for selection, and cells were cloned by serial dilution method.

**Reporting summary.** Further information on research design is available in the Nature Research Reporting Summary linked to this article.

**Data availability**

RNA-seq data produced in this study have been deposited on NCBI GEO under the accession number GSE123752. The source data underlying Figs. 1c, 2b–f, 3a, b, d, 4a, c–f, 5a–d, 6a–f, and Supplementary Figs. 1, 2b–d, 3a, b, e, 4a–g, 5a–d, and 6a–c are provided as a Source Data file. All data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. Source data are provided with this paper.

**Code availability**

FaRline is publicly available (http://fasterdb.ens-lyon.fr/FARLINE.tgz). Other custom codes used in this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

Received: 25 March 2019; Accepted: 26 May 2020;

Published online: 16 June 2020

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