# Interactions of the LINE-1 encoded ORF1p with proteins and chromatin converge on a role in neuronal physiology

Sandra Sinnassamy, Olivia Massiani Beaudoin, Bérangère Lombard, Damarys

Loew, Tom Bonnifet, Magali Fradet, Héloïse Monnet, Thomas Cailleteau,

Nicolas Servant, Rajiv L Joshi, et al.

► To cite this version:

Sandra Sinnassamy, Olivia Massiani Beaudoin, Bérangère Lombard, Damarys Loew, Tom Bonnifet, et al.. Interactions of the LINE-1 encoded ORF1p with proteins and chromatin converge on a role in neuronal physiology. 2025. hal-05339118

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1 Interactions of the LINE-1 encoded ORF1p with proteins and chromatin

2 converge on a role in neuronal physiology

3

4 Sandra Sinnassamy¹, Olivia Massiani Beaudoin¹, Berangère Lombard², Damarys

5 Loew², Tom Bonnifet¹, Magali Fradet³, Héloïse Monnet³, Thomas Caille³, Nicolas

6 Servant⁴, Rajiv L. Joshi¹* & Julia Fuchs¹*

7

8

9 ¹CIRB, Collège de France, Université PSL, INSERM, CNRS, 75005, Paris, France

10 ²Institut Curie, Université PSL, CurieCoreTech Mass Spectrometry Proteomics, Paris,

11 France

12 ³Orion Technological Core, CIRB, Collège de France, Université PSL, INSERM,

13 CNRS, 75005, Paris, France

14 ⁴Institut Curie, INSERM U900, Mines Paris Tech, Université PSL, Paris, France

15

16

17 **Keywords:** LINE-1, ORF1p, transposable elements, retrotransposons, chromatin,

18 transcriptome, neurite morphology, mass spectrometry, dopaminergic neurons,

19 human post-mortem brain

20 *For correspondence:

21 J.F. (julia.fuchs@college-de-france.fr); R.L.J. (rajiv.joshi@college-de-france.fr)

22
23 **Abstract**

24 Retrotransposons are emerging as novel regulators of embryonic and brain 25 development. We recently demonstrated that the LINE-1-encoded protein ORF1p is 26 abundantly expressed in adult mouse and human neurons, although its function 27 remains unclear. Here, we characterize the ORF1p interactome in differentiated 28 mouse and human neurons using mass spectrometry and identify novel partners 29 implicated in gene regulation and neuron-specific processes. ORF1p localizes not 30 only to neuronal nuclei, where it associates with chromatin under steady-state 31 conditions, but also to neurites, supporting a role in neuronal physiology. To further 32 explore its nuclear functions, we sorted human post-mortem neurons with high or low 33 nuclear ORF1p levels and performed ORF1p knockdown in cultured human neurons, 34 followed by chromatin accessibility assays. Both approaches revealed consistent 35 patterns of differential chromatin accessibility dependent on ORF1p. Loss of ORF1p 36 also led to the downregulation of long, neuron-specific genes and altered neurite 37 morphology. Together, these findings point to a physiological role of ORF1p in post-38 mitotic neurons, mediated through converging interactions with proteins and 39 chromatin. 40

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46

## 47 Introduction

Transposable elements (TEs), once considered junk DNA, are gaining interest as physiological modulators of genome architecture¹ and early embryonic ² as well as neurodevelopment³, but also pathological drivers of aging-related⁴ and neurogenerative processes. Half of the human genome is composed of TEs and 17% of the Long Interspersed Nuclear Element-1 (LINE-1 or L1)⁵ TE family. Between 100 and 150 LINE-1⁶,⁷ are considered full-length L1 elements (fLINE-1). They are the only autonomous mobile elements in the human genome and are characterized by a bidirectional RNA polymerase II promoter in the 5'UTR and intact open reading frames encoding the canonical L1 proteins, ORF1p and ORF2p, essential for LINE-1 mobilization. ORF1p and ORF2p, translated from a bicistronic mRNA, associate with *cis-preference* to the LINE-1 mRNA to form a ribonucleoprotein (RNP). Once the LINE-1 RNP gets access to the nucleus, a new copy is pasted into the genome through target-primed reverse transcription (TPRT). ORF1p is an RNA binding protein and a nucleic acid chaperon and ORF2p has endonuclease and reverse transcriptase activities. When abnormally expressed, LINE-1 may represent a threat to genome and cellular integrity by generating DNA double strand breaks and triggering inflammatory pathways⁸. Consequently, LINE-1 expression is rigorously controlled through multiple mechanisms at the transcriptional and post-transcriptional levels⁹. Nevertheless, LINE-1 has been shown to exert physiological functions especially during early embryonic¹⁰,¹¹and neuronal development³,¹²⁻¹⁴. Intriguingly, recent and optimized next generation sequencing data have highlighted the abundant expression of LINE-1 in adult human brain¹⁵,¹⁶, revealing significant enrichment of LINE-1 expression, including evolutionary young L1HS and L1PA2 elements, in adult
human neurons with no or limited expression of LINE-1 in non-neuronal cells at steady-state<sup>12,15,16</sup>. Indeed, on the protein level, LINE-1 encoded ORF1p expression was observed predominantly in neurons throughout the adult mouse brain<sup>17</sup> and in several regions of the human brain<sup>17,18</sup> as well as in human dopaminergic neurons<sup>17-19</sup>. The presence of ORF1p in adult neurons is a surprising observation given the deleterious consequences to cellular integrity attributed to LINE-1 expression and led us to test the hypothesis of a physiological function of this protein in the adult brain.

To this end, we used a combination of mass spectrometry, chromatin accessibility assays and transcriptomics. We found an important protein interaction network of endogenous ORF1p in adult neurons encompassing proteins related to RNA metabolism, as expected for an RNA binding protein, but also proteins related to nuclear functions including chromatin regulation and gene expression as well as proteins with neuron-specific functions. This finding and the presence of ORF1p in chromatin fractions led us to analyse chromatin accessibility in nuclei with high or low ORF1p content using sorted *post-mortem* neurons or ORF1p loss-of-function (ORF1p-LOF) in differentiated human neurons in which we also assessed transcriptomic changes. Characteristics of differentially accessible regions were significantly different between nuclei with high or low ORF1p content and these differences were highly similar across two different experimental systems involving gene loci related to genes important for neuronal functions. ORF1p-LOF led to the downregulation of particularly long, protein-coding genes enriched in neuronal functions. Functionally, we observed morphological changes in neurites of adult mouse neurons upon ORF1p-LOF. Together, these data suggest a role of ORF1p in the maintenance and integrity of neuronal function in adult neurons via converging actions on chromatin organization, gene transcription and protein interactions.
96

## 97 Results

### 98 Identification of protein partners of endogenous ORF1p in mouse and human neurons

100 We and others have previously observed a widespread expression of ORF1p in mouse and human neurons at steady-state¹⁷⁻²⁰. This somewhat surprising presence of ORF1p in neurons evokes the question of a possible physiological role of ORF1p specific to these cells. In a first attempt to address this question, we identified, using a quantitative mass spectrometry approach, protein partners of endogenous ORF1p in mouse (differentiated MN9D) and human (differentiated LUHMES¹⁹,²¹) dopaminergic neurons (Fig. 1A) using well characterized and specific antibodies to ORF1p¹⁷. Just as differentiated human dopaminergic LUHMES neurons¹⁹,²¹, mouse dopaminergic MND9 precursor cells can be differentiated into mature, post-mitotic neurons (Suppl. Fig. 1A) which express ORF1p (Suppl. Fig. 1B) along with the neuronal marker β-III tubulin (TUJ1, Suppl. Fig. 1C) and the dopaminergic marker tyrosine hydroxylase (TH, Suppl. Fig. 1C). As expected, the pattern of ORF1p staining was mainly cytoplasmic, but we also observed, as in human dopaminergic LUHMES¹⁹, a readily detectable nuclear staining (Suppl. Fig. 1B). It is important to note that human and mouse ORF1p differ significantly in their amino acid composition (31% identity, NCBI Needleman-Wunsch Global Align), but share the same protein domains²² which is reflected in very similar Alpha Fold predicted 3D protein structures for mouse²³ and human²⁴ ORF1p. Following immunoprecipitation (IP) of endogenous ORF1p or isotype control IgG in human (LUHMES) and mouse (MN9D) dopaminergic neurons (n=5 IPs per condition), we performed liquid chromatography coupled to mass spectrometry (LC-MS/MS) followed by a
quantitative analysis (Fig. 1A). ORF1p was strongly enriched in the immunoprecipitated material, with a high IP-ORF1p/IP-IgG ratio (mouse ORF1p: 3.76 log2(ratio), p= 4.16E-05, Fig. 1B; human ORF1p: 5.35 log2(ratio), p= 8.73E-07, Fig. 1C). 348 proteins were detected as ORF1p partners in MN9D (Fig. 1B) and 267 proteins were identified as ORF1p partner proteins in LUHMES neurons (Fig. 1C). As some proteins detected in the ORF1p IP were completely absent from the control IP-IgG and, as a consequence, no ratio could be calculated, these putative ORF1p protein partners are represented separately as peptides/100aa (Fig. 1B, C, right panels). Next, we confronted ORF1p protein partners identified in neurons with those found in previous studies²⁵⁻³⁴ including our previous study of ORF1p interactors in the mouse brain¹⁷. Nine proteins were identified in all studies (Suppl. Fig. 1D), namely RPL19, IGF2BP3, LARP1, ATXN2, RPL24, YBX1, DDX6, PABPC1 and RPL8, representing thus high-confidence, highly conserved and non-cell-type specific ORF1p protein partners most of which were RNA binding proteins (RBPs), just as ORF1p itself (Suppl. Fig. 1H, I). 49 proteins were shared between ORF1p partners identified in various human cell types and ORF1p partners identified in human LUHMES neurons (Suppl. Fig. 1E), among them: HNRNPU, NCL and IGF2BP2 and of those 14 proteins were overlapping in addition with ORF1p protein partners identified in mouse MN9D neurons, including RPL19, IGF2BP3, RALY, LARP, ATXN2, RPL24, YBX1, CLASP1, DDX6, UBAP2L, DDX17, ELAVL1, PABPC1 and RPL8 (Suppl Table. 1). Similarly, 70 proteins among which GPHN, ELAVL2, ARID1B, FXR1, POLR2A, MAP1B, EEA1, ARID1A and HELZ were in common between previously identified mouse ORF1p partners and those identified in MN9D neurons (Suppl. Fig. 1F). Focusing on overlapping ORF1p protein partners in mouse and human neurons (Suppl. Fig. 1G), the 41 proteins that were shared mostly identified
146 as "RNA processing" and/or "RNA binding" in a STRING analysis visualized by
147 Cytoscape (Suppl. Fig. 1G). To identify conserved categories of ORF1p protein
148 partners between mouse and human neurons, we inquired for shared significant GO
149 terms. As expected, among the top shared categories, many GO terms were related
150 to RNA metabolism (Fig. 1D, cat.III = category 3 interactors). However, two
151 unexpected categories emerged: first, in both mouse and human neurons, ORF1p
152 protein partners were enriched in GO terms related to nuclear presence and function
153 (cat.I = category I interactors), namely "gene expression" (107 proteins in LUHMES
154 (fold enrichment (FE): 3,25; FDR:1,61E-26), 76 in MN9D (FE: 1,76; FDR: 0,0001), 23
155 in common) and "nucleus" (186 proteins in LUHMES (FE: 1,37; FDR: 3,07E-05), 162
156 in MN9D (FE: 1,82; FDR: 3,47E-22), 23 in common). In this category I of ORF1p
157 protein partners, 31 unique proteins were identified, among which 17 ribosomal
158 proteins with non-canonical functions, in addition to DDX6, FXR1, GSN, IGF2BP3,
159 KHDRBS1, LARP1, MLF2, MYH9, PABPC1, PRKRA, YBX1, HNRNPU, YTHDC1
160 and RCOR2, the last three of which have chromatin-related functions. In LUHMES
161 cells, ORF1p interacted with proteins related to chromatin organization and
162 epigenetic regulation (H1-2, H1-4, H1-10, MACROH2A1, MACROH2A2, CHD4,
163 CBX8, ASH1L, SUPT16H, DPF2, SMARCA4, MORC2), proteins related to DNA
164 damage and repair (TOP2B, PARP1, TP53BP1, XRCC6 (Ku70) and GADD45GP1),
165 proteins involved in transcriptional regulation (CTNNB1, YBX1, ZC3H11A, THRAP3,
166 SNW1, BCLAF1, PSIP1, TFAP2D), proteins associated nuclear structure and
167 envelope (LMNA, LBR, TMPO, EMD, NUP153, NUP214, RANBP2, NUMA1) and
168 nuclear transport and signalling (PAWR, FXR1 and FXR2) in addition to many more
169 involved in RNA processing, RNA metabolism and ribosome biogenesis (Suppl Table
170 1). In MN9D neurons, ORF1p interacted with chromatin remodelers (CHD7, ARID1B,
171 ARID1A), transcription factors (ZFHX3, STAT2, ZNF638, ZNF281, TCF4), with RNA
172 polymerase subunits or associated proteins (POLR2M, POLR2A, POLR2B, POLR2C,
173 RPAP2) and nuclear transport proteins (NUP50 and KPNA3) in addition to many
174 RNA metabolism related proteins (Suppl Table 1). In the second category (cat.II
175 interactors), ORF1p protein partners were enriched for GO terms related to neuron
176 morphology like "synapse" (LUHMES; FE: 1,92; FDR: 001; MN9D; FE: 3,62;
177 FDR:13E-32), "neuron projection" (LUHMES; FE: 1,85; FDR: 0,01; MN9D; FE: 2,46;
178 FDR: 6,35E-10), "dendrite" (LUHMES; FE: 2,15; FDR: 0,0313; MN9D; FE: 3,13; FDR:
179 1,93E-09), "axo-dendritic transport" (LUHMES; FE: 5,84; FDR: 0,04; MN9D; FE: 5,53;
180 FDR: 0,008), "cytoskeleton protein binding" (LUHMES; FE: 2,73; FDR: 1,12E-05;
181 MN9D; FE: 4,04; FDR: 1,01E-19) and "actin binding" (LUHMES; FE: 3,91; FDR:
182 7,70E-06; MN9D; FE: 4,91; FDR: 3,10E-12) (Fig. 1D). STRING analysis of physical
183 protein-protein interactions of ORF1p partners in mouse (Fig. 1E) and human (Fig.
184 1F) neurons revealed the interaction profile of these proteins among each other
185 according to their functional category (colours), confidence in physical interaction
186 (thickness of edges) and known interactors (yellow nodes). The emergence of
187 categories related to nuclear functions, including gene expression, was particularly
188 intriguing in light of observations of a nuclear localization of ORF1p at steady-state in
189 human neurons¹⁹. Indeed, while ORF1p has been previously observed in the nucleus
190 in different cell lines²⁸,³⁵⁻⁴¹ and in human post-mortem neurons¹⁷,¹⁸, the role of
191 nuclear ORF1p has not been previously addressed except with regard to
192 retrotransposition efficiency⁴². We therefore asked the question whether nuclear
193 ORF1p might have a role in mature neurons, potentially independent of the LINE-1
194 life cycle.

195
196 **ORF1p is located in the nucleus and associated to chromatin in post-mitotic**
197 **neurons**

198 Based on the identification of chromatin-related proteins as ORF1p protein partners,
199 we characterized the subcellular localization of ORF1p in mouse and human
200 neurons. Using immunofluorescence, we observed a predominant cytoplasmic
201 expression of ORF1p in both, LUHMES and MN9D neurons, but also distinct nuclear
202 dots in both species (Fig. 2A). Cytoplasmic and nuclear fractionation confirmed the
203 presence of ORF1p in the nuclear fraction of mouse and human post-mitotic neurons
204 (Fig. 2B). To gain a more precise understanding of the subcellular localization of
205 ORF1p, we performed a more detailed biochemical fractionation, which confirmed
206 that ORF1p is predominantly located to the cytoplasm, but also present in the
207 membrane-bound fraction (Fig. 2C-E). Furthermore, ORF1p was localized to the
208 soluble nuclear fraction (=nucleoplasm) and, importantly, reliably detected as
209 chromatin-bound in the chromatin fraction of mouse neurons (Fig. 2C), human
210 neurons (Fig. 2D) and in human post-mortem tissue extracts from the cingulate gyrus
211 of a neurologically healthy individual (Fig. 2E). We confirmed the separation of all
212 fractions using dedicated marker proteins and verified that stress granules were not
213 contaminating the chromatin-bound fraction using the stress granule marker G3BP1
214 (Fig. 2C, D). The presence of ORF1p on chromatin is in line with previous
215 observations indicating the association of ORF1p with chromatin independently of
216 ORF2p in cancer cells<sup>43</sup>. Together, our observations suggest a close proximity and
217 potentially functional relation of ORF1p with chromatin in neurons.
218

219 **Changes in nuclear ORF1p levels correlate with alterations in chromatin**
220 **accessibility patterns in neurons**
In order to explore whether ORF1p might have a functional impact on chromatin organization, we turned to an assay for transposase-accessible chromatin followed by sequencing (ATAC-seq). As we had previously shown that ORF1p was present in neurons in the human cingulate gyrus¹⁷ and established its presence in the chromatin-bound fraction in this brain region (Fig. 2E), we isolated nuclei from frozen human post-mortem cingulate gyrus tissues of six individuals (n=3 neurologically-healthy individuals and n=3 individuals with Parkinson disease (PD), Suppl Table 2).

We then sorted them as schematized in Figure 3A using two antibodies, the neuronal marker NeuN, separating neurons from non-neuronal nuclei, and ORF1p, separating nuclei with high levels of ORF1p (ORF1p-high; P3, Fig. 3B) and those with lower levels of ORF1p including those not expressing ORF1p (ORF1p-low; P4, Fig. 3B).

The corresponding isotype control profiles are shown in Suppl. Fig 2A. Confirming our previous observations of a predominant neuronal expression of ORF1p in the mouse and human brain¹⁷, non-neuronal nuclei were ORF1p negative, while neurons contained varying levels of nuclear ORF1p, allowing us to separate three distinct populations, NeuN-/ORF1p-minus; NeuN+/ORF1p-high and NeuN+/ORF1p-low, which we also verified by Western blot (Fig. 3C). This confirmed the absence of ORF1p in non-neuronal nuclei and the successful sorting of two neuronal populations with either high or low levels of nuclear ORF1p. We then performed high-throughput chromatin profiling on those three populations. In order to exclude that ORF1p-high and ORF1p-low nuclei were assimilated to a specific neuronal subtype, we plotted total ATAC-seq reads on promoter regions (<1kb) for cell type specific markers selected from Single Cell Base

(https://biomarkerres.biomedcentral.com/articles/10.1186/s40364-023-00523-3)

which showed a clear separation of NeuN- and NeuN+ accessibility profiles as
expected and, importantly, no overt difference between accessibility profiles on neuronal subpopulation marker genes between ORF1p-high and -low nuclei (Suppl. Fig. 2B). Based on the number of normalized peaks, we determined differentially accessible regions (DARs). The Volcano plot in Figure 3D depicts all significantly enriched DARs, with 406 DARs more accessible in the NeuN+/ORF1p-high population (DAR+), and 379 DARs less accessible in NeuN+/ORF1p-high nuclei (DAR- ≈ more accessible in NeuN+/ORF1-low neurons; log2 fold-change□□≥□1 / ≤ -1, p-value□≤□0.01). We next analysed genomic features associated with each DAR. DARs were mostly located in introns (first and others) as well as in distal intergenic and, to a lesser extent, in promoter regions (Fig. 3E). However, there were significant differences in the distribution of DARs over genomic features between NeuN+/ORF1p-high and NeuN+/ORF1p-low nuclei. NeuN+/ORF1p-high/DAR-regions were enriched in introns (q = 0.0004; Fisher's Exact test followed by Benjamini, Krieger and Yekutieli post-hoc test) compared to NeuN+/ORF1p-high/DAR+ regions, which were more frequently located in distal intergenic regions (q = 0.0004; Fisher's Exact test followed by Benjamini, Krieger and Yekutieli post-hoc test). To gain a more precise idea about the functional features that DARs in both conditions were associated to, we overlapped DARs with regulatory features stemming from a ChromHHM annotation, which defines and characterizes chromatin states across the genome based on experimental data of a given tissue, in this case of human *post-mortem* cingulate gyrus (https://pmc.ncbi.nlm.nih.gov/articles/PMC8734071/; Fig. 3F). Less accessible chromatin (DAR-) in ORF1p-high nuclei coincided with enhancers (Enh), weak repressed polycomb (ReprPCWk) domains and domains associated with weak transcription (TxWk), while more accessible chromatin (DAR+) in ORF1p-high nuclei
271 was mostly located in weak repressed polycomb (ReprPCWk) and weak transcription
272 (TxWk) domains (Fig. 3F). The distribution of DARs between ORF1p-high and
273 ORF1p-low nuclei was significantly different, with an overrepresentation of enhancers
274 (Enh, q=0.0001) and active transcriptional start sites (TssA, q=0.0013) among DAR-
275 regions and an overrepresentation of repressed polycomb regions (ReprPCWk,
276 q=0.0001) and heterochromatin domains (Het, q=0.001) in DAR+ regions in ORF1p-
277 high nuclei (Fisher's Exact test followed by Benjamini, Krieger and Yekutieli post-hoc
test). This indicated the existence of two different profiles of chromatin accessibility
278 between these two nuclei populations. ORF1p-low nuclei were characterized by more
279 accessible chromatin in intronic regions related to active transcription and enhancer
280 activity and ORF1p-high nuclei showed open chromatin in repressed or
281 heterochromatin regions rather located in distal intergenic genomic regions. We next
282 visualized the top two candidates of each category using IGV. Tracks of DLX1 (distal-
283 less homeobox 1) show an example of DAR+ regions in ORF1p-low neurons (Fig.
284 3G). DLX1 is a member of the homeobox transcription factor family, predicted to be a
285 transcriptional regulator for TGF beta signalling and linked to autism⁴⁴. The DAR in
286 the DLX1 gene is annotated as a “flanking bivalent transcriptional start site/enhancer”
287 and “bivalent enhancer” in adult cingulate gyrus ChromHMM annotations and
288 overlaps with a CpG island. There are two other DARs in this region, one intergenic
289 in-between DLX1 and DLX2 and one in the promoter region of DLX2, both
290 characterized by bivalent enhancer or bivalent TSS annotations. These loci thus
291 provide examples of the overrepresentation of DARs in ORF1p-low chromatin in
292 intronic and promoter regions and transcriptional start sites and enhancers as
293 depicted and quantified in Figure 3F. IGV tracks of SPACA3 (Sperm Acrosome
294 Associated 3) show an example of a DAR+ region in ORF1p-high neurons (Fig. 3H).
295
SPACA3 is a sperm-specific gene and the DAR+ peak in ORF1p-high chromatin overlaps with a "weak repressed polycomb" annotation reflects the overrepresentation of ORF1p-high DAR+ regions in repressive chromatin regions (Fig. 3F). Using ChIP-seq data from sorted human post-mortem neurons<sup>45</sup> for tri-methylated Histone H3 at lysine 4 (H3K4me3), which marks transcriptional start sites of active genes, tri-methylated Histone H3 at lysine 27 (H3K27me3), a histone modification associated with polycomb-mediated heterochromatin regions and acetylated Histone H3 at lysine 27 (H3K27ac), a marker for active enhancers, we observed significant differences in proportions of DAR+ regions overlapping with all three histone profiles. H3K27me3 was overrepresented in ORF1p-high nuclei (q=0.0003) in line with the repressive polycomb signature observed in the ChromHMM data, whereas H3K4me3 (q=0.0007) and H3K27ac (q=0.0003) were significantly overrepresented over DAR+ regions in ORF1p-low nuclei (Suppl. Fig. 2C, Fisher's Exact test followed by Benjamini, Krieger and Yekutieli post-hoc test), in line with the active transcription and enhancer profile observed in the ChromHMM data (Fig. 3F). Together, this data indicated a distinct genomic and functional genomic profile of DARs dependent on ORF1p nuclear content, suggesting a regulatory function of nuclear ORF1p on chromatin dynamics. We then asked which transcription factor (TF) binding sites were overrepresented among the identified DARs and performed transcription factor motif enrichment analysis with HOMER (Suppl. Fig. 2D, E). In DAR+ regions in neuronal nuclei with a high ORF1p content, predicted TF motifs were predominantly showing members of the highly conserved basic leucine zipper (bZIP) transcription factor family like ATF3, involved in axonal regeneration<sup>46</sup> and three members of the FOS TF family (FOSL2, FRA1 and FOS) which are TFs orchestrating the conversion of short-term extracellular signals into
321 long-term functional changes via the activation of dedicated gene expression
322 programs as members of the immediate early genes including roles in memory
323 formation<sup>47</sup>. We also identified signatures of another immediate early gene, the zinc
324 finger TF EGR1, within DAR+ regions in NeuN+/ORF1p-high nuclei. Interestingly,
325 EGR1 is also involved in memory formation<sup>47</sup>. TF signatures on ORF1p-low DAR+
326 regions were mainly composed of homeobox genes like Engrailed-1 (En1) (Suppl.
327 Fig. 2E) which have important functions in brain patterning<sup>48</sup> but also in the
328 maintenance of adult neurons<sup>48</sup>.

329

330 LINE-1 knock-down induces changes in chromatin organization

331 Having established significant changes in chromatin accessibility in nuclei differing in
332 the levels of ORF1p content in the human cingulate gyrus, we then turned to human
333 LUHMES neurons in which we experimentally manipulated ORF1p steady-state
334 levels using LINE-1 siRNAs targeting evolutionary young LINE-1 elements. LUHMES
335 neurons were transfected at day three and retrieved at day seven of differentiation.
336 Efficient knock-down of ORF1p was verified by immunostaining with a significant
337 reduction in the cytoplasmic and the nuclear compartment (Fig. 4A) and by Western
338 blot (Suppl. Fig. 3A) while LINE-1 RNA was slightly but not significantly reduced
339 (Suppl. Fig. 3B). We also verified by Western blot the loss-of-function (LOF) of
340 ORF1p after LINE-1 knock-down (KD) in the chromatin-bound fraction (Suppl. Fig.
341 3C). Using this knock-down protocol, we performed ATAC-seq on LINE-1 siRNA and
342 control siRNA (Ctrl) transfected neurons. The MA-plot in Figure 4B shows the DARs
343 (log2 fold-change□ ≥ □1 / ≤ -1, p-value□≤ 0.05), with 432 regions significantly more
344 accessible (DAR+) after ORF1p-LOF and 917 less accessible regions (DAR-)
345 compared to control. Similarly to human post-mortem nuclei with low ORF1p levels,
346 DAR+ regions in the ORF1p-LOF condition were mostly located in introns and DAR-
347 regions in distal intergenic regions, but with no significant overrepresentation when
348 comparing the two conditions (Fig. 4C). When analysing the regulatory regions
349 overlapping DAR+/DAR- regions in ORF1p-LOF nuclei (Fig. 4D) using a ChromHMM
350 collection from human substantia nigra as a suitable reference for differentiated
351 LUHMES cells which are dopaminergic⁴⁹, we identified a very similar profile as in
352 ORF1p-high and ORF1p-low nuclei in the cingulate gyrus (Fig. 3E). Indeed, just as in
353 ORF1p-low nuclei, DAR+ regions in ORF1p-LOF nuclei were enriched on active
354 transcriptional start sites (q=0.0001) and enhancers (q=0.03, Fisher's Exact test
355 followed by Benjamini, Krieger and Yekutieli post-hoc test). And similar to ORF1p-
356 high nuclei, ORF1p-Ctrl DAR+ regions were overrepresented on repressed chromatin
357 domains associated with polycomb (ReprPCWk, q=0.0001; ReprPC, q=0.005) and
358 zinc finger genes/repeat associated genomic regions (ZNF/Rpts, q=0.001; Fisher's
359 Exact test followed by Benjamini, Krieger and Yekutieli post-hoc test, Fig. 4D). Again,
360 using histone ChIP-seq data from sorted post-mortem neurons pooled from 5
361 different brain regions⁴⁵, we also found a significant overrepresentation in the fraction
362 of DAR+ regions in ORF1p-LOF nuclei over H3K4me3 domains (q=0.0001), while
363 DAR- regions were overrepresented in H3K27me3 (q=0.0001) domains (Suppl. Fig.
364 3D). We then chose representative examples for DAR+ regions in ORF1p-Ctrl (Fig.
365 4E) and ORF1p-LOF (Fig. 4F) for visualization. WDR74 is an example of a DAR+
366 region in ORF1p-LOF neurons and is located in the promoter region overlapping with
367 an active transcription start site (TssA) in line with the preferential location of DARs in
368 ORF1p-LOF promoters depicted in Figure 4C and the overrepresentation of TssA
369 regulatory regions more accessible in ORF1p-LOF (Fig. 4D). ROCK1P1, a
370 pseudogene, contains two DARs overlapping with "ZNF/Rpts", "Het" and "TxWk"
371 annotations in line with regions overrepresented in ORF1p-LOF DAR- regions (Fig 372 4D). Next, we investigated the transcription factor binding sites enriched in those 373 DAR. In DAR+ regions in Ctrl neurons, the family of basic helix-loop-helix (bHLH) 374 TFs (Suppl. Fig. 3E) was strongly represented. These TFs have essential regulatory 375 functions for neural cell fate specification and in embryonic development$^{50,51}$. 376 Interestingly, analogous to the TF profile overrepresented on DAR+ regions in 377 ORF1p-low nuclei (Suppl. Fig. 2F), DAR+ regions in ORF1p-LOF nuclei were 378 enriched in homeobox genes (Suppl. Fig. 3F). Therefore, low ORF1p nuclear content 379 might favour accessibility to genomic regions regulated by homeobox proteins which 380 are essential for specifying cell identities and positioning during embryonic 381 development but also play a role in the adult brain related to neuronal maintenance$^{52}$. 382 Taken together, when comparing features across these two independent ATAC-seq 383 experiments in human neurons (post-mortem cingulate gyrus neurons and 384 differentiated dopaminergic neurons in culture) with high or low nuclear ORF1p 385 content, we made similar observations; nuclei containing high levels of ORF1p 386 showed more accessible chromatin preferentially in distal intergenic regions 387 associated with polycomb-associated repressive domains and nuclei with low ORF1p 388 content were predominantly characterized by more accessible DARs in intronic 389 regions associated with enhancer functions and active transcription containing 390 signatures of homeobox transcription factor binding sites (Suppl Table 3). This might 391 suggest, that LINE-1 regulate cell identity in post-mitotic neurons. 392

393 Nuclear ORF1p modulates chromatin accessibility on genes related to neuron-
394 specific processes
We next compared the actual overlap of DAR-associated genes between both experimental systems. Of the 648 DAR-associated genes identified in cingulate gyrus neurons and the 1,189 DAR-associated genes identified in LUHMES neurons, 71 events were found to be shared between the two (Fig. 5A). This overlap represents 10.96% of the CG DARs and 5.97% of the LUHMES DARs. While this is only a modest overlap (Jaccard similarity coefficient 4.02%), GO analysis of these genes revealed five significant biological processes: axonogenesis, CNS neuron development, axon guidance, neuron recognition and neuron projection guidance (Fig. 5B). The GOChord plot in Figure 5B shows the eight genes sharing these GO terms. IGV tracks of two of them highlighted the presence of DARs on two enhancers in introns of the zinc finger transcription factor GLI2 and on a putative alternative promoter of XR_007087219 adjacent 5' to GLI2 (ATAC-hu and ATAC-lu DARs) and an exonic DAR in a putative alternative promoter in the ROBO1 gene (ATAC-hu DAR) along an intronic DAR (chr3:79052746-79057310; ATAC-lu DAR) (Fig. 5C). These findings reinforce the possibility of a role of nuclear ORF1p in neuron-specific functions.

411

412 **Knock-down of ORF1p affects the neuronal transcriptome, including**
413 **downregulation of long genes related to neuron-specific functions**

414 A way to interrogate changes in neuronal cell identity and function is via the
415 characterization of the transcriptome. Therefore, we performed RNA-seq analysis
416 after ORF1p-LOF in differentiated LUHMES neurons. Principal component analysis
417 (PCA) separated ORF1p-Ctrl and ORF1-LOF transcriptomes (Fig. 6A). Using
418 DESeq2, we identified 962 differentially expressed genes (DEG) (log2FC≥0.5 / 
419 ≤ -0.5, p-value ≤ 0.01), of which 264 were upregulated and the majority, 698 genes,
420 were significantly downregulated as shown in the Volcano plot in Figure 6B.
421 Characterization of major "gene biotypes" using Ensembl annotations (hg38)
422 revealed a similar pattern between all LUHMES expressed transcripts (total=56861)
423 and transcripts upregulated upon ORF1p-LOF (total=264) with almost equal
424 proportions of protein-coding transcripts (34% ORF1p-Ctrl; 29% ORF1-LOF) and
425 long non-coding transcripts (30% ORF1p-Ctrl; 34% ORF1-LOF). However, ORF1p-
426 LOF downregulated transcripts showed a significant different repartitioning of
427 transcripts. Of all downregulated transcripts, 73 % were protein-coding and only 13%
428 IncRNA transcripts (Chi-square test, observed versus expected, DF=4, p>0.0001).
429 Similarly, when evaluating the fraction of long (>100kB) versus short genes (<100kB)
430 differentially regulated upon ORF1p-LOF, we found that downregulated genes, but
431 not upregulated genes, were particularly long (>/=100kB). Compared to the fraction
432 of long genes among all expressed transcripts in differentiated LUHMES neurons
433 (9,69% of a total of 56861 transcripts) or genes upregulated upon ORF1p-LOF
434 (10,61% of a total of 264 transcripts), downregulated genes upon ORF1p-LOF
435 belonged more than two-times more frequent to the category of >100kB genes
436 (21,78% of a total of 698 transcripts) (Fig. 6C, D). This might indicate that LINE-1 is
437 implicated in the regulation of long gene expression. Interestingly, long genes have
438 been associated with neuron-specific functions⁵³,⁵⁴. GO analysis of upregulated
439 genes after ORF1p-LOF identified "odorant binding" and olfactory receptor activity as
440 a significantly enriched (Suppl. Fig. 4A). More interestingly, downregulated genes
441 were enriched in highly significant GO terms related to synapse (FDR: 2,53e-011,
442 FE: 2.32; GO CC), neuron projection morphogenesis (FDR: 0,000425, FE: 2,72; GO
443 BP), nervous system development (FDR: 0,000275, FE: 1,73; GO: BP) and synapse
444 organization (FDR: 0.0005, FE: 3,01; GO BP) (Fig. 6E). Protein-protein interaction
445 (PPI) network analysis of significant downregulated genes upon ORF1p-LOF
446 contained in the GO CC term "Synapse" (GO:0045202; 50 genes) using STRING
447 with a stringent cutoff (log2 fold change ≤ -0.5 / ≤ -0.5, p-value ≤ 0.005) showed a
448 highly interconnected protein interaction network (Fig. 6F). A large proportion of
449 these proteins were implicated in pre- and post-synapse, axon guidance, regulation
450 of neurotransmitter levels and synapse organization. In line with this, KEGG pathway
451 analysis identified "axon guidance" as one of the most significant pathways enriched
452 in downregulated transcripts (Suppl. Fig. 4B). We independently tested four
453 downregulated genes with functions related to neurons by RT-qPCR (DLG5, NEGR1,
454 EFNA5 and GABRB3) and validated DLG5, NEGR1 and EFNA5 to be significantly
455 downregulated after ORF1p-LOF (Fig. 6G).

456 DARs identified in ATAC-seq are associated to the closest gene, although this might
457 not be always biologically relevant as open chromatin regions, especially outside
458 proximal promoter regions, do not necessarily regulate proximal genes but might act
459 at greater distances. We nevertheless overlapped RNA-seq DEGs and ATAC-seq
460 DARs obtained from LUHMES neurons and identified an overlap of 26 genes (Suppl.
461 Fig. 4C). Interestingly, GO analysis of these genes (APBB2, CACNA2D1, EVC,
462 DUSP22, ENOX2 TMEM120B, ATF7, NMNAT2, USP13, SEPTIN9, SPATS2L,
463 MDGA1, SMAD2, EFNA5, PSMB11, CPVL-AS2, UXS1, PPM1L, PCDH8, SDC3,
464 PTBP2, PTPN3, MARCHF1, AUTS2, SATB1, TMTC2) revealed that they belonged to
465 neuron specific pathways such as "axon development", "regulation of synaptic
466 membrane adhesion" and "neuron migration" (Suppl. Fig. 4C). Interestingly, three of
467 these genes with neuronal functions, namely AUTS2, PTBP2 and EFNA5, also
468 contained DARs in cingulate gyrus neurons. AUTS2 (Activator Of Transcription And
469 Developmental Regulator) is a component of the polycomb group multiprotein
470 complex 1 with regulatory functions in neurite morphology and is associated with
471 autism spectrum disorders, intellectual disability and developmental delay⁵⁵. PTBP2
472 (Polypyrimidine Tract Binding Protein 2) is predominantly present in the brain and
473 involved in splicing regulation⁵⁶. EFNA5 (Ephrin-A5) is involved in migration,
474 repulsion and adhesion, notably in axon guidance during neuronal development⁵⁷.
475 The gene locus of EFNA5 with ATAC-seq and RNA-seq tracks is shown in Suppl.
476 Figure 4D.

477 Taken together, these transcriptomic changes upon ORF1p-LOF suggest a role of
478 steady-state ORF1p in the homeostasis and maintenance of post-mitotic neurons.

480 ORF1p loss of function leads to changes in neurites morphology

481 In addition to the fact that nuclear ORF1p dependent changes in chromatin
482 accessibility and in the transcriptome converged upon the notion that ORF1p might
483 be involved in regulating the homeostasis and maintenance of adult neurons at
484 steady-state, the mass spectrometry analysis of protein interactors of endogenous
485 ORF1p in both, mouse and human neurons, had revealed proteins related to neuron-
486 specific functions with high-confidence (cat.II ORF1p interacting proteins, Fig. 1D-F).
487 Cat.II ORF1p interactors belonged to GO categories like "neuron projections" (33
488 proteins in LUHMES, 65 in MN9D; 5 in common), "synapse" (42 proteins in LUHMES,
489 111 in MN9D, 15 in common), "dendrites" (18 in LUHMES, 43 in MN9D, 5 in
490 common) and "axo-dendritic transport" (6 in LUHMES, 8 in MN9D, 1 in common;
491 Suppl Table 1). Among these cat.II protein interactors of endogenous mouse and
492 human ORF1p in neurons, 18 common proteins emerged; 9 ribosomal proteins and 2
493 myosin proteins with non-canonical functions in addition to YBX1, HNRNPU, MLF2
494 and FXR1 and two microtubule-associated proteins with canonical functions in
neurite morphology, namely two microtubule-associated proteins, MAP6 (dendrite morphogenesis, axonal growth and transport⁵⁸, and MAP1B (neurite extension⁵⁹).

These findings again suggest a possible role of ORF1p specific to neurons which converge towards a pathway related to neurites (Fig. 1D). Moreover, this idea was further supported by the presence of ORF1p itself in neurites as revealed by immunofluorescence stainings of endogenous ORF1p in MN9D and LUHMES (Fig. 7A). Using TUJ1 to identify neurites, immunostaining of ORF1p showed its localization in neurites in both, human and mouse dopaminergic neurons (Fig. 7A).

Additionally, we transfected MN9D neurons with human ORF1p-HA and detected ORF1p by HA immunostaining, as expected in the cytoplasm and the nucleus, but also localized to neuronal extensions (counterstained with TUJ1), including axons (counterstained with NEFL) (Fig. 7B). This indicated a possible colocalization of ORF1p with proteins related to neurite functions in the neuritic compartment itself.

We verified the co-localization and co-immunoprecipitation of PABPC1, an interesting candidate with regard to neurite physiology⁶⁰,⁶¹ and protective against dopaminergic neurodegeneration⁶², which we had identified as interacting with ORF1p in both, human and mouse neurons by mass spectrometry (Suppl Table 1), and others in different cultured cell types²⁶⁻³⁰,³³,⁶³. Co-immunoprecipitation (Fig. 7C) and colocalization (Fig. 7D) in mouse neurons validated the interaction and identified that both proteins colocalize in the cytoplasm but also in neurites. We thus hypothesized, that both, nuclear ORF1p-induced chromatin and transcriptional changes and ORF1p protein-protein interactions in the cytoplasm and in neuronal extensions might affect neurite morphology. In order to test this hypothesis, we carried out ORF1p-LOF in MN9D neurons at seven days of differentiation and collected them at day 10 to perform neurite staining. ORF1p-LOF in mouse neurons was efficient as verified by
520 Western blot (Fig. 7E). Using an automated, non-biased image quantification
521 workflow, we measured neurite morphology via measures of neurite diameter and
522 area (Fig. 7F, G). Quantifications of images illustrated in Figure 7F revealed a
523 significant loss of the mean diameter of neurites after ORF1p-LOF accompanied by a
524 decrease in the total neurite area (Fig. 7G). In summary, these data suggest that
525 ORF1p plays a role in the maintenance of neurite morphology possibly by converging
526 pathways mediated by protein interactions and by modulating chromatin accessibility
527 and transcriptomic landscapes.

528

529 Discussion

530 Starting from the surprising observation that the LINE-1 encoded ORF1p protein is
531 expressed at steady-state in various neuronal subtypes in the adult brain of mice and
532 humans<sup>17,18</sup>, we addressed here the hypothesis that the sustained presence of
533 ORF1p in an important fraction of neuronal cells in the brain<sup>17</sup> might be a requirement
534 for these neurons to maintain specific functions. Using quantitative mass
535 spectrometry, confocal imaging, chromatin accessibility and transcriptomics on
536 human post-mortem neurons and differentiated mouse and human neurons in culture
537 with varying levels of nuclear ORF1p, we provide evidence that ORF1p interacts with
538 chromatin-binding proteins, binds to chromatin, modulates chromatin accessibility
539 and maintains neuronal gene expression. Loss of function of ORF1p leads to the
540 downregulation of genes related to neuronal processes and alters neurite
541 morphology. In addition, ORF1p directly binds to proteins implicated in neurite
542 morphology, indicating that it might be the converging interaction of ORF1p with
543 proteins and chromatin that mediate this effect. To our knowledge, this data
544 represents the first demonstration of a role of a fLINE-1 encoded protein in the
homeostasis of mature neurons and indicates a possible exaptation of a
retrotransposon-encoded protein for physiological functions in the adult brain. This
data thus adds to the growing evidence for the exaptation of retrotransposon-derived
RNA and proteins, for now documented during early development<sup>2,10,11,64</sup>, including in
the developing brain<sup>3</sup>. These studies have focused on the role of LINE-1 RNA. During
early preimplantation development, LINE-1 RNA transcription leads to global
chromatin relaxation and ribosomal DNA transcription promoting blastocyst
formation<sup>10,11</sup>. However, accumulating evidence also suggests that LINE-1, not
necessarily full-length and coding, interfere in the differentiation of the neuronal
lineage. LINE-1 RNA was identified as a structural organizer of chromatin
organization<sup>65</sup>, activators of neuronal gene expression in ESCs<sup>66</sup>, in human neuronal
progenitors<sup>67</sup> and cerebral organoids<sup>12,68</sup>, as well as in embryonic and adult
neurogenesis<sup>3</sup> and as regulators of mouse brain corticogenesis<sup>14</sup>. Importantly, one
study reported that downregulation of LINE-1 expression in cis using CRISPRi in
cerebral organoids led to the upregulation of genes linked to neuronal growth and
neurogenesis and resulted in reduced cerebral organoid size<sup>68</sup>. Another study using
shRNA to knock-down LINE-1 in trans reported the downregulation of neuronal and
synaptic genes<sup>14</sup>, similar to what we observed in post-mitotic neurons here. Mangoni
et al. showed that LINE-1 act as a long non-coding (Inc) RNA and as such
epigenetically mediates the deposition of H3K27me3 on these downregulated genes
regulating cell differentiation and neuronal migration in the developing mouse brain.
They did not investigate, however, whether during corticogenesis, ORF1p was
present on chromatin and/or attached to the LINE-1 RNA. While this would be
interesting to pursue, both sets of data suggest a function of LINE-1 and/or LINE-1
encoded ORF1p in regulating parts of the neuronal transcriptome, either during
570 cortical development¹⁴, or, as we show here, in the maintenance of a stable post-
571 mitotic phenotype. Further, both studies link LINE-1 activity, either mediated through
572 the binding of the LINE-1 RNA to polycomb complexes¹⁴ or via nuclear ORF1p
573 mediating differential accessibility on polycomb-repressed genome domains itself
574 (this study: higher nuclear levels of ORF1p, both in human post-mortem and in
575 LUHMES neurons, are associated with more accessible chromatin on weak
576 polycomb repressive domains), by affecting chromatin accessibility on the PRC-
577 related gene AUTS2 or by interacting with the PRC1 protein CBX8 (this study). PRC1
578 is involved in chromatin silencing following PRC2 mediated tri-methylation of histone
579 H3 at lysine 27⁶⁹,⁷⁰. Notably, Polycomb complex proteins are important for cellular
580 identity in development⁷¹ and in neuronal morphology⁷² through the epigenetic
581 silencing of target genes⁷³. LINE-1 RNA and PRC2 interact also outside the nervous
582 system maintaining nucleolar organization and an embryonic stem cell identity⁷⁴. The
583 interaction of LINE-1 RNA with PRC2 or the differential accessibility of PRC
584 chromatin domains depending on nuclear ORF1p content and the interaction of
585 ORF1p with the PCR1 protein CBX8 could thus suggest a global and important role
586 in maintaining cell identities, possibly including that of post-mitotic neurons. While the
587 analysis identifies a clear association between ORF1p levels and chromatin
588 accessibility patterns, other factors such as disease status may also contribute to the
589 observed differences. However, the consistency between post-mortem findings
590 and controlled in vitro experiments suggests a robust relationship between ORF1p
591 and chromatin accessibility independent of these potential confounders and provides
592 cross-validation across different experimental systems, supporting the specificity of
593 ORF1p's effect on chromatin accessibility.
One important question which arises in this context is that of the molecular form of LINE-1 associated with the observed cellular and molecular changes upon knock-down, that is whether ORF1p is acting alone, ORF1p is associated to LINE-1 RNA, to other cellular RNAs and/or to ORF2p. As introduced above, ORF1p is a nucleic acid chaperone and a RNA binding protein and known to be, due to its cis-preference, associated to LINE-1 RNA but also to other cellular RNAs prone to forming a LINE-1 RNP<sup>75-79</sup>. We have shown here that ORF1p is present in the nucleus and bound to chromatin in post-mortem human neurons and in differentiated mature mouse and human neurons in culture, an observation that had been already made earlier in other cell types and *in vitro*<sup>80-82</sup>. In dividing cells, the LINE-1 RNP is supposed to enter the nucleus passively when the nuclear envelope breaks down during mitosis<sup>35,82</sup> before ORF1p re-exits the nucleus through an active process<sup>35</sup>. In non-dividing somatic cells<sup>38</sup> and in G<sub>0</sub>-arrested cells like neurons<sup>37</sup>, where retrotransposition events can occur, ORF1p supposedly enters the nucleus through binding to import proteins and active nuclear transport<sup>41,83</sup> including in post-mitotic neurons<sup>19</sup>. Whether once in the nucleus, it associates with RNA or not to bind chromatin, is an open question, which remains to be addressed. It is however possible, that the presence of steady-state nuclear ORF1p could be a particular feature of post-mitotic neurons providing a unique nuclear function. As we have shown here, nuclear chromatin containing higher or lower levels of ORF1p showed differences in chromatin accessibility with similar characteristics in two different neuronal systems, suggesting a functional role of ORF1p on chromatin which might or might not be involving (LINE-1) RNA. While the overlap of genomic sites of differential chromatin accessibility in the cingulate gyrus and in LUHMES neurons was rather low, both systems recapitulated specific features of chromatin accessibility
619 depending on nuclear ORF1p content. High nuclear ORF1p led to more accessible
620 chromatin on weak repressed polycomb domains and predominantly in distal
621 intergenic regions while low nuclear ORF1p was associated with more accessible
622 chromatin over enhancers and flanking active transcription sites in intronic regions.
623 How exactly nuclear ORF1p can regulate chromatin accessibility remains to be
624 investigated. One possibility is through the association with proteins involved in
625 chromatin-related functions. Indeed, we identified many nuclear proteins with
626 chromatin-related function interacting with ORF1p. These protein interactors of
627 ORF1p provide an extensive network of proteins related to chromatin organization
628 and transcription regulation. Three protein interactors of ORF1p stood out as they
629 were identified as common ORF1p interacting proteins in both, mouse and human
630 neurons: RCOR2 (corepressor 2 of REST), a transcriptional repressor required for
631 mouse ESC differentiation⁸⁴ and neurogenesis⁸⁵,⁸⁶, YTHDC1, a protein implicated in
632 m6A-dependent RNA metabolism⁸⁷ and HNRNPU, a DNA and RNA binding protein
633 associated with various neurodevelopmental disorders⁸⁸. HNRNPU is involved in
634 chromatin organization, transcription regulation and many other nuclear processes⁸⁹
635 and has been previously identified as an ORF1p interactor in cancerous cell lines³³.
636 Interestingly, YTHDC1 has been shown to bind m6A-modified LINE-1 RNA in order to
637 form a protein-RNA complex which represses the 2-cell program in ESCs⁹⁰ but might
638 also be involved in neuronal survival⁹¹ and cerebral stress response⁹². It would be
639 interesting to test the functional relevance of the interaction of ORF1p with these
640 proteins in mediating chromatin accessibility in neurons. While the functional impact
641 of these protein-protein interactions remains to be determined, their identification
642 reinforces the hypothesis that it might be ORF1p that modulates chromatin
643 accessibility in mature neurons, either directly or indirectly through the binding to
these proteins and/or to RNA. Given the extensive list of ORF1p protein interactors with functions in RNA metabolism, we cannot exclude that ORF1p might also regulate gene expression post-transcriptionally. While many of these interactions were consistent with previously identified protein partners of ORF1p in human cancer cell lines, mouse brain tissues and sperm and were identified in well-established neuronal cell lines, future studies in primary neurons or brain tissue will be important to confirm their relevance in vivo and fully characterize the specificity and strength of these associations.

A combination of observations suggests a role of ORF1p in neuron-specific functions. (i) shared genes containing DARs in both neuronal systems were enriched in gene categories related to axonogenesis and neuron projection. (ii) Transcriptomic changes observed after ORF1p-LOF indicated that downregulated transcripts were particularly long, protein-coding and related to neuron-specific functions like synapse, neuron projection and axon guidance. (iii) Endogenous and HA-tagged ORF1p localized to neurites, both in mouse and human neurons and (iv) mass spectrometry identified axono-dendritic ORF1p interacting partners. (iv) Functionally, ORF1p-LOF induced morphological changes in neurites. Indeed, effects of LINE-1 on neurite morphology have been previously observed during mouse and human brain development. LINE-1 KD in vitro and in vivo induced morphological NPC differentiation and increased the number of dendritic branches, length and complexities¹³. This was accompanied by LINE-1-KD DEGs enriched in functional categories including morphogenesis and projection, similar to what we identified here. Morphological changes during neurodevelopment were characterized by an increase in neurite branches, while we observed decrease in neurite diameters. This suggest that fILINE-1 activity acts on neuronal morphology in post-mitotic neurons.
Interestingly, while Toda et al. did detect LINE-1 RNA on chromatin, there was no correlation of the presence of RNA and DEG gene loci as described during zygotic development¹⁰, which could suggest that the presence of ORF1p is mandatory for specificity in chromatin targeting in neurons. Indeed, during pre-implantation development, ORF1p is exclusively cytoplasmic¹⁰, while in neurons, ORF1p is readily detectable in the nucleus at steady-state. This data suggests a dual role of ORF1p converging on the regulation of specialized neuronal functions. While future work will be required to demonstrate the direct causal links between these sequential events, the consistent enrichment for neuron-specific biological processes such as 'axon guidance' in the proteomic, chromatin, and transcriptomic analyses provides strong correlative support for a unified functional pathway.

Together with mounting evidence of the importance of LINE-1 activity during embryonic and adult neurogenesis² and neurodevelopment³, our work thus contributes to the increasing evidence of the exaptation of what was once called "selfish elements" for important physiological brain functions and beyond. This is important to take into consideration when targeting LINE-1 activity for developing novel therapies for diseases like cancer, autoimmune and neurodegenerative diseases. We have shown previously, that excessive ORF1p and its translocation to the nucleus alters nuclear membrane integrity, transport and heterochromatin organization¹⁹, indicating that increased nuclear levels of ORF1p might be deleterious. Our present study adds to the understanding of LINE-1 biology by showing that mature neurons depend on ORF1p for the fine-tuning of chromatin organization, neuron-related gene expression and neurite morphology. Taken together, we propose a threshold model in which intermediate levels of ORF1p are necessary for maintaining neuronal morphology and function while levels going
694 below or beyond a physiological steady-state expression window might be harmful to
695 neurons.

696

697 **Material and Methods**

698 *Ethical approvals*

699 Reglementary authorizations to use human cell lines and human samples were
700 obtained from the French Ministry of Higher Education, Research, and Innovation
701 (authorization number DC-2020-4013). Experiments using siRNA in human cells
702 (LUHMES) were authorized according to regulatory procedures defined by the
703 French Ministry of Higher Education, Research, and Innovation (OGM n°8273 and
704 OGM n°10463).

705

706 **Cell culture**

707 Lund Human Mesencephalic (LUHMES) cells were ordered from ATCC (ATCC-CRL-
708 2927). Cell culture dishes were pre-coated with 50 µg/mL poly-L-ornithine (Merck, #
709 P4957) diluted (1:2) in DPBS (Gibco, # 14190144) with additionally 1 µg/mL human
710 plasma fibronectin (Sigma, # F0895) at 37°C for at least 3 hours and then rinsed 2
711 times with DPBS. Proliferating LUHMES were cultured using Advanced DMEM/F12
712 (Gibco, #12634028) added with 1% N-2 supplement (Gibco, # 15410294), GlutaMax
713 (Gibco, # 35050061) and 40 ng/mL human recombinant FGF (Peprotech, # 100-18B)
714 at 37C° in 5 % CO₂ incubator. LUHMES cells were differentiated into mature
715 dopaminergic neurons. For this purpose, neurons were cultured in Advanced
716 DMEM/F12 added with 1% N-2 supplement and GlutaMax, supplemented with 1
717 µg/ml doxycycline (Sigma), 2 ng/ml recombinant human GDNF (Peprotech, # 450-10)
and 1mM cAMP (Sigma, # D0627). ORF1p immunoprecipitation was done on day 5 of differentiation and all ORF1p knock-down experiences were conducted on day 7 of differentiation, when LUHMES cells are mature dopaminergic neurons⁹³,⁹⁴. Medium was changed every two days. Cells used in this study had a maximum of 15 passages.

The MN9D cell line was purchased from Merck (# SCC281). Cell culture plasticware was coated with 1 mg/mL poly-lysine (Merck, # P4707) for 2 hours and then rinsed 2 times with DPBS. Cells were cultured in DMEM High Glucose (Sigma, # D5796) supplemented with 10% fetal bovine serum (FBS) (Gibco, #10270098) in 5% CO₂, 37°C incubator. MN9D were differentiated for 10 days with the following medium: DMEM High Glucose (Sigma, # D5796) supplemented with 10% fetal bovine serum (FBS) (Gibco, #10270098) with 1 mM n-butyrate (Sigma, # B5887) and 1 mM dibutyryl cAMP (Sigma, # D0627). Media was changed every two days. Experiments were performed on day 10 of differentiation. Cells used in this study had a maximum of 15 passages.

## SiRNA delivery

Around 150 000 cells/well of LUHMES or MN9D were plated. Media was freshly replaced 2 hours before. Cells were lipofected using RNAiMAX (Invitrogen, # 13778150) at day 3 of differentiation for LUHMES and day 7 of differentiation of MN9D, with 100 nM of siRNA.

siRNA Ctrl: TAATGTATTGGAACGCATA

siRNA Orf1⁹⁵ (human ORF1 - LUHMES): AAGAAGGCTTCAGACGATCAA

siRNA Orf1²⁰ (mouse ORF1 - MN9D): CTATTACTCTGATACCTAAAC
743 Plasmid delivery

744 MN9D were plated at the density of 200 000 cells/well into a pre-coated 4-well plate.

745 On day 7 of differentiation, new pre-warmed media was added 2 hours before and
746 cells were transfected with 1 µg of plasmid/well (pcDNA3-ORF1-HA, kind gift from Dr.

747 John and Dr. Ariumi⁹⁶) or pmaxGFP (Lonza) using Lipofectamin 3000 (Thermo Fisher
748 Scientific, # L3000015) according to manufacturer's instruction.

749

750 Western Blot

751 Cells were scraped at day 7 of differentiation (LUHMES) and at day 10 of

752 differentiation (MN9D) in RIPA buffer (10mM Tris-HCl pH 8.0, 150mM NaCl, 1mM

753 EDTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS). Laemmli buffer

754 was added, and samples were boiled 10 min at 95°C before loading on a 1.5mm

755 NuPAGE 4-12% Bis-Tris polyacrylamide gel (Invitrogen, #NP0336BOX) for the

756 Western blotting. Gels migrated in NuPAGE™ MES SDS Running Buffer (Invitrogen,

757 #NP0002) for 1h10 minutes at 175mV. Gels were transferred onto a methanol-

758 activated PVDF membrane (Immobilon, # IPVH00010) in transfer buffer (25 mM Tris

759 pH:8,3 and 192 mM Glycine) during 1h30 at 400 mA. Membranes were blocked in

760 5% milk TBST (0,2% Tween 20, 150 mM NaCl, 10 mM Tris pH:8) for 1 hour. Primary

761 antibodies were diluted in 5% milk TBST, and membranes were incubated o/n at 4°C.

762 After 3 x 10 min washing in TBST, membranes were incubated for 1h with the

763 respective secondary antibodies diluted at 1/2000 in 5% milk TBST, followed by 3 x

764 10 min washes in TBST. Membranes were revealed by the LAS-4000 Fujifilm system

765 using Clarity Western ECL Substrate (Bio Rad) or Maxi Clarity Western ECL

766 Substrate (Bio Rad) according to the sensitivity needed. Quantifications were

767 performed using LI-COR Image Studio Lite (v.5.2.5).
768

769 **Immunofluorescence**

770 Cells were fixed using 4% methanol-free formaldehyde (Sigma, # 28908) diluted in PBS for 20 min at RT and washed 3 times with PBS. Cells were permeabilized with 0.5% Triton X-100 PBS 20 min and washed twice with PBS. 100mM glycine was added for 20 min and after washing 3 times with PBS, cells were incubated with blocking buffer (10% FBS, 0.5% Triton X-100, PBS) for 1 hour. Primary antibodies in blocking buffer were added overnight at 4°C. The next day, following 3 washes with PBS for 10 min, cells were incubated with secondary antibodies diluted in blocking buffer for 1 hour at RT. Finishing with 3 PBS washes, slices were mounted with Fluoromount (Invitrogen, # 00-4958-02). Images were obtained from a Spinning-disk W1 (Yokogawa) or Spinning-disk X1 (Yokogawa) and viewed in Fiji Image J (v.2.16.0).

781

782 **Image Analysis**

783 Neurite network analysis was conducted using a custom macro developed for the Fiji software⁹⁷, incorporating functionalities from the Bio-Formats⁹⁸, Analyse Skeleton⁹⁹ and Local Thickness¹⁰⁰ plugins. Code is openly accessible at: https://github.com/orion-cirb/Axon_Skel_Analyzer.

787 Nuclei were segmented and counted in the DAPI channel using the following sequence of operations: maximum intensity z-projection, median filtering (radius = 10 pixels), Huang automatic thresholding, hole filling, watershed splitting, object labelling, and filtering based on morphological criteria (minimum area = 40 µm², minimum circularity = 0.7). Cell bodies were segmented in the ORF1p channel by successively applying sum slices z-projection, median filtering (radius = 50 pixels),
793 Huang thresholding, morphological opening (20 iterations) and dilation (5 iterations),
794 hole filling, and size filtering (minimum area = 80 µm2).

795 The TUJ1 channel was segmented via maximum intensity z-projection, background
796 subtraction (rolling ball radius = 50 pixels), median filtering (radius = 2 pixels), Huang
797 thresholding, morphological closing (4 iterations), an additional median filtering
798 (radius = 2 pixels), selective hole filling (holes < 10 µm²), and size filtering (minimum
799 area = 20 µm²). To isolate neurites, cell bodies segmented from the ORF1p channel
800 were subtracted from the TUJ1 channel binary mask. The resulting neuronal
801 processes were then skeletonized and branches shorter than 4□µm were excluded
802 to minimize artifacts. This processing pipeline enabled local thickness analysis to
803 quantify the distribution of branch diameters.

804 The analysis of ORF1p intensity after ORF1p-LOF in LUHMES cells was performed
805 using the Nuclei_ORF1p plugin¹⁹ developed for the Fiji software. This tool enabled
806 quantification of ORF1p signal specifically in the nucleus versus the cytoplasm of
807 cells.

809 Subcellular fractionation

810 Subcellular fractionation was performed with 10⁶ cells using the Subcellular Protein
811 Fractionation kit (Thermo Fisher Scientific, # 78840) according to manufacturer's
812 recommendations. For cytoplasm and nucleus fractionation, cells were washed in
813 PBS and resuspend in lysis buffer (10mM HEPES ph7.9, 40mM KCL, 5% glycerol,
814 3mM MgCl2, 0.2% NP40, EDTA-free protease inhibitor cocktail and phosphatase
815 inhibitors) and incubated for 30 min at 4°C. Lysates were passed 20 times in a
816 Dounce homogenizer (loose) and then left for 30 min on a wheel at 4°C. After
817 centrifugation at 4°C at 500g for 5 min, the supernatants were kept as the cytosolic
818 fraction, and the pellets are washed once in lysis buffer and resuspend in nuclear
819 lysis buffer (20mM HEPES ph7.9, 420mM NaCl, 25% glycerol, 1.5mM MgCl2, EDTA-
820 free protease inhibitor cocktail and phosphatase inhibitors) and rotated for 30
821 minutes on a wheel at 4°C. Finally, lysates were centrifuged for 10 min at 14000g at
822 4°C and the supernatant was collected as nuclear extract.

823

824 RNA extraction

825 3.5 x 10⁵ cells were scrapped in TRIzol reagent (Invitrogen, # 15596026) and total
826 RNA was extracted using Direct-zol™ RNA Microprep (Zymo, # R2062) according to
827 manufacturer's instructions. All samples were subjected to DNase I treatment during
828 extraction.

829

830 RT-qPCR

831 300 ng RNA was subject to reverse transcription using All-In-One 5X RT MasterMix
832 kit (Abm, # G592). qPCR was performed in duplicate for each sample and each gene
833 with SsoAdvanced Universal SYBR Green (BioRad, #1725274) with the following
834 protocol: a first heat up at 95°C, followed by the denaturation step at 95°C, annealing
835 at 57°C and finally extension at 72°C for a total of 40 cycles, on a CFX Opus 384
836 Real-Time PCR System (BioRad, # 12011452). qPCR was analysed by CFX
837 Maestro 2.0 software (v5.0.021.0616) (BioRad) and each gene of interest was
838 normalized to three housekeeping genes: ENO2, TBP and HPRT.

839 Following primers were used: HPRT (F: CAGCCCTGGCGTCGTGATTAGT; R:
840 CCAGCAGGTCAGCAAAGA AT); TBP (F: CAGCATCACTGTTTCTTGGCGT; R:
841 AGATAGGGATTCCGGGAGTCAT); ENO2 (F: CGTTACTTAGGCAAAGGTGTCC; R:
842 CTCCAGCATCAGGTTGTCCAGT); DLG5 (F: GCCTTCAGAGTCTGCCTTGT; R:
843 CAGGCTGGAAGACAGGAAGG); NEGR1 (F: TTAAGAGGCAGGCTCGCATT; R:
844 TACCAACGTGACACAGGAGC); EFNA5 (F: AGCGGTCCATTTGGAGAGTG; R:
845 ATGAGGACTCCGTCCCAGAA); GABRB3 (F: GCCTTGGCTGTCTTTTCTGC; R:
846 ACCTTATGGGCTGCTTCGTC).

847

848 Immunoprecipitation and sample preparation for mass spectrometry

849 Magnetic beads were coupled with ORF1p (MN9D: Abcam, #ab245124) (LUHMES: Abcam, #ab245249) or IgG rabbit (Abcam, #ab172730) antibodies using the Dynabeads® Antibody Coupling Kit (Thermo Fisher Scientific, # 14311D) according to the manufacturer's recommendations. After two washes in DPBS, cells were scraped in lysis buffer (10mM Tris HCl pH:8, 150mM NaCl, 0,5% NP40, protases and phosphatase inhibitors), incubated on ice for 15 min and sonicated (Bioruptor UCD-200) for 15 min (30s on/ 30s off) on ice at medium power. Lysates were incubated with coupled beads and diluted in wash buffer (10 mM Tris HCl pH8, 150 mM NaCl, protases and phosphatase inhibitors) overnight on a wheel at 4°C. Samples were washed 4 times with wash buffer and were resuspended in 100 µl of 25 mM NH4HCO3. IP samples were then washed twice with 100 µL of 25 mM NH4HCO3 (ABC) and proteins were trypsin/lysC (0.2 µg, Promega) digested directly on beads in a total volume of 100µL of 25 mM ABC for one hour at 37°C while vortexed at 1000rpm. Samples were then cleaned on homemade C18 StageTips (AttractSPE Disk Bio C18-100.47.20 Affinisep), before being eluted using 40/60 CH3CN/H2O☐+☐0.1% formic acid and vacuum concentrated to dryness.

865

866 LC-MS/MS Analysis
867 LUHMES liquid chromatography was performed with an RSLC nano system (Ultimate
868 3000, Thermo Scientific) coupled online to an Orbitrap Exploris 480 mass
869 spectrometer (Thermo Scientific). Peptides were trapped on a C18 column (75 µm
870 inner diameter × 2 cm; nanoViper Acclaim PepMap™ 100, Thermo Scientific) with
871 buffer A (2/98 CH3CN/H2O in 0.1% formic acid) and separation was performed on a
872 50 cm × 75 µm C18 column (nanoViper Acclaim PepMap™ RSLC, 2 µm, 100Å,
873 Thermo Scientific) with a linear gradient of 3% to 29% buffer B (100% CH3CN in
874 0.1% formic acid, A': 100% H2O in 0.1% formic acid) at a flow rate of 300 nL/min
875 over 91 min. For Data dependent acquisition (DDA), MS full scans were performed in
876 the ultrahigh-field Orbitrap mass analyser in ranges m/z 375–1500 with a resolution
877 of 120□000 at m/z 200. The top 20 intense ions were subjected to Orbitrap for further
878 fragmentation via high energy collision dissociation (HCD) activation and a resolution
879 of 15□000 with the intensity threshold kept at 1.3 × 10⁵. We selected ions with
880 charge state from 2+ to 6+ for screening. Normalized collision energy (NCE) was set
881 at 27 and the dynamic exclusion of 40s.

882 MN9D LC-MS/MS analysis was performed, as previously, in data independent
883 acquisition (DIA) mode and by using a linear gradient of 2% to 30% buffer B. MS full
884 scans were performed in the ultrahigh-field Orbitrap mass analyser in range m/z 375–
885 1500 (120□000 resolution; IT 100 ms; AGC 300%). 40 DIA scan of 15 Da isolation
886 window with 1 Da overlap between 400 to 1000 m/z (15 000 resolution; NCE 25;
887 AGC 3000; IT auto) were set.

888 For protein identification, LUHMES DDA data were searched against the Homo
889 Sapiens (UP000005640_9606) UniProt database using Sequest HT through
890 proteome discoverer (PD version 2.4). Enzyme specificity was set to trypsin and a
891 maximum of two miss cleavages sites were allowed. Oxidized methionine, Met-loss,
892 Met-loss-Acetyl and N-terminal acetylation were set as variable modifications.

893 Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions
894 and 0.02 Da for MS/MS peaks.

895 MN9D DIA data were searched against the Mus Musculus (UP000000589) Uniprot
896 database using Spectronaut v18.6 (Biognosys) by directDIA+ analysis using default
897 search settings. Enzyme specificity was set to trypsin and a maximum of two missed
898 cleavage site were allowed. N-terminal acetylation and oxidation of methionine were
899 set as variable modifications.

900 The resulting files were further processed using myProMS v3.10.0
901 (https://github.com/bioinfo-pf-curie/myproms)¹⁰¹. FDR calculation used Percolator¹⁰²
902 and was set to 1% at the peptide level for the LUHMES data. The label free
903 quantification was performed by peptide Extracted Ion Chromatograms (XICs) and
904 DDA were reextracted by conditions and computed with MassChroQ version
905 2.2.21¹⁰³ (LUHMES data). For protein quantification, XICs from proteotypic peptides
906 shared between compared conditions (TopN matching) with missed cleavages were
907 used. Median and scale normalization at peptide level was applied on the total signal
908 to correct the XICs for each biological replicate (N=5). To estimate the significance of
909 the change in protein abundance, a linear model (adjusted on peptides and biological
910 replicates) was performed, and a two-sided t-test was applied on the fold change
911 estimated by the model. The p-values were adjusted using the Benjamini-Hochberg
912 FDR procedure.

913 The mass spectrometry proteomics raw data have been deposited to the
914 ProteomeXchange Consortium via the PRIDE¹⁰⁴ partner repository under the
915 accession number PXD066047.
917 **RNA sequencing and analysis**

918 Four samples for each condition (siRNA-Ctrl and siRNA-Orf1) were sequenced
919 (Novogene Europe). Quantity and quality were assessed using Nanodrop and
920 Bioanalyzer RNA 6000 Pico Kit (Agilent Technologies, # 5067-1513). Libraries
921 including rRNA removal were generated by RNA Library Prep Kit for Illumina
922 following manufacturer's recommendations and sequenced as pair-end (PE) 150 bp
923 using Novaseq x plus sequencer. Mapping was performed by Hisat2 (v2.0.5) on
924 human genome (hg38/GRCh38 assembly). Differential enrichment analysis was done
925 with DESeq2 (v1.46.0) and data were normalized using DESeq2 scaling and size
926 factors method. Genes where the sum of reads from all samples was under ten reads
927 were removed. Genes with no reads for at least 6 samples on 8 were discarded.
928 Volcano plot was generated by considering as significatively upregulated (264
929 genes), genes with log2 fold change ≥ 0.5 and p ≤ 0.01; significatively
930 downregulated (698 genes), genes with log2 fold change ≤ -0.5, p ≤ 0.01). Another
931 more stringent threshold was fixed for defining downregulated genes (log2 fold
932 change ≤ -0.5, p ≤ 0.005), and among those, genes forming a network involved in
933 synapse GO (50 genes) were visualized by STRING database (v.12.0). Gene
934 annotations were retrieved from hg38 RefSeq_all and long genes were defined as >
935 100kB. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was
936 conducted with R function enrichKEGG from clusterProfiler (v.4.14.4). Gene Ontology
937 (GO) analysis was executed using the PANTHER GO database (release 19.0). p-
938 values were adjusted using the Benjamini-Hochberg method.

939

940 Human Samples
941 Cingulate gyrus brain tissue from control (n=5) and Parkinson disease patients (n=5)
942 were provided by the Biobank Neuro-CEB and conserved at -80°C. Tissues
943 underwent one manual physical shock and were then disposed in precooled Tissue-
944 Tube bags (Covaris) and dry cryo-pulverized with a level 6 impact using the
945 CryoPrep Dry Pulveriser (Covaris). All steps were done on dry ice.

946

947 Isolation and sorting of nuclei (FANS)

948 Nuclei from pulverized tissues of human patients were resuspend in homogenization
949 buffer (HB) (0.25M sucrose, 0.5mM spermidine, 0.15nM spermine, NP40 0.3%,
950 protease inhibitors, 1mM DTT). Tissues were grinded mechanically using a Dounce
951 homogenizer and passed through a 40µm cannula. The recovered cells were then
952 put on an OptiPrep gradient (Sigma, # D1556) and centrifuged for 15 minutes at
953 10000g. Pellets were dried and put in PBS spermine 0.15nM. Human nuclei were
954 then labelled 1h on a wheel at 4°C using the following antibodies: NeuN PE (Merck, #
955 FCMAB317PE), ORF1p AF647 (Abcam, # ab314927) and control antibodies: IgG1
956 mouse-PE (Beckman Coulter, # A07796) and IgG rabbit-AF647 (Abcam, #
957 ab199093). Nuclei sorting was performing using a FACSARIA II cell sorter (Beckton
958 Dickinson) equipped with three lasers (405, 488 and 640nm), a 70µm nozzle and
959 FACSDiva software (version 6.1.2). Nuclei were detected according to their forward
960 scattering light (FSC) and gated using forward and side scatter light signals.
961 Aggregates were excluded by forward scatter area versus height pulse
962 measurement. Finally, PE and AF647 fluorescence were respectively measured
963 through a 585/42 and 660/20nm bandpass filters. P3 corresponds to all nuclei
964 (defined as ORF1p-high) which fluoresce beyond the maximum intensity of nuclei in
965 the control (non-relevant antibody that fluoresces in the same channel as ORF1 (x-
axis, 647nm)), while P4 is everything below this threshold (ORF1p-low nuclei). As in the control (non-relevant antibody that fluoresces in the same channel as NeuN (y-axis, NeuN-PE)), autofluorescent nuclei form a diagonal, we excluded nuclei in this range and defined as NeuN-positive all nuclei above this autofluorescent window. 50 000 nuclei were recorded. Nuclei were collected in PBS buffer in Eppendorf tubes.

971

972 **ATAC sequencing and analysis**

973 After FANS, 50,000 nuclei per condition were centrifuged at 4°C, 500g for 10 min.
974 ATAC-seq was then performed using a commercial kit (Diagenode Cat.
975 No.C01080006 for human tissue and Cat. No.C01080001 for LUHMES) according to
976 the instructions of the manufacturer. Profiles of libraries were verified using the
977 Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). The library pool was
978 quantified by qPCR using the KAPA library quantification kit (Roche). Sequencing
979 was carried out on the NovaSeq 6000 instrument from Illumina using paired-end 2 x
980 100 bp sequencing. ATAC-seq data were analyzed using the Curie Institute ATAC-
981 seq pipeline (v1.02, DOI: 10.5281/zenodo.7576558 and available at
982 https://github.com/bioinfo-pf-curie/ATAC-seq/releases/tag/v1.0.2). Briefly, sequencing
983 reads were aligned after adapter removal on the Human reference genome (hg38)
984 with bwa-mem. Aligned reads were filtered to remove mitochondrial reads, PCR
985 duplicates, singleton and reads aligned with a mapping quality lower than 20.
986 Genomic sites from the ENCODE blacklist regions were also discarded from the
987 analysis. The peak calling was then performed using the MAC2 software with the
988 following parameters "-f BAMPE --nomodel -q 0.01". DeepTools (v3.5.4) command
989 multiBamSummary was used to count reads from all peaks. All peaks were kept and
990 peaks with the sum of reads from all samples smaller than ten reads were removed.
991 Differential accessible peaks were determined with DESeq2 (v1.46.0). For LUHMES,
992 we compared siRNA-Ctrl (n=2) and siRNA-ORF1 (n=2) and peaks were considered
993 as significant with a p-value ≤ 0.05 and log2FC ≥ and ≤ 0.5. For human samples
994 (NeuN- = 4 samples, NeuN+ ORF1p low = 6 samples and NeuN+ ORF1p high = 6
995 samples), peaks were considered as significant with a p-value ≤ 0.01 and log2FC ≥
996 and ≤ 1. Peaks were annotated to the nearest gene with the function annotatePeak
997 from ChIPseeker R package (v.1.42.1), using
998 TxDb.Hsapiens.UCSC.hg38.knownGene and TSSregion set to ±3kb. Motif
999 enrichment analysis was performed with HOMER (v5.1), using findMotifsGenome.pl,
1000 with default parameters. GO analysis was conducted on the PANTHER GO database
1001 (release 19.0). Significative DAR association with known chromatin states was
1002 investigated using available ChromHMM annotations (retrieved from Roadmap=
1003 https://pmc.ncbi.nlm.nih.gov/articles/PMC8734071/) from adult cingulate gyrus
1004 (ATAC human brain) and substantia nigra (ATAC LUHMES). 71 significant genes
1005 were overlapping between ATAC-seq in human post mortem tissues and LUHMES.
1006 GO BP analysis of those proteins were carried out in R using the clusterProfiler
1007 (v.4.14.4). package and represented as a GOChord plot (package GOplot v.1.0.2).
1008 Bigwig tracks were visualized using the Integrated Genome Viewer (IGV) browser
1009 (v2.19.1).

1010

1011 **Statistics**

1012 Statistical analyses were done with Graph Pad PRISM software (v10.4.1). The
1013 significance threshold was defined as p<0.05 except stated otherwise. We assessed
1014 the distribution of differentially accessible regions (DARs) across genomic features or
1015 regulatory regions using two-sided Fisher's exact tests. To ensure statistical
robustness, categories with low total counts (<5 DARs) were excluded from these analyses. False discovery rate (FDR) correction with reported q-values was applied to p-values of the remaining comparisons using the Benjamini-Hochberg procedure (Q =1%). Gene biotype and long gene overrepresentation analyses were done using a Chi-Square test with observed = number of feature counts in ORF1p-LOF and expected = all RNA-seq transcript counts.

1022

1023 Acknowledgements

SS acknowledges Université PSL for a Ph.D. fellowship and was enrolled with the Ecole Doctorale ED3C. This work was supported by the Agence Nationale de la Recherche (National French Agency for Research; ANR-20-CE16-0022 NEURAGE), the "Fondation du Collège de France" (to J.F.), the "Fondation NRJ/Institut de France" (to J.F.), the "Fondation Alzheimer" (to J.F.) and the "Fédération pour la Recherche sur le Cerveau" (FRC, to J.F.). High-throughput sequencing was performed by the ICGex NGS platform of the Institut Curie supported by the grants ANR-10-EQPX-03 (Equipex) and ANR-10-INBS-09-08 (France Génomique Consortium) from the "Agence Nationale de la Recherche" ("Investissements d'Avenir" program), by the ITMO-Cancer Aviesan (Plan Cancer III) and by the SiRIC-Curie program (SiRIC Grant INCa-DGOS-465 and INCa-DGOS-Inserm_12554). Data management, quality control and primary analysis were performed by the Bioinformatics platform of the Institut Curie. The LSMP thanks Patrick Poullet from the bioinformatics platform of the Institut Curie U1331 for the continuous development of myProMS. We thank the "The Brainbank Neuro-CEB Neuropathology Network" for the human *post-mortem* tissues. The Neuro-CEB Neuropathology network includes: Dr Franck Letournel (CHU Angers), Dr Marie-
1041 Laure Martin-Négrier (CHU Bordeaux), Dr Maxime Faisant (CHU Caen), Pr Catherine
1042 Godfraind (CHU Clermont-Ferrand), Pr Claude-Alain Maurage (CHU Lille), Dr
1043 Vincent Deramecourt (CHU Lille), Dr Mathilde Duchesne (CHU Limoges), Dr David
1044 Meyronnet (CHU Lyon), Dr André Maues de Paula (CHU Marseille), Pr Valérie Rigau
1045 (CHU Montpellier), Dr Fanny Vandenbos-Burel (Nice), Pr Charles Duyckaerts (CHU
1046 PS Paris), Pr Danielle Seilhean (CHU PS, Paris), Dr Susana Boluda (CHU PS,
1047 Paris), Dr Isabelle Plu (CHU PS, Paris), Dr Serge Milin (CHU Poitiers), Dr Dan
1048 Christian Chiforeanu (CHU Rennes), Dr Florent Marguet (CHU Rouen), Dr Béatrice
1049 Lannes (CHU Strasbourg). We would also like to thank the following patient
1050 organisations which support the Neuro- CEB brainbank: ARSLA, CSC, France DFT,
1051 Fondation ARSEP, Fondation Vaincre Alzheimer and France Parkinson. We also
1052 thank the Fondation Bettencourt Schueller for their support. We especially thank Ariel
1053 Di Nardo and Alain Prochiantz for their continuous support.

1054

1055 Contributions

1056 S.S. carried out the majority of the experimental work and collected and analyzed the
1057 data. O.M.B. designed and performed the FANS analysis with M.F. and S.S., carried
1058 out the subcellular fractionations with S.S. and prepared the IP samples for mass
1059 spectrometry (LUHMES). B.L. carried out the LC-MS/MS experimental work and D.L.
1060 supervised MS and MS data analysis which was followed up by S.S and T.B.. H. M.
1061 and T.C. set up the script for image analysis, N.S. supervised the bioinformatical
1062 analyses done by S.S.. S.S., R.L.J. and J.F interpreted the data, S.S. and J.F. wrote
1063 the manuscript with input from all authors. J.F. and R.L.J. conceived and supervised
1064 the project.

1065
1066 **Competing interests**

1067 The authors declare no competing interests.

1068

1069 **Figure Legends**

1070

1071 **Figure 1: Quantitative mass spectrometry analysis of protein partners of endogenous ORF1p in human and mouse dopaminergic neurons**

1073 **A.** Graphical representation of the experimental protocol used to identify protein partners of endogenous ORF1p in mouse and human neurons. Endogenous ORF1p was immunoprecipitated (IP) from human (LUHMES) and mouse (MN9D) differentiated dopaminergic neurons, respectively. Enrichment was verified by Western blot analysis and lysates were analysed by LC-MS/MS followed by the identification and quantification of protein partners.

1079 **B.** Specific protein partners of ORF1p found by proteomic mass spectrometry in MN9D neurons, presented according to their fold change (ratio IP-ORF1p/IP-IgG) and adjusted p-value. Proteins identified in the IP ORF1p (ab216324) by LC-MS/MS were compared to IP IgG. For each condition (IP IgG, IP ORF1p) 5 independent immunoprecipitations were performed. Proteins with at least 2 distinct proteotypic peptides in all 5 replicates in the IP ORF1p condition with a ratio IP ORF1p/IP IgG ≥ 2 and a p-value ≤ 0.05 were considered as ORF1p interacting proteins. In the right panel, partners of ORF1p, which were not detected in the IP IgG condition (and therefore have no fold-change or p-value) are shown according to the number of peptides per 100 amino acids identified.

1089 **C.** Specific protein partners of ORF1p identified by proteomic mass spectrometry in LUHMES neurons, presented according to their fold change (ratio) and adjusted p-value.
1091 value. For each condition (IP IgG, IP ORF1p) 5 independent immunoprecipitations
1092 were performed. Proteins identified in the IP ORF1p (ab49) by LC-MS/MS were
1093 compared to IP IgG. Proteins with at least 1 distinct proteotypic peptide in at least 3
1094 replicates identified in the IP ORF1p condition with a fold change ≥ 2 and p-
1095 value ≤ 0.05 were considered as ORF1p interacting proteins. In the right panel,
1096 partners of ORF1p, which were not detected in the IP IgG condition (and therefore
1097 have no fold-change or p-value) are shown according to their number of peptides per
1098 100 amino acids identified.

1099 D. Top common GO categories (CC, MF, BP) of ORF1p protein partners in mouse
1100 (MN9D) and human (LUHMES) dopaminergic neurons

1101 E. Physical protein-protein interactions (PPI) network of ORF1p protein partners
1102 identified by mass spectrometry in MN9D neurons using the STRING database and
1103 visualized with Cytoscape. ORF1p protein partners belong to different functional and
1104 enriched GO terms (rings) : nucleus (dark blue), negative regulation of gene
1105 expression (blue), RNA polymerase II, core complex (light blue), synapse (red),
1106 neuron projection (pink) and RNA binding (green). Interactors previously identified
1107 are highlighted in yellow nodes. The thickness of the edges is proportional to the
1108 confidence in the physical interaction. Only proteins with physical interactions are
1109 shown.

1110 F. Physical protein-protein interactions (PPI) network of ORF1p partners found in
1111 LUHMES neurons by mass spectrometry using the STRING database and visualised
1112 via Cytoscape. Partners belong to different functional enriched GO terms (rings):
1113 gene expression (dark blue), nucleus (blue), axon guidance (red), mRNA splicing
1114 (light blue) and RNA binding (green). Interactors previously identified are highlighted
1115 in yellow nodes. The thickness of the edges is proportional to the confidence in the
1116 physical interaction. Only proteins with physical interactions are shown.

1117

1118 **Figure 2: ORF1p is located to the nucleus and associated to chromatin in post-**
1119 mitotic neurons

1120 **A.** Immunostainings of endogenous ORF1p in LUHMES and MN9D neurons. Nuclear
1121 localisation of endogenous ORF1p is indicated by white arrows (antibody human
1122 ORF1p: Millipore MABC1152; antibody mouse ORF1p: Abcam ab216324) Scale bar
1123 is 10µm.

1124 **B.** Biochemical separation of the nuclear and cytoplasmic compartments of two
1125 independent samples followed by Western blot using an ORF1p antibody and two
1126 markers, one for the nuclear compartment (H3K9me3) and one for the cytoplasmic
1127 compartment (eIF4E). ORF1p is present in the cytoplasmic and the nuclear fraction
1128 of LUHMES and MN9D neurons. A representative experiment is shown (n=3
1129 independent experiments). *C: Cytoplasm, N: Nucleus*.

1130 **C.** Subcellular fractionation of the cytoplasm (CE), the membrane bound fraction
1131 (MB), the nucleoplasm (NP), the chromatin-bound fraction (CB) and the pellet (PE) of
1132 cell lysates of MN9D neurons followed by Western blotting using an ORF1p antibody
1133 and different protein markers (CE marker: eIF4E; MB marker: EGFR, NP marker:
1134 SP1; CB marker: H3K9me3; stress granule marker: G3BP1). ORF1p is present in all
1135 compartments, including the chromatin-bound fraction. A representative experiment
1136 is shown (n=3 independent experiments).

1137 **D.** Subcellular fractionation of the cytoplasm (CE), the membrane bound fraction
1138 (MB), the nucleoplasm (NP), the chromatin-bound fraction (CB) and the pellet (PE) of
1139 cell lysates of LUHMES neurons followed by Western blotting using an ORF1p
antibody and different protein markers (CE marker: eiF4E; MB marker: EGFR, NP marker: SP1; CB marker: H3K9me3; stress granule marker: G3BP1). ORF1p is present in all compartments, including the chromatin-bound fraction. A representative experiment is shown (n=3 independent experiments).

**E.** Subcellular fractionation of the cytoplasm (CE), the membrane bound fraction (MB), the nucleoplasm (NP), the chromatin-bound fraction (CB) and the pellet (PE) of lysates of human post-mortem cingulate gyrus tissue of a neurologically-healthy individual followed by Western blotting using an ORF1p antibody and different protein markers (CE marker: eiF4E; MB marker: EGFR, NP marker: SP1; CB marker: H3. PE marker: Vimentin1. ORF1p is present in all compartments, including the chromatin-bound fraction. A representative experiment is shown (n=3 independent experiments).

**Figure 3: Nuclear ORF1p levels are associated with chromatin landscape variations in neurons**

**A.** Graphical representation of the experimental set-up using fluorescent-activated nuclei sorting (FANS) to isolate neuronal nuclei with high or low nuclear ORF1p content from human post-mortem cingulate gyrus. Nuclei were extracted from pulverized frozen tissues of post-mortem cingulate gyrus of neurologically-healthy individuals (n=3) and individuals diagnosed with Parkinson's disease (n=3). Nuclei were sorted with an antibody against NeuN to separate neurons (NeuN+) from non-neuronal cells (NeuN-) and an antibody against ORF1p to separate neuronal nuclei into two populations: those containing high amounts of ORF1p (ORF1p-high) and those containing no or low amounts of ORF1p (ORF1p-low). Sorted populations were then subjected to ATAC-sequencing.
1165 **B.** Representative fluorescent-activated nuclei sorting (FANS) profile showing the separation of non-neuronal cells (NeuN-; P6), NeuN+ cells (P3+P4) with either low (P4) or high (P3) nuclear ORF1p levels.

1168 **C.** Western blot of nuclei sorted by FANS. ORF1p is not detectable in non-neuronal cells (NeuN-). Higher amounts of ORF1p are present in the sorted population of ORF1p-high neuronal nuclei compared to ORF1p-low or ORF1p-negative neuronal nuclei. NeuN was used as a control for the separation of neuronal from non-neuronal nuclei. H3 was used as a loading control.

1173 **D.** Volcano-plot of differentially accessible regions (DARs) identified by ATAC-seq comparing ORF1p-high and ORF1p-low human post-mortem neurons LUHMES. Red: significantly more accessible regions (DAR+) in NeuN+ ORF1p-high; Blue: significantly less accessible regions in NeuN+ ORF1p-high (DAR-) (log2 fold change ≤ -1, pvalue ≤ 0.01).

1178 **E.** Genomic features associated with differentially-accessible-region (DAR) in NeuN+/ORF1p-high and NeuN+/ORF1p-low.

1180 **F.** Annotation of functional genomic features overlapping significant DARs using human post-mortem cingulate gyrus ChromHMM data retrieved from Roadmap (https://pmc.ncbi.nlm.nih.gov/articles/PMC873).

1184 **G.** IGV track of an example of DAR+ regions in ORF1p-low neuronal nuclei in the DLX1/2 genomic locus.

1186 **H.** IGV track of an example of a DAR+ region in ORF1p-high neuronal nuclei in the SPACA3 genomic locus.

<caption>1189 **Figure 4: ORF1p knock-down induces changes in chromatin organization**</caption>
1190 **A.** Representative image of ORF1p knock-down by immunofluorescence (left) and quantification of ORF1p staining intensity (right; each dot on the dot blot represents one image). Two-tailed Mann–Whitney *U* test. Error bars are represented as mean ± SEM. *p* ≤ 0.05; **p** ≤ 0.01; ***p** ≤ 0.001; ****p ≤ 0.0001.

1194 **B.** MA-plot of differentially accessible regions (DAR) identified by ATAC-seq after ORF1p-LOF in human neurons (log2 fold change ≤ -0.5, p-value ≤ 0.05).

1196 **C.** Genomic features associated with differentially-accessible-regions (DARs) more (DAR+) or less accessible (DAR-) in ORF1p-LOF neurons.

1198 **D.** Annotation of functional genomic features overlapping significant DARs using human post-mortem *substantia nigra* ChromHMM data retrieved from Roadmap (https://pmc.ncbi.nlm.nih.gov/articles/PMC873).

1202 **E.** IGV track of a DAR+ region in ORF1p-LOF neuronal nuclei as identified by ATAC-seq in the WDR74 genomic locus.

1204 **F.** IGV track of DAR- regions in neurons after ORF1p-LOF in the ROCK1P1 gene locus.

1206

1207 **Figure 5: Nuclear ORF1p modulates chromatin accessibility on genes related to neuron-specific processes**

1209 **A.** Venn diagram showing the overlap between ATAC-seq DARs from human neuronal nuclei containing high or low amounts of ORF1p (1260 DARs) and ATAC-seq DARs from control human dopaminergic neurons (LUHMES) compared to neurons after ORF1p-LOF (719 DARs). 71 genes are associated to DARs in both conditions.
1214 B. GO analysis of 71 genes which are associated with at least one DAR in both
1215 ATAC-seq analyses. GO biological process complete; Fisher's Exact test with FDR
1216 correction. 52 out of 71 were unambiguously mapped. Axon guidance (fold
1217 enrichment 11.92, FDR: 0.02, 7 genes).

1218 C. IGV tracks of 2 genes identified in the GO analysis in Figure 5B sharing DARs
1219 between human cingulate gyrus ORF1p-high/-low and human neurons Ctrl/ORF1p-
1220 LOF. Shown are genomic loci around the genes GLI2 and ROBO1.

1222 **Figure 6: Loss of ORF1p leads to transcriptomic changes in neuron-specific**
1223 **genes**

1224 A. Principal component analysis (PCA) of RNA-seq data showing the separation of
1225 control neurons (blue) and ORF1p-KD neurons (red; n=4 samples per condition,
1226 LUHMES).

1227 B. Volcano plot showing differentially expressed genes (DEGs) in LUHMES neurons
1228 upon ORF1p-LOF. 698 genes were significantly upregulated (in red: log2 fold change
1229 ≥ 0.5, p-value ≤ 0.01); 264 genes were significantly downregulated (in blue: log2 fold
1230 change ≤ -0.5, p-value ≤ 0.05).

1231 C. Parts of whole graph showing the distribution of gene biotypes extracted from
1232 NCBI RefSeq among all expressed transcripts present in the RNA-seq analysis (left)
1233 or upregulated (middle) or downregulated transcripts (right) upon ORF1p-LOF.
1234 Distributions of gene biotypes among expressed or upregulated transcripts were
1235 similar while downregulated transcripts were mainly protein-coding transcripts. Gene
1236 biotype overrepresentation analysis was done using a Chi-Square test with observed
1237 = number of feature counts in ORF1p-LOF and expected = all RNA-seq transcript
1238 counts.
1239 **D.** Gene length analysis of all expressed transcripts in human LUHMES neurons (left), upregulated (middle) or downregulated transcripts (right) upon ORF1p-LOF.
1240
1241 Genes were separated in two categories; long genes (>100kB) and short genes (<=100kB). The fraction of long genes among all and upregulated transcripts was similar (around 10%) but was significantly higher among downregulated transcripts (22%). Long gene overrepresentation analysis was done using a Chi-Square test with observed = number of feature counts in ORF1p-LOF and expected = all RNA-seq transcript counts.
1242
1243
1244
1245
1246

1247 **E.** GO analysis of significant downregulated genes from ORF1-LOF vs Ctrl. FDR was calculated from p-values (Fisher's exact test) by the Benjamini-Hochberg procedure.
1248
1249 CC: Cellular Component, BP: Biological process and MF: Molecular Function.
1250

1251 **F.** Protein-protein interaction (PPI) network of significantly downregulated genes upon ORF1p-LOF (log2 fold change ≤ -0.5, p-value ≤ 0.005) illustrated for the GO Cellular Component term “Synapse” GO:0045202 (50 genes). Genes are coloured according to their associated GO term.
1252
1253

1254 **G.** Quantitative RT-PCR depicting the relative mRNA expression of DLG1, NEGR1, EFNA5 and GABRB3 normalized to ENO2, TBP and HPRT in LUHMES neurons treated with siRNA Ctrl or siRNA ORF1 (n=3 per condition). Two-tailed Student's t-test. Data is represented as mean± SEM *p* ≤ 0.05; **p** ≤ 0.01; ***p* ≤ 0.001; ****p* ≤ 0.0001.
1255
1256
1257
1258
1259

1260 **Figure 7: ORF1p knock-down induces phenotypic changes of neurites**

1261 **A.** Representative images showing ORF1p expression (red) in neurites (green; TUJ1) in mouse (upper panel) and human (lower panel) dopaminergic neurons. Scale bar is 10µm.
1262
1263
1264 **B.** Expression of plasmid encoded human ORF1p tagged with HA and labelled with a HA antibody (green) in differentiated mouse dopaminergic neurons (MN9D). Neurons were transfected at day 10 of differentiation. Neurites were counterstained with NEFL or TUJ1 (white) as indicated. ORF1p is readily detectable in TUJ1-positive neurites and NEFL-positive axons. Scale bar 10µm.

1265
1266
1267
1268

**C.** Co-immunoprecipitation of ORF1p with PABPC1-antibody coated beads. Western blot confirming efficient immunoprecipitation of PBPC1 and specific co-immunoprecipitation of ORF1p.

1272 **D.** Immunofluorescence staining of ORF1p (red) and PABPC1 (green) in differentiated mouse neurons. Co-localization is visible in yellow.

1274 **E.** Western blot analysis and quantification of ORF1p knock-down in differentiated MN9D (n = 6, one dot in the dot plot corresponds to one well; Mann Whitney test, ****p ≤ 0.0001) (n=3 independent experiments).

1277 **F.** Immunofluorescent staining of the neuritic network of mouse dopaminergic neurons using TUJ1. siRNA Ctrl neurons are shown on the left and siRNA ORF1p neurons on the right. Scale bar 10µm. Masks were established using a Fiji-operated script for the quantification of neurite morphology.

1281 **G.** Quantifications of neurite morphology based on TUJ1 staining of neurites. The mean diameter of neurites (in µm) was quantified using Fiji. Each point on the graph corresponds to the mean diameter value of an independent experiment. Neurite total area (µm²) was quantified in siRNA Ctrl and siRNA-ORF1p MN9D neurons; n=9 independent experiments.

1286

1287 **Supplementary Figures**

1288 **Suppl. Figure 1:**
1289 **A.** Scheme of the differentiation protocol of MN9D cells into mature mouse
1290 dopaminergic neurons. All experiments (including quantitative mass spectrometry)
1291 were performed at day 10 of differentiation.

1292 **B.** Immunofluorescence staining of endogenous ORF1p (ab216324) in differentiated
1293 MN9D neurons.

1294 **C.** Immunofluorescence staining of MN9D at 10 days of differentiation expressing the
1295 neuronal marker TUJ1 (green, left) and the dopaminergic marker tyrosine
1296 hydroxylase (TH, red, right).

1297 **D.** Venn Diagram of proteins identified by quantitative mass spectrometry of
1298 immunoprecipitated endogenous ORF1p from mouse dopaminergic neurons (MN9D)
1299 and human dopaminergic neurons (LUHMES) compared to mouse and human
1300 published ORF1p interactors¹⁷,²⁶⁻³⁴,⁶³.

1301 **E.** Venn Diagram of proteins identified by quantitative mass spectrometry of
1302 immunoprecipitated endogenous ORF1p from human dopaminergic neurons
1303 (LUHMES) compared to previously published mouse and human ORF1p
1304 interactors²⁶⁻³⁴.

1305 **F.** Venn Diagram of proteins identified by quantitative mass spectrometry of
1306 immunoprecipitated endogenous ORF1p from mouse dopaminergic neurons (MN9D)
1307 compared to previously published mouse and human ORF1p interactors¹⁷,⁶³.

1308 **G.** Common partners of ORF1p in LUHMES and MN9D neurons and their physical
1309 interactions identified by quantitative mass spectrometry.

1310

1311 **Suppl. Figure 2:**

1312 **A.** Representative fluorescent-activated nuclei sorting (FANS) profile showing the
1313 isotype controls for the NeuN and the ORF1p antibody used in Figure 3B.
1314 **B.** Heatmap of brain cell type markers and ATAC seq promoter peak numbers (<1kB)

1315 in NeuN-, NeuN+ ORF1p-low and NeuN+ ORF1p-high populations. NeuroD6, TH,

1316 PAX2, GABRA1, CAMK2A, GRIA1, DBH and RBFOX3 (= NeuN) and SST are
1317 markers of specific neuronal populations

1318 (https://biomarkerres.biomedcentral.com/articles/10.1186/s40364-023-00523-3) and
1319 show a high accessibility on promoters in both, ORF1p-high and ORF1p-low nuclei.

1320 OLIG1, SEMA3C, MBP, S100B, SLC6A1, GFAP and OLIG2 are glia-cell markers
1321 and show higher chromatin accessibility in promoter regions in non-neuronal (NeuN-)
1322 nuclei.

1323 **C.** Heatmap of differentially accessible regions in ORF1p-high and ORF1p-low nuclei
1324 and their overlap with the histone marks H3K4me3, H3K27me3 and H3K27ac.

1325 Histone post-translational modification profiles were retrieved from a previously
1326 published dataset¹⁰⁵

1327 **D-E.** Motif enrichment analysis of significant DARs in ORF1p-high and ORF1p-low
1328 neuronal populations.

1329

1330 **Suppl Figure 3:**

1331 **A.** Western Blot and quantification showing ORF1p-LOF in differentiated LUHMES
1332 neurons (n = 4 wells; three independent experiments, day 7 post-differentiation).

1333 Two-tailed Mann-Whitney *U* test. Data are represented as mean±SEM.

1334 **B.** Subcellular fractionation of control (siRNA-Ctrl) or ORF1p-LOF (siRNA ORF1)
1335 LUHMES neurons. Shown are the cytoplasm (CE; marker: eIF4E), the membrane-
1336 bound fraction (MB), the nucleoplasm (NP; marker: SP1), the chromatin-bound
1337 fraction (CB; marker: H3K9me3) and the pellet (PE).
1338 **C. LINE-1 5'UTR RT-qPCR of control (siRNA-Ctrl) and ORF1p-LOF (siRNA ORF1)**
1339 LUHMES neurons.

1340 **D. Heatmap of differentially accessible regions in Ctrl and ORF1p-LOF nuclei and their overlap with the histone marks H3K4me3, H3K27me3 and H3K27ac. Histone post-translational modification profiles were retrieved from a previously published dataset¹⁰⁵.**

1344 **E-F. Motif analysis of significant DARs identified in ATAC-seq in Control and ORF1p-LOF human dopaminergic neurons (LUHMES).**

1347 **Suppl Figure 4:**

1348 **A. GO analysis of significantly upregulated transcripts in ORF1-LOF vs Ctrl neurons.**
1349 FDR was calculated from p-values (Fisher's exact test) by the Benjamini-Hochberg procedure. MF: Molecular Function.

1351 **B. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly downregulated genes upon ORF1p-LOF.** P-value was calculated using one-sided Fisher's exact test and the p-value was adjusted for multiple testing by the Benjamini-Hochberg method.

1355 **C. Venn diagram of common differentially accessible regions (DARs) in ATAC-seq (1230 total) and differentially regulated genes (DEGs) in RNA-seq (962 total) from human neurons (LUHMES).** GO (biological process, BP) of the 26 common genes.

1358 **D. IGV track of an example of the neuron-specific gene EFNA5 with DARs in human post-mortem cingulate gyrus (ORF1p-high versus ORF1p-low) and human LUHMES neurons (Ctrl versus ORF1p-LOF).** EFNA5 was significantly downregulated upon ORF1p-LOF.
1363 **Suppl. Table 1.** ORF1p protein partners in LUHMES and MN9D as identified by
1364 quantitative mass spectrometry.

1365

1366 **Suppl Table 2.** Metadata of post-mortem cingulate gyrus tissues used in
1367 FANS/ATAC-seq

1368

<table><thead><tr><td>Diagnosis</td><td>Gender</td><td>Age</td><td>PMI (hrs)</td><td>Braak Stage</td></tr></thead><tbody><tr><td>PD</td><td>F</td><td>62</td><td>-</td><td>-</td></tr><tr><td>PD</td><td>F</td><td>79</td><td>-</td><td>II</td></tr><tr><td>PD</td><td>M</td><td>83</td><td>20</td><td>II</td></tr><tr><td>non-PD</td><td>F</td><td>92</td><td>21</td><td>I</td></tr><tr><td>non-PD</td><td>M</td><td>84</td><td>15</td><td>-</td></tr><tr><td>non-PD</td><td>M</td><td>73</td><td>10</td><td>II</td></tr></tbody></table>

1369

1370 PD: Parkinson disease

1371 PMI: post-mortem interval

1372

1373 **Suppl Table 3.** Characteristics of significantly enriched "differentially accessible
1374 regions" more accessible (DAR+) in neurons with either high or low nuclear ORF1p
1375 content identified by ATAC-seq in human post-mortem cingulate gyrus neurons and
1376 human dopaminergic neurons in culture (LUHMES).

1377 In red overlapping characteristics in both experimental systems are highlighted.

1378
differentially **more** accessible regions (DAR+)

<table><thead><tr><th></th><th>ORF1p-high</th><th>ORF1p-low</th></tr></thead><tbody><tr><th>human brain</th><td></td><td></td></tr><tr><th>genomic region</th><td>distal intergenic</td><td>intronic</td></tr><tr><th>functional feature</th><td>heterochromatin</td><td>flanking active TSS</td></tr><tr><th></th><td>weak repressed polycomb</td><td>enhancer</td></tr><tr><th>histone profile</th><td>H3K27me3</td><td>H3K4me3, H3K27ac</td></tr><tr><th>TF binding sites</th><td>bZIP, ZF</td><td>Homeobox, bZIP</td></tr></tbody></table>

differentially **more** accessible regions (DAR+)

<table><thead><tr><th></th><th>Control</th><th>ORF1p-LOF</th></tr></thead><tbody><tr><th>LUHMES</th><td></td><td></td></tr><tr><th>genomic region</th><td></td><td></td></tr><tr><th>functional feature</th><td>repressed polycomb</td><td>flanking active TSS</td></tr><tr><th></th><td>weak repressed polycomb</td><td>enhancer</td></tr><tr><th></th><td>ZNF/Repeats</td><td></td></tr><tr><th>histone profile</th><td>H3K27me3</td><td>H3K4me3</td></tr><tr><th>TF binding sites</th><td>bHLH</td><td>Homeobox</td></tr></tbody></table>

in red= overlapping terms in both ATAC-seq experiments

1379

1380

1381

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<caption>Figure 1</caption>

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

A

<figure><img src="image_2.png" /></figure>

C

human dopaminergic neurons (LUHMES)

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

E

Network of ORF1p interactors (MN9D)

<figure><img src="image_6.png" /></figure>

F

Network of ORF1p interactors (LUHMES)

<figure><img src="image_7.png" /></figure>
<figure><img src="image_8.png" /><figcaption>Figure 2</figcaption></figure>

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_9.png" /><figcaption>Figure 3</figcaption></figure>
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

<figure><img src="image_10.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Figure 5</figcaption></figure>

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_12.png" /><figcaption>Figure 6</figcaption></figure>
Figure 7

A preprint is currently under review. The version posted September 09, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_13.png" /><figcaption>Figure 7</figcaption></figure>
<caption>Suppl Figure 1</caption>

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>
<caption>Suppl Figure 2</caption>

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

A

<figure><img src="image_21.png" /></figure>

B

<figure><img src="image_22.png" /></figure>

C

<figure><img src="image_23.png" /></figure>

D

TF motifs enriched in NeuN+/ ORF1p-high

<table><thead><tr><th>Motifs</th><th>TF</th><th>q-value</th></tr></thead><tbody><tr><td>SAATGACTCACTC</td><td>Fos12 (bZIP)</td><td>0.0018</td></tr><tr><td>TGCGTGGGTG</td><td>Egr1 (Zf)</td><td>0.0018</td></tr><tr><td>TAATGACTCACTC</td><td>Fra1 (bZIP)</td><td>0.0123</td></tr><tr><td>SAATGACTCACTC</td><td>Atf3 (bZIP)</td><td>0.0123</td></tr><tr><td>ATATGACTCACTC</td><td>Fos (bZIP)</td><td>0.0123</td></tr></tbody></table>

E

TF motifs enriched in NeuN+/ ORF1p-low

<table><thead><tr><th>Motifs</th><th>TF</th><th>q-value</th></tr></thead><tbody><tr><td>SAATGACTCACTC</td><td>En1 (Homeobox)</td><td>0.0029</td></tr><tr><td>TGCGTGGGTG</td><td>LHX9 (Homeobox)</td><td>0.0029</td></tr><tr><td>TAATGACTCACTC</td><td>DLX2 (Homeobox)</td><td>0.0029</td></tr><tr><td>SAATGACTCACTC</td><td>Gsx2 (Homeobox)</td><td>0.0029</td></tr><tr><td>ATATGACTCACTC</td><td>AP-1 (bZIP)</td><td>0.0029</td></tr></tbody></table>
Suppl Figure 3

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_24.png" /><figcaption>TF motifs enriched in ORF1p-Ctrl</figcaption></figure>

<table><caption>TF motifs enriched in ORF1p-LOF</caption><thead><tr><th>Motifs</th><th>TF</th><th>q-value</th></tr></thead><tbody><tr><td>CCATCTGTT</td><td>NeuroD1 (bHLH)</td><td>0.0003</td></tr><tr><td>CCATTTGCT</td><td>Mesp1 (bHLH)</td><td>0.0064</td></tr><tr><td>CACCTGTCTAAACAG</td><td>Forkhead, bHLH</td><td>0.0219</td></tr><tr><td>TGACAGTCGTT</td><td>Atoh7(bHLH)</td><td>0.0348</td></tr><tr><td>ATACAGCTGCG</td><td>Atoh1(bHLH)</td><td>0.0946</td></tr></tbody></table>

<table><thead><tr><th>Motifs</th><th>TF</th><th>q-value</th></tr></thead><tbody><tr><td>ATGACTCAGT</td><td>HNF6 (Homeobox)</td><td>0.0000</td></tr><tr><td>TCGTGGCGT</td><td>Cux2 (Homeobox)</td><td>0.0000</td></tr><tr><td>ATGACTCATG</td><td>Emx2 (Homeobox)</td><td>0.0000</td></tr><tr><td>ATGACTCATGC</td><td>Hnf6b (Homeobox)</td><td>0.0000</td></tr><tr><td>ATGACTCATGC</td><td>Gsx2 (Homeobox)</td><td>0.0000</td></tr></tbody></table>
Suppl Figure 4 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<figure><img src="image_25.png" /><figcaption>Suppl Figure 4</figcaption></figure>