Author response: Polysome-CAGE of TCL1-driven chronic lymphocytic leukemia revealed multiple N-terminally altered epigenetic regulators and a translation stress signature (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of 'closed chromatin' epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc, targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis. Editor's evaluation Results presented in this article suggest that T-Cell Leukemia/Lymphoma 1 (TCL1) protein promotes alternative transcription site selection in chronic lymphoid leukemia, resulting in the production of N-terminally truncated versions of chromatin regulators and induction of expression of transcription factors including c-MYC. Finally, the authors provide evidence that TCL1 drives translational reprogramming akin to that observed under metabolic stress. Notwithstanding technical limitations that obscured direct detection of N-terminally truncated protein variants, it was found that this study is of broad potential interest inasmuch as it provides previously unappreciated evidence that TCL1 orchestrates epigenetic, transcriptional, and translational rewiring in leukemic cells. Based on this, the study by Ogran et al. should be of significant interest across a number of biomedical research disciplines ranging from regulation of gene expression to cancer research. https://doi.org/10.7554/eLife.77714.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Chronic lymphocytic leukemia (CLL) accounts for 25–30% of all leukemias in the Western countries, with incidence rates ranging from 3.65 to 6.75 cases per 100,000 population per year (Siegel et al., 2019; Sant et al., 2010). CLL is characterized by an outgrowth of malignant CD5 positive B cells, mainly residing in the peripheral blood (PB), bone marrow, and lymphoid organs, and by a high biological heterogeneity reflected in clinically different outcomes including disease progression, therapy response, and relapse (Kröber et al., 2002; Zenz et al., 2010). The hallmark of the CLL disease is mainly decreased apoptosis resulting from overexpression of anti-apoptotic proteins and resistance to apoptosis in vivo. The Tcl1 oncogene is highly expressed in CLL cells, and its expression level correlates with the CLL aggressiveness (Herling et al., 2006). Tcl1 was initially discovered as a gene involved in the rearrangement of the T-cell leukemia/lymphoma 1 locus at 14q32.1, the most common chromosomal aberrations detected in mature T-cell leukemias (Virgilio et al., 1994). The central role of TCL1 in CLL was demonstrated in transgenic mice overexpressing the human Tcl1 gene under the control of the immunoglobulin promoter and enhancer (Eµ-Tcl1) (Bichi et al., 2002). These mice develop normally into adulthood but develop CLL that is highly similar to the human disease and is characterized by CD5+ cells, which accumulate in the spleens, livers, and lymph nodes. The association of high TCL1 expression and malignant transformation is linked to its involvement in several major oncogenic pathways, including the enhancement of AKT activity (Herling et al., 2008; Noh et al., 2012; Patil et al., 2020; Pekarsky et al., 2010); interaction with the DNA repair ataxia–telangiectasia-mutated (ATM) factor (Gaudio et al., 2012), and the transcription factor CREB (Pekarsky et al., 2008) as well as in contributing to accelerated tumorigenic and the prosurvival NF-κB signaling (Gaudio et al., 2012). Inhibition of activator protein 1 (AP-1) transcriptional activity via interaction of TCL1 with the AP-1 complex represents another mechanism to antagonize the expression of pro-apoptotic factors (Gaudio et al., 2012; Motiwala et al., 2011). TCL1 was also shown to contribute to epigenetic reprogramming via interacting with the de novo DNA methyltransferase 3A (DNMT3A) and reducing its enzymatic activity (Palamarchuk et al., 2012). Considering the central role of TCL1 in tumor initiation, progression, and maintenance, it is reasonable that its oncogenic functions involve additional, yet to be discovered, activities. The transcription initiation of a specific gene can occur from more than one position in the DNA, creating several mRNA isoforms. Alterations in transcription start site (TSS) selection and alternative promoter (AP) usage increase transcriptome diversity and regulation. For example, the level of transcription initiation can vary between different TSSs under different growth conditions, in response to a specific signal, or different cell types and tissues. In addition, mRNA isoforms with different 5′ leaders can vary in their translation efficiency (TE) or half-lives. Likewise, AP usage can lead to the generation of protein isoforms that differ in their N termini and as a result can have different or even opposite biological functions. Recent large-scale promoter analysis in hundreds of human and mouse primary cell types shed light on the prevalence of AP usage in mammals (Forrest et al., 2014) as well as in multiple cancer types (Demircioğlu et al., 2019). Several studies have examined the translation and stability of transcript isoforms of the same gene and the contribution of AP usage to the translational response to stress (Floor and Doudna, 2016; Arribere and Gilbert, 2013; Wang et al., 2016; Tamarkin-Ben-Harush et al., 2017). However, presently little is known about the impact of TSSs and AP usage changes on the protein content of cells undergoing cancer progression. Considering that even transient cellular stress is associated with dramatic changes in the landscape of TSSs and AP usage that are coordinated with translation (Tamarkin-Ben-Harush et al., 2017), it is reasonable to assume that such alterations can affect translatability in cells undergoing oncogenic transformation. Some of the new cancer-specific protein isoforms generated by AP usage can help maintain the transformed phenotype. Presently, the vast majority of studies measured global changes in mRNA abundance and transcript translatability in cancer cells, while the potential effect of variations in TSSs selection and AP usage on translation was hardly addressed. This study examined the changes in TSS selection and AP usage and their impact on the translatome during malignant transformation of B cells by the Tcl1 oncogene. Using healthy and Eµ-Tcl1 transgenic mice, we compared the TSSs landscape of normal and CLL B cells. Our findings revealed a marked elevation of APs in malignant CLL B cells with a particularly high prevalence of intra-genic promoters that are predicted to generate N-terminally truncated or N-terminally modified proteins. The induced cryptic promoters are driven by nearby enhancers and upregulation of their cognate transcription factors, including the c-Myc oncogene. A promoter shift in the opposite direction accounts for the induced expression of the immune checkpoint ligand PD-L2. Notably most of the intra-genic-generated transcripts are also efficiently translated. These remarkable changes of promoters and TSSs are linked to the loss of function of many 'closed chromatin' epigenetic regulators via induction of inactive isoforms by promoter shifts or by reduced expression, resulting in a feed-forward loop. The activation of intra-genic cryptic promoters and the c-Myc oncogene elevation are mediated, at least in part, by the Tcl1 oncogene itself and are therefore intrinsic to the CLL B cells. We further found a dramatic and specific translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing energy stress. These findings explored the contribution of 5′ end mRNA isoforms and their associated translational changes during cellular transformation and uncovered novel Tcl1 oncogenic pathways. Results Global analysis of TSSs revealed extensive induction of promoter shifts in CLL B cells To address the potential contribution of AP to CLL we used the Eµ-Tcl1 transgenic mice overexpressing the human Tcl1 gene under the control of the immunoglobulin heavy chain (IgH) variable region promoter (VH) and immunoglobulin heavy chain enhancer (Eµ-Tcl1) (Bichi et al., 2002). Like the human CLL, Eµ-Tcl1 mice accumulate transformed CD5+ B cells in the spleens, livers, and lymph nodes during adulthood, reproducing CLL (Pekarsky et al., 2008). Splenocytes from healthy and Eµ-Tcl1 mice were subjected to affinity-based capture using CD19 magnetic beads to isolate B cells and CLL B cells, respectively. Total RNA was extracted and subjected to Cap Analysis of Gene Expression (CAGE) (Carninci et al., 2006; Takahashi et al., 2012) library preparation and Illumina deep sequencing of the first 27 nucleotides CAGE tags from the 5′ ends (Figure 1A). An average of 10.625 million CAGE tags per sample was aligned to a total of 2.67 million CAGE-derived TSSs (CTSSs) that were mapped and clustered to a consensus set of 28,616 promoters that correspond to a total of 11,515 genes. Using this high-throughput data from two independent biological replicates, we determined the global landscape of TSSs and their relative abundance in healthy and CLL B cells. Metagene analysis of both WT and Eµ-Tcl1 CAGE data showed almost perfect alignment to the annotated TSS atlas of the FANTOM5 project (Lizio et al., 2015; Figure 1B), indicating the proper calculation of the TSS positions at a single-base resolution. The CAGE libraries were assessed with coefficient scores calculated using the Pearson pairwise-correlation test and the reproducibility of the replicates were found to be high (Figure 1—figure supplement 1A). As expected, in both WT and Eµ-Tcl1 samples, most of the TSSs with the highest expression level were mapped to the core promoter region (Figure 1C). A significant fraction of the TSSs was found within introns and intergenic regions. Since the CAGE method captures all capped RNAs, these data also detect enhancer RNAs (eRNAs), a class of short noncoding RNAs resulting from a balanced bidirectional transcription from enhancers that are unstable and nonpolyadenylated (Andersson et al., 2014; Kim et al., 2010) (examples of an enhancer and super-enhancer are shown in Figure 1—figure supplement 1A, C). We examined the possibility that part of the intronic and intra-genic CAGE reads are derived from active eRNAs. Based on the criteria of balanced bidirectional read mapping, we calculated the frequencies of eRNAs in introns and intergenic regions and found that most of the intronic TSSs (85% in Eµ-Tcl1 and 79% in WT) and a significant fraction of the intra-genic TSSs (62% in Eµ-Tcl1 and 50% in WT) are indeed derived from eRNAs (Figure 1C). The ability of CAGE to detect lowly expressed genes makes it crucial to reduce the mean–variance relationship of count data prior to differential expression (DE) analysis. A 'blind' analysis of the variance stabilizing transformation was performed and summarized by a principle component analysis (PCA). PCA1 explained 85% of the variance and distinguished between WT and Eµ-Tcl1, where WT samples are tightly clustered and Eµ-Tcl1 are separated by 13% of the variance, which is explained by PCA2 (Figure 1—figure supplement 1D). As no systemic effect on samples was found, we decided to fit a DE model without using a correction factor. Out of the 28,616 consensus TSSs, we found in Eµ-Tcl1 3337 upregulated and 1829 downregulated TSSs (Figure 1D), suggesting an overall upregulation of many promoters in CLL. To test the contribution of eRNA to the differentially expressed TSSs in Eµ-Tcl1, we used an established method to predict eRNA–TSS interactions based on genomic distance and expression-based correlation (Andersson and Sandelin, 2020; Thodberg and Sandelin, 2019). We found 255 upregulated eRNAs, of them 106 were positively correlated to the 203 upregulated TSSs, while out of 179 downregulated eRNAs, 86 were positively correlated to the 331 downregulated TSSs (Figure 1D). Lengths of the TSS clusters were calculated by the interquartile range (IQR) of highly expressed (>10 tag per million [TPM]) TSSs. TSS clusters lengths holding 10–90% of pooled TSSs expression were found to distribute into sharp and broad promoters bimodally (Figure 1—figure supplement 1E). Consistent with known promoter architecture (Carninci et al., 2006; Suzuki et al., 2001), general TSSs are characterized by YR at the (−1) and (+1) positions (Figure 1—figure supplement 1E). Promoters with broad TSS distributions tend to be surrounded by CpG islands, while promoters with sharp TSSs are defined by the presence of a TATA box around position −30 (Figure 1—figure supplement 1F). Separately plotted LOGOs of differentially expressed TSSs, showed enrichment of poly-pyrimidine sequence in the upregulated sharp TSSs at the positions of +2 to +12 (Figure 1E). Figure 1 with 1 supplement see all Download asset Open asset Global analysis of transcription start sites (TSSs) in healthy and chronic lymphocytic leukemia (CLL) B cells. (A) A scheme of the Cap Analysis Gene Expression (CAGE) in healthy (WT) and CLL B cells (Eµ-Tcl1). WT and Eµ-Tcl1 splenic B cells were isolated using CD5 and CD19 magnetic beads. Total RNA samples were subjected to the CAGE as described in Materials and methods. CAGE-derived TSSs (CTSSs) were mapped and clustered into genomic blocks referred as tag clusters (blue bar) with a peak of most recovered CTSS (blue tick) indicates the TSSs in a single-base resolution. (B) Metagene analysis of Eµ-Tcl1 (orange) and WT (gray) CAGE libraries aligned to annotated TSS atlas of the FANTOM5 project. (C) The frequency (left) and expression level (right, log2 TPM) of CAGE tag clusters in Eµ-Tcl1 (orange) and WT (gray) mice by gene locations. The percentage values on the columns of intronic and intergenic locations refer to predicted enhancer RNA (eRNA) TSSs. (D) Differentially expressed (DE) TSSs. Left circle indicates unchanged (light gray) and significantly DE (brown) TSS between Eµ-Tcl1 and WT mice. Right circle details frequencies of Eµ-Tcl1 upregulated genic and eRNA TSSs (orange and dark orange, respectively) and downregulated genic and eRNA TSSs (gray and dark gray). Subgrouping of DE TSSs that are positively correlated with enhancers are indicated. (E) Sequence LOGOs (−40 to +30 relative to the TSS) of Eµ-Tcl1 upregulated (lower panel) and downregulated (upper panel) sharp promoters where TATA box situated between −31 and −24 sites and cap-proximal region (12 bases) are boxed in a dashed line. A marked elevation of new CLL-specific protein isoforms generated by intra-genic TSSs When an alternative TSS is located at the promoter or at the 5′ UTR regions, the original ORF is retained. However, new intra-genic TSSs located in introns or CDS are predicted to give rise to protein isoforms bearing alternative or truncated N termini and, consequently, potential different or even opposite biological functions (illustrated scheme in Figure 2A). We analyzed AP usage in our data and found that 2530 genes (22%) contain more than one promoter that contributes over 10% of their total gene expression (Figure 2B), consistent with previous studies (Tamarkin-Ben-Harush et al., 2017). To determine differential TSS usage (DTU), we examined the multipromoter subset and found 550 significant DTUs from 489 genes where the alternative TSS is upregulated in Eµ-Tcl1 and 373 DTUs from 328 genes with downregulated alternative TSS. The 47% more cases of upregulated alternative TSS indicate that DTU during CLL transformation is gaining more new isoforms than losing the canonical ones (Figure 2C). The Eµ-Tcl1 upregulated alternative TSSs were enriched at the core promoter, 5′ UTR, CDS, and intron regions but not at distal regions (Figure 2C). Median expression (log2 TPM) of alternative TSS in all locations ranged between 1.6 and 2.7 with no apparent differences between up- and downregulated alternative TSS (Figure 2—figure supplement 1A). By analyzing the correlation between alternative TSSs and nearby active enhancers, which are marked by eRNAs, a clear elevation in eRNA frequencies was found in Eµ-Tcl1 upregulated alternative TSSs (Figure 2—figure supplement 1B). These findings suggest the significant role of active enhancers in the activation of DTUs in Eµ-Tcl1. Figure 2 with 1 supplement see all Download asset Open asset Analysis of differential TSS usage (DTU) reveals alternative 5′ UTRs and new protein isoforms derived from cryptic promoters in chronic lymphocytic leukemia (CLL). (A) Scheme of five alternative TSSs (ATSSs) generating isoforms with alternative 5′ UTR regions (Siegel et al., 2019; Sant et al., 2010; Kröber et al., 2002; Zenz et al., 2010; Herling et al., 2006) and modification of the N′ termini for example, new chimeric CDS (Kröber et al., 2002) and N′-terminal truncation (Herling et al., 2006). (B) The frequency and the percentage of uni-TSS genes and multi-TSS genes including only TSS clusters that constitute over 10% of total gene expression. (C) Frequencies of alternative TSSs that were upregulated (orange) and downregulated (gray) in Eµ-Tcl1, grouped by gene-structure locations. (D) Gene track showing differential TSS usage (DTU) in Pdcd1lg2 (PD-L2) in Eµ-Tcl1 and WT mice. Canonical promoter (P3) and intra-genic promoter (P4, located just upstrem to the last CDS of PD-L2) are up- and downregulated in Eµ-Tcl1, respecively. Both, located on the plus strand (blue). Bellow, is a scheme illustrating the P4-induced truncation of PD-L2 lossing most of its functional domains. (E) Chd1 protein truncation in Eµ-Tcl1 resulted by induction of cryptic promoter. In the upper track, two promoters on the plus strand (blue ticks) presented along to a partial gene-structure scheme. While the canonical promoter located upstream to the annotated 5′ UTR is similarly expressed in Eµ-Tcl1 and WT, an Eµ-Tcl1-specific induction of an intra-genic alternative TSS located in the 16th intron resulting in N′ terminus truncation of Chd1 lacking several active domains as illustrated below. (F)Fbxo5 5′ UTR shorthening resulted by DTU in Eµ-Tcl1. On the upper track, two alternative promoters located on the minus strand shown as short red bars with indicator tick of TSS. Coverage peaks in the two lower tracks (WT and Eµ-Tcl1) showing differential expression of the two promoters where the nearest alternative TSS to the ORF is upregulated in Eµ-Tcl1. (G) Dot plot of ORF-retaining alternative TSS (ATSS) in peach and ORF-truncating intra-genic alternative TSS (turquoise) presenting expression (log2 TPM) against ATSS log FC evaluating the degree of promoter shifting (DTU). Positive and negative ATSS log FCs refer to Eµ-Tcl1 up- and downregulated ATSS, respectively. Dot sizes correspond to the FDR statistical significance of the DTU analysis. To explore the consequences of DTUs in CLL, we first analyzed several DTU events (Figure 2D–F). An intriguing example of DTU resulting in an N-terminally truncated isoform was found in programmed cell death 1 ligand 2 (Pdcd1lg2/PD-L2). This gene has an intra-genic alternative TSS generating a protein isoform lacking the Ig-like extracellular domains of PD-L2 (Latchman et al., 2001; Li et al., 2018; Zak et al., 2017), specifically expressed in WT B cells and is predicted to be inactive. In contrast, the full-length protein is exclusively expressed in the CLL cells via promoter shift and likely contributes to immunosuppression (Figure 2D). An opposite example is chromodomain 1 (Chd1), in which promoter shifting in Eµ-Tcl1 is within an intra-genic alternative TSS that results in truncation of several critical active domains, presumably leading to loss of function (Figure 2E). The F-box only protein 5 (Fbxo5) has two promoters located upstream to the main ORF differing only by their 5′ UTR length resulting in 490 nt shorter 5′ UTR in the Eµ-Tcl1-specific transcript isoform (Figure 2F). Global analysis of the promoter shifts levels and its significance of all DTUs revealed that the upregulated alternative TSS isoforms predicted to give rise to proteins with alternative/truncated N termini are dramatically in the Eµ-Tcl1 samples compared to WT B cells (Figure This a previously unappreciated diversity of the CLL Gene analysis of genes by AP usage enrichment in critical CLL pathways, for example, NF-κB signaling (Pekarsky et al., signaling et al., signaling and signaling et al., chromatin and 2 the derived from mouse associated with the DTU gene such as and consistent with Tcl1 promotes chromatin induction of intra-genic cryptic and activation of the c-Myc oncogene To the for the broad induction of intra-genic cryptic we previous and studies transcription initiation within regions as a of in the of chromatin regulators that maintain chromatin or et al., et al., 2019; et al., 2015; and 2013; et al., 2020; et al., 2012). Our Eµ-Tcl1 data found chromatin with ORF-retaining or alternative TSSs that be into factors associated with or chromatin of them were upregulated in Eµ-Tcl1 (Figure most of the upregulated alternative TSS within regions of chromatin such as the known epigenetic such as and are predicted to truncated proteins that loss of critical domains due to DTU (Figure 3A and To the CAGE we chromatin and analyzed them by 5′ using RNA samples from mice CLL B cells. The results of the 5′ dramatic upregulation of the isoforms by alternative TSS of the and (Figure supplement In to the truncated chromatin epigenetic regulators of chromatin are downregulated These findings revealed chromatin in Eµ-Tcl1 B cells that can cryptic Figure with 1 supplement see all Download asset Open asset promoters of chromatin epigenetic regulators and the role of (A) Dot plot of ORF-retaining and ORF-truncating alternative transcription start sites (TSSs) within genes associated with or chromatin For evaluating the degree of promoter shifting (DTU), the data are presented by expression (log2 TPM) against log FC of alternative TSS Positive and negative ATSS log FCs refer to Eµ-Tcl1 up- and downregulated ATSS, respectively. (B) mouse cell were by with and as (C) Exogenous expression of cryptic A scheme illustrating the canonical and cryptic promoter and the of to the 5′ UTR by canonical TSS and intra-genic alternative TSS. The results are presented as the between cryptic and canonical significant differences are by as and Figure data 1 Western of human Download 1 expressed genes. protein protein methyltransferase methyltransferase function The induction of intra-genic promoters can be driven by TCL1 or by associated with cancer progression. To between these we established mouse human and determined by the relative expression of the alternative intra-genic TSS of genes shown in the We observed a significant elevation of the intra-genic TSS of and but not These findings suggest that Tcl1 itself can promote of the chromatin and promoter at least in genes. of the chromatin the of transcription factor in promoter and enhancer regions. Since CAGE provides expression data of genes along with their promoter and enhancer regions, we the contribution of to the activation of cryptic promoters in Eµ-Tcl1. By generating position frequency of et al., mouse core we the apparent differences in the of in ORF-retaining and alternative TSSs, differentially in Eµ-Tcl1 (Figure by frequencies mainly differ between up- and downregulated ORF-truncating alternative of the (Figure Using we found enriched in the promoters of the upregulated alternative TSSs compared to the promoters of the canonical upregulated TSS in Eµ-Tcl1 (Figure When we these data with the of upregulated in Eµ-Tcl1 we found that upregulated levels of the and c-Myc correspond to the highly enriched and (Figure Figure Download asset Open asset of targeting cryptic promoters and enhancers. (A) A principle component analysis analysis of of transcription sites et al., mouse core in promoters upstream and to transcription start site of upregulated and downregulated ORF-retaining or ORF-truncating alternative TSSs. PCA1 and PCA2 and of the total respectively. (B) enrichment analysis of alternative promoters upstream and to TSS) relative to the overall the enriched and c-Myc levels are upregulated in Eµ-Tcl1 and are indicated. (C) Analysis of the c-Myc expression in the mouse by significant differences are marked with (D) enrichment analysis of enhancers associated with alternative promoters relative to overall induced enhancers. The levels of enriched are upregulated in Eµ-Tcl1 of the c-Myc oncogene is the hallmark of many et al., As c-Myc is the upregulated in our CAGE data of Eµ-Tcl1 CLL and is predicted to intra-genic we Tcl1 itself can Using the MEFs we found that the levels c-Myc are dramatically upregulated in all the indicating that its activation in Eµ-Tcl1 B CLL is most likely As many of the upregulated alternative TSSs in Eµ-Tcl1 are associated with active enhancers (Figure 2—figure supplement 1B), we analyzed enrichment also in the upregulated enhancers and found enriched (Figure the linked to these enriched are all upregulated in the Eµ-Tcl1 and and These findings suggest that these enhancers and their