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  1. Evaluation Summary:

    In this study, Ogran and colleagues provide evidence suggesting that T-Cell Leukemia/Lymphoma 1 (TCL1) protein may promote alternative transcription site selection and promoter usage in chronic lymphoid leukemia. It is further proposed that these TCL1-dependent alterations lead to the production of N-terminally truncated versions of proteins including chromatin regulators while bolstering expression of transcription factors including MYC. Collectively, it was found that these results are of broad interest inasmuch as they suggest previously unappreciated rewiring of epigenetic, transcriptional, and translational programs in leukemic cells. To this end, this article should be of significant interest across a variety of fields of biomedical research ranging from regulation of gene expression to cancer research. The paper would be strengthened by mechanistic data linking TCL1 to alterations in transcription site selection and/or alternative promoter usage and by stronger validation of the expression of N-truncated proteins and their functional consequences.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    In this article, Ogran et al set out to map the alterations in transcription start sites (TSS) and/or the alternative promoter usage in T-Cell Leukemia/Lymphoma 1 (TCL1)-driven chronic lymphoid leukemia and their impact on the collection of efficiently translated mRNAs. To achieve this, the authors employed an Eu-TCL-1 mouse model from which they derived CLL cells and compared them to normal B cells. This revealed profound differences in transcription start site selection and alternative promoter usage in CLL vs. normal B cells using a battery of genomic analyses. Some evidence is provided that these effects are coordinated with translational programs via orchestration of the alterations in chromatin modifiers and transcription factors including c-MYC. Finally, the authors show that the forced expression of TCL-1 in an unrelated cell line (mouse embryonic fibroblasts) causes similar effects as in CLL cells, thus suggesting that their observations are not limited to Eu-TCL-1 CLL cell line. Overall, this study provides initial insights into the mechanisms that may coordinate TCL-1-dependent epigenetic, transcriptional, and translational programs and should thus be of significant interest to the broad spectrum of researchers from those focusing on gene expression to those studying hematological malignancies.

    Strengths: This study employs powerful genomic approaches (e.g., polysome-CAGE) to address an important gap in knowledge related to the coordination of epigenetic, transcriptional, and translational programs in neoplasia. Overall, it was thought that most of the studies were well executed and that provided results support most of the author's conclusions.

    Weaknesses: The major weaknesses of the study were related to the relative lack of validation and biochemical and functional characterization of the large-scale studies. To this end, the impact of the alterations in TSS and alternative promoter selection on corresponding protein stoichiometry and function remains unclear. Moreover, it was thought that more mechanistic detail linking the alterations in ORF length of chromatin modifiers and alterations in chromatin in the context of TCL-1 is required to support the author's model.

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  3. Reviewer #2 (Public Review):

    Chronic lymphocytic leukemia (CLL) is a major form of leukemia lacking a cure. Ogran et al. focus on a mouse model of CLL where overexpression of Tcl1 under the immunoglobulin promoter and enhancer leads to the development of a disease which resembles CLL in humans. While previous studies of Tcl1 have focused on a range of pathways associated with cancer, Ogran assesses how Tcl1 affects transcription start-site usage. It is unclear why this was the focus in the studies of Tcl1. To assess transcription start-site (TSS) usage between control and Tcl1 overexpressing CD19+ cells, CAGE is used. In aggregate, this is an interesting study reporting on a new mechanism downstream of Tcl1.

    Major concerns:

    1. There are merely two replicates performed. While each replicate is sequenced at a high depth, additional replicates would likely have improved the study.

    2. The CAGE approach does not appear to employ unique molecular identifiers (UMNs). UMIs allow the removal of RNA sequencing reads arising from the PCR step during library preparation. Therefore, each read does not necessarily arise from a unique RNA molecule. Accordingly, comparisons of signals of TSS usage may be compromised.

    3. Some additional quality control information would have helped in understanding the reproducibility across the replicates of the CAGE data. For example, what proportion of the TSS were supported by both replicates? What proportion was supported by all 4 experiments?

    4. There are two different analyses of Tcl1-dependent changes in TSS. One is presented in figure 1D and seems to correspond to alterations that indicate a change in expression rather than TSS usage. The second in figure 2D is for analysis of differential TSS usage. Notably, the former (i.e., alterations in mRNA levels) seems to be substantially more common but is not pursued in this study. For both these analyses, there are no indications of what thresholds were used for differential expression. It would also have been informative to see the p-value distributions. Finally, the methods section could be clearer in describing these analyses.

    5. In the plots of the CAGE data, it is not clear how reproducible the signals were across the replicates. Also, including false-discovery rates for the apparent differences would be beneficial.

    6. It is unclear how the analysis handles missing data.

    From the analysis, the authors identify ample alterations in TSS associated with Tcl1 over-expression. Although the CAGE approach has been validated in the past, there is no validation of the mRNAs encoding truncated proteins using e.g., 5'RACE. Such validation would have further supported the main conclusion of the study. The authors next make the observation that many of the truncated proteins encode epigenetic regulators which lead to a model where both primary and secondary effects of Tcl1 over-expression target the chromatin giving rise to a more open structure and thereby allowing alternative TSSs to give rise to mRNAs encoding truncated peptides. Yet, the authors do not present data that there are alterations to chromatin.

    To show that Tcl1 directly affects TSS usage independent of the oncogenic process, MEFs overexpressing Tcl1 are used. In these experiments, primers targeting introns are compared to those targeting exons. These experiments support that mRNAs with introns are more common in cells expressing Tcl1. However, these may be un-spliced. In this context, it would also be interesting to learn about the relative levels of the intronic relative to the exonic signals to see if the relative abundances of these variants are similar and if they match the CAGE data set.

    The authors continue the study by applying CAGE on mRNA associated with polysomes. A detailed approach is used whereby the polysome fractions are used to generate several pools. However, it seems that this experiment was only performed one time. The resulting data set is analyzed using a ratio approach (comparing translated signal to free signal) across the alternative TSSs. This approach will potentially lead to the introduction of spurious correlations which may lead to false-positive findings. The false discovery rate for these comparisons is unknown. As mRNAs encoding truncated versions of the protein are also associated with polysomes, these experiments support that these truncated proteins are synthesized. Yet, there is no support that these proteins are present in the cell as no assessments of expression of truncated proteins were made.

    In the final part of the manuscript, the authors assess how the first nucleotide(s) affect translation. This leads to the conclusion that mRNAs starting with a C are translated more poorly. This agrees with the suppressed translation of mRNAs with TOP motifs under cellular stress. Next, the authors make more detailed claims regarding the first 3 nucleotides. In this context, it would have been interesting to see if this prediction is supported by functional experiments. Finally, the authors test a few drugs targeting epigenetic modifiers and an inhibitor to eIF4E is also used (unclear to me why). The argument is that inhibition of these factors does not seem to affect the Tcl1 over-expression cells, and that accumulation of truncated proteins would therefore not be detrimental (while leading to alternative TSSs producing mRNAs encoding truncated proteins). Overall, it seems that this final section may aim to link the identified truncated proteins to pro-cancer properties. Indeed, although clearly challenging to assess, the manuscript does not include data supporting that the described effects on TSS leading to truncated proteins contribute to Tcl1's pro-cancer activity.

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  4. Reviewer #3 (Public Review):

    In this manuscript, Dikstein and colleagues perform CAGE-Seq from total and polysome-associated RNA to detect alternative promoter usage in B-cells from Eµ-Tcl1 mice. They find considerable alternative transcription start site (TSS) usage compared with B-cells from wt mice, including intragenic events ultimately potentially resulting in N-terminally truncated proteins. The authors propose that there is a feed-forward mechanism by which Tcl1-promoted alternative TSS usage in genes encoding chromatin regulators contributes to the 'openness' of chromatin and to transcriptional alterations (including overexpression of Myc) observed in Eµ-Tcl1 cells. These results are interesting and provide a solid analysis of TSS usage in B-cells. However, the extensive bioinformatics analysis provided in this manuscript is often not followed by validation. The manuscript would benefit from validation to support the major conclusions, including assessment of stable expression of N-terminally truncated products, assessment of chromatin accessibility, and a deeper understanding of the alternative TSS events that are directly due to Tcl1 over-expression.

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