Low-level repressive histone marks fine-tune gene transcription in neural stem cells

  1. Arjun Rajan
  2. Lucas Anhezini
  3. Noemi Rives-Quinto
  4. Jay Y Chhabra
  5. Megan C Neville
  6. Elizabeth D Larson
  7. Stephen F Goodwin
  8. Melissa M Harrison
  9. Cheng-Yu Lee

  • Curated by eLife

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    eLife assessment

    This important work shows how a transcription factor, fruitless isoform C, fine-tunes the expression of stemness genes to maintain proper stem-cell identity during neurogenesis in Drosophila. The rationale of the work is well-thought-out, conclusions are supported by compelling evidence, and experiments are performed in a rigorous manner, although additional statistics would improve the conclusions. Overall, this work will be of interest to those working on chromatin regulation, transcription regulation, and stem-cell biology.

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Abstract

Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor Fruitless C (Fru C ) binds cis -regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fru C function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. Fru C negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis -regulatory regions. Identical to fru C loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans.

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  1. Version published to 10.7554/elife.86127 on eLife
  2. eLife

    eLife assessment

    This important work shows how a transcription factor, fruitless isoform C, fine-tunes the expression of stemness genes to maintain proper stem-cell identity during neurogenesis in Drosophila. The rationale of the work is well-thought-out, conclusions are supported by compelling evidence, and experiments are performed in a rigorous manner, although additional statistics would improve the conclusions. Overall, this work will be of interest to those working on chromatin regulation, transcription regulation, and stem-cell biology.

  3. eLife

    Reviewer #1 (Public Review):

    The goal of this study is to identify transcription factors that mediate stem cell transitions during differentiation. To achieve this, the authors examine the type II Drosophila neuroblast lineage, using single-cell RNA sequencing to examine all cell types in the type II lineage. There are known patterns of expression for neurons in this lineage, so they can identify clusters in their data set that are in the developmental state of transitioning from neuroblast to immature intermediate neuronal progenitor. They have outlined a set of expression criteria for transcription factors that are candidates for fine-tuning stem cell fate. They find that an isoform of Fruitless, called FruC, is a candidate transcription factor. Using microscopy and several genetic perturbation conditions the authors find that FruC is expressed in neuroblasts and can alter the number of cells in the lineage. To determine the mechanism that FruC uses to influence stemness the authors examine genomic occupancy of FruC, changes in histone modifications in FruC loss-of-function studies, and examination of DNA occupancy of proteins that function in chromatin modification. The authors argue that FruC functions to promote low-level H3K27me3 enrichment to maintain stemness based on comparisons across these data sets. The identification of transcription factors and the mechanisms used to maintain or differentiate stem cells is an important goal and is still a fundamental question in biology. The Drosophila model is poised for this type of analysis, given the knowledge of gene expression across cell fate that the authors use in this study.

    Comments the authors should address:
    This is a valuable study that relies on several state-of-the-art genomic data sets to examine the mechanism that drives stemness. However, the authors should be using statical approaches to support their major conclusion regarding FruC and the role of H3K27me3. The study presents peak data in genome browser tracks of a handful of loci in the Notch pathway that show the pattern of reduced HK3K27me3 and not the other modifications they examine. However, it is not clear if the majority of FruC target genes in the genomic analyses have this pattern, though they argue they do. The major conclusion that FruC promotes a stem cell fate is based on the overlap between the list of genes they identify bound by FruC and the lists of genes that have changes in histone modifications (H3K27ac, H3K4me3, and H3K27me3). The limited use of statistical approaches to draw these conclusions is a weakness of the study. The authors do not use statistics to find changes in chromatin modification at loci, instead relying on 2-fold change calculations. Furthermore, the authors don't indicate if the genes with altered histone modification/binding peaks are significantly enriched (or not) with FruC targets, with no quantitative assessments of these data. The data in Figures 5,6 and S4 should have statistics/quantification to support the major conclusions of their study that FruC targets differ in H3K27me3, but not H3K27ac, and H3K4me3.

  4. eLife

    Reviewer #2 (Public Review):

    This study addresses the molecular mechanism by which the FruC transcription factor regulates neurogenesis in Drosophila. The authors combine genetics and genomics to profile FruC genomic binding along with that of trithorax-like (Trl) and Su(H) and several histone modifications including H3K27me3. They propose that Fru acts to fine-tune the expression of Notch effector genes and they show that this regulation does not involve changes in H3K27ac, nor H3K4me3, but rather that FruC-regulated gene expression is correlated with changes in H3K27me3 levels at Fru target genes. While the study is well conducted and combines state-of-the-art techniques, there are several aspects that could be improved. The authors propose that Fru fine-tunes the expression of Notch effector genes, but they do not directly measure gene expression in any of the genetic backgrounds. It is important to do this to have some type of precise measure of transcriptional changes (what does 'fine tune' really mean), as the authors' model is based on subtle changes in H3K27me3. It would be important to quantify and correlate both processes more precisely. Similarly, the authors claim that Fru promotes 'low levels' of H3K27me3 at its bound loci throughout the genome, but they do not describe the criteria that define 'low levels' versus high levels of HK27me3.

    In the authors' model, FruC likely functions together with PRC2 to regulate gene expression, and local low-level enrichment of repressive histone marks act to fine-tune gene expression. However, in the absence of experiments directly addressing the molecular mechanisms by which Fru regulates transcription, it would be more accurate to claim that changes in H3K27me3 correlate with altered gene expression.

  5. eLife

    Reviewer #3 (Public Review):

    Rajan et al. used scRNAseq to identify transcription factors responsible for fine-tuning stemness gene expression in neural stem cells (neuroblasts), identifying Fruitless (fru) as a putative regulator of this process. Specifically, loss of the fru isoform C (fruc) results in increased stemness gene expression, while its overexpression leads to the opposite effect. Consistently, overexpression of fruc in a brat-null neuroblast-tumor background is sufficient to partially restore differentiation. Furthermore, by performing extensive genome-wide binding studies, the authors show that Fruc preferentially binds to cis-regulatory elements of stemness genes, with evidence that this transcription factor regulates the Notch-pathway via co-binding with Notch-target genes. The overall impact of FruC on transcription was not assessed.

    Their data also shows that instead of regulating the deposition of histone marks associated with active transcription, such as H3K27ac or H3K4me3, loss of fruc results in decreased levels of the repressive mark H3K27me3, namely in the Notch locus or in Notch downstream effector genes, indicating that FruC fine-tunes the expression of their bound genes through maintenance of low-levels of repressive marks at cis-regulatory elements of its target genes. Given the extensive binding profile of FruC the effects promoted by its misexpression in neuroblasts are likely multifactorial.

    In addition, the authors also show that PRC2 subunits, Caf1 and Su(z)12, the multisubunit complex responsible for catalyzing H3k27me3 deposition, (1) co-localize with Fru in Fruc-bound regions and (2) their loss partially phenocopies the previous results obtained for fruc depletion. The authors propose a model in which Fruc, via synergistic work with PRC2, is capable of fine-tuning the expression of stemness genes, in particular, Notch and Notch targets in neuroblasts by promoting low levels of transcriptionally repressive histone marks at their target cis-regulatory elements. If FruC and PRC2 functionally interact, and if the recruitment of one factor affects the binding of the other remains unknown.

    The authors present an assortment of results that will be useful for those working in transcription and chromatin regulation, namely in the field of Drosophila neural stem cells (neuroblasts). Specifically, the authors provide robust single-cell RNA sequencing results and analysis that can be used by researchers interested in trying to understand the transcriptional state of neuroblasts and their progeny. Additionally, genome-wide binding studies for FruC or PRC2 subunits, together with the profiling of active/repressive histone marks, offer new insights regarding transcription factor or transcriptional repressor binding and the respective read-out in terms of histone modifications. Moreover, the authors propose an interesting model via which transcription regulation of Notch and Notch downstream effectors is rendered via fine-tuning of the transcriptional output. Hence, FruC restrains and limits the levels of its target genes within neuroblasts, avoiding the segregation of high levels of stemness-associated proteins to the progeny, which would incur in fate and differentiation defects. The model proposed here highlights how transcription regulation by histone marks is much more dynamic and layered, other than being dictated only by the mutually exclusive presence of either active or repressive marks.

  6. Version published to 10.1101/2022.11.18.517130v1 on bioRxiv