Protein arginine methyltransferase 5 (Prmt5) localizes to chromatin loop anchors and modulates expression of genes at TAD boundaries during early adipogenesis
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How the genome is folded three dimensionally is thought to control gene regulatory pathways during critical processes such as cellular differentiation. Using multiple assays, the authors of this important study probe topologically associated domains (TADs) and report involvement of a protein arginine methyltransferase 5 in chromatin organization and transcriptional regulation. The data gathered are generally solid and broadly support the role of this protein in organization of the adipocytic lineage, but additional control experiments would make the inferences stronger.
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Abstract
Protein arginine methyltransferase 5 (Prmt5) is an essential regulator of embryonic development and adult progenitor cell functions. Prmt5 expression is mis-regulated in many cancers, and the development of Prmt5 inhibitors as cancer therapeutics is an active area of research. Prmt5 functions via effects on gene expression, splicing, DNA repair, and other critical cellular processes. We examined whether Prmt5 functions broadly as a genome-wide regulator of gene transcription and higher-order chromatin interactions during the initial stages of adipogenesis using ChIP-Seq, RNA-seq, and Hi-C using 3T3-L1 cells, a frequently utilized model for adipogenesis. We observed robust genome-wide Prmt5 chromatin-binding at the onset of differentiation. Prmt5 localized to transcriptionally active genomic regions, acting as both a positive and a negative regulator. A subset of Prmt5 binding sites co-localized with mediators of chromatin organization at chromatin loop anchors. Prmt5 knockdown decreased insulation strength at the boundaries of topologically associating domains (TADs) adjacent to sites with Prmt5 and CTCF co-localization. Genes overlapping such weakened TAD boundaries showed transcriptional dysregulation. This study identifies Prmt5 as a broad regulator of gene expression, including regulation of early adipogenic factors, and reveals an unappreciated requirement for Prmt5 in maintaining strong insulation at TAD boundaries and overall chromatin organization.
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eLife assessment
How the genome is folded three dimensionally is thought to control gene regulatory pathways during critical processes such as cellular differentiation. Using multiple assays, the authors of this important study probe topologically associated domains (TADs) and report involvement of a protein arginine methyltransferase 5 in chromatin organization and transcriptional regulation. The data gathered are generally solid and broadly support the role of this protein in organization of the adipocytic lineage, but additional control experiments would make the inferences stronger.
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Reviewer #1 (Public Review):
In this study, the authors strive to characterize the role of protein arginine methyltransferase 5 (Prmt5) in chromatin organization and transcriptional regulation during adipogenesis. Their main aim is to delve into Prmt5's function during the differentiation of preadipocytes by conducting genome-wide analyses, including ChIP-Seq, RNA-Seq, and Hi-C experiments. They hypothesize and present evidence for Prmt5's broad regulatory effect on gene expression and its role in maintaining topologically associating domain (TAD) boundaries and overall chromatin organization.
Strengths of the study include its genome-wide approach, which provides a comprehensive perspective on Prmt5's potential involvement in adipogenesis. These methods yield a large dataset and offer novel insights into Prmt5's possible function in …
Reviewer #1 (Public Review):
In this study, the authors strive to characterize the role of protein arginine methyltransferase 5 (Prmt5) in chromatin organization and transcriptional regulation during adipogenesis. Their main aim is to delve into Prmt5's function during the differentiation of preadipocytes by conducting genome-wide analyses, including ChIP-Seq, RNA-Seq, and Hi-C experiments. They hypothesize and present evidence for Prmt5's broad regulatory effect on gene expression and its role in maintaining topologically associating domain (TAD) boundaries and overall chromatin organization.
Strengths of the study include its genome-wide approach, which provides a comprehensive perspective on Prmt5's potential involvement in adipogenesis. These methods yield a large dataset and offer novel insights into Prmt5's possible function in adipogenesis.
However, there are a few areas where the methodology and interpretation of results fall short. One noticeable gap is the absence of a comprehensive control in the ChIP-Seq experiments. Specifically, a control such as ChIP in Prmt5 knockdown cells would have been valuable to account for potential non-specific binding. This lack of a robust control raises questions about the confidence in the detected Prmt5 peaks.
Moreover, the knockdown experiments predominantly employ a single siRNA. Given the well-known off-target effects of siRNA-mediated RNA interference, this approach might cast doubts on the reliability of the results derived from these experiments. Therefore, the inclusion of multiple siRNAs in each assay would greatly strengthen the data.
Lastly, the authors' assertion of Prmt5's influence on the weakening of TAD boundaries and transcriptional dysregulation could benefit from further experimentation. As it stands, this finding is merely correlative, not causative, and represents a very minor fraction of Prmt5 occupied sites. Hence, the evidence provided does not overwhelmingly support the authors' conclusions regarding Prmt5's role in chromatin organization.
This research is potentially important for our understanding of adipogenesis and the role of Prmt5, which is already known for its diverse roles in cellular processes. However, while the authors have taken on an ambitious research question, there are some areas where the study falls short in substantiating the broad conclusions it draws.
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Reviewer #2 (Public Review):
This study by Syed et al identifies Prmt5 as a novel and broad modulator of gene expression and genome architecture during the early stages of adipogenesis. Specifically, Prmt5 is reported to be required to maintain strong insulation at TAD boundaries.
This is a logically and clearly conducted study that relies on the integration of public datasets (PCHi-C) to identify chromatin loops, with its own new genomics datasets, including Prmt5 ChIPseq and Hi-C data in control and Prmt5 kd cells. Despite showing relatively model effects of Prmt5 kd on genome architecture, the results are informative and contribute to advancing our knowledge of chromatin-linked processes during early adipogenesis.
The manuscript would benefit from incorporating ATACseq data (public or own) to better appreciate binding profiles of …
Reviewer #2 (Public Review):
This study by Syed et al identifies Prmt5 as a novel and broad modulator of gene expression and genome architecture during the early stages of adipogenesis. Specifically, Prmt5 is reported to be required to maintain strong insulation at TAD boundaries.
This is a logically and clearly conducted study that relies on the integration of public datasets (PCHi-C) to identify chromatin loops, with its own new genomics datasets, including Prmt5 ChIPseq and Hi-C data in control and Prmt5 kd cells. Despite showing relatively model effects of Prmt5 kd on genome architecture, the results are informative and contribute to advancing our knowledge of chromatin-linked processes during early adipogenesis.
The manuscript would benefit from incorporating ATACseq data (public or own) to better appreciate binding profiles of Prmt5 at H3K27ac sites. A more detailed analysis of these relative enrichments would also be useful, particularly if linked to a transcription factor footprint from ATAC data.
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