Joydeb Sinha
Postdoctoral Scholar, Bioengineering
All Publications
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The H3.3K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation.
Molecular cell
2024
Abstract
Histone H3.3 is frequently mutated in tumors, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the epigenetic landscape, its effects on gene expression dynamics remain unclear. Here, we use a synthetic reporter to measure the effects of H3.3K36M on silencing and epigenetic memory after recruitment of the ZNF10 Kruppel-associated box (KRAB) domain, part of the largest class of human repressors and associated with H3K9me3 deposition. We find that H3.3K36M, which decreases H3K36 methylation and increases histone acetylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a modelfor establishment and maintenance of epigenetic memory, where the H3K36 methylation pathway is necessary to maintain histone deacetylation and convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.
View details for DOI 10.1016/j.molcel.2024.09.015
View details for PubMedID 39368466
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The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation.
bioRxiv : the preprint server for biology
2023
Abstract
Histone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the cellular epigenetic landscape, it remains unclear how it affects the dynamics of gene expression. Here, we use a synthetic reporter to measure the effect of H3.3K36M on silencing and epigenetic memory after recruitment of KRAB: a member of the largest class of human repressors, commonly used in synthetic biology, and associated with H3K9me3. We find that H3.3K36M, which decreases H3K36 methylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a new model for establishment and maintenance of epigenetic memory, where H3K36 methylation is necessary to convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.
View details for DOI 10.1101/2023.10.13.562147
View details for PubMedID 37873347
View details for PubMedCentralID PMC10592807
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Single-cell chromatin state transitions during epigenetic memory formation.
bioRxiv : the preprint server for biology
2023
Abstract
Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.
View details for DOI 10.1101/2023.10.03.560616
View details for PubMedID 37873344
View details for PubMedCentralID PMC10592931
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High-throughput functional characterization of combinations of transcriptional activators and repressors.
Cell systems
2023
Abstract
Despite growing knowledge of the functions of individual human transcriptional effector domains, much less is understood about how multiple effector domains within the same protein combine to regulate gene expression. Here, we measure transcriptional activity for 8,400 effector domain combinations by recruiting them to reporter genes in human cells. In our assay, weak and moderate activation domains synergize to drive strong gene expression, whereas combining strong activators often results in weaker activation. In contrast, repressors combine linearly and produce full gene silencing, and repressor domains often overpower activation domains. We use this information to build a synthetic transcription factor whose function can be tuned between repression and activation independent of recruitment to target genes by using a small-molecule drug. Altogether, we outline the basic principles of how effector domains combine to regulate gene expression and demonstrate their value in building precise and flexible synthetic biology tools. A record of this paper's transparent peer review process is included in the supplemental information.
View details for DOI 10.1016/j.cels.2023.07.001
View details for PubMedID 37543039
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The circadian clock mediates daily bursts of cell differentiation by periodically restricting cell-differentiation commitment.
Proceedings of the National Academy of Sciences of the United States of America
2022; 119 (33): e2204470119
Abstract
Most mammalian cells have an intrinsic circadian clock that coordinates metabolic activity with the daily rest and wake cycle. The circadian clock is known to regulate cell differentiation, but how continuous daily oscillations of the internal clock can control a much longer, multiday differentiation process is not known. Here, we simultaneously monitor circadian clock and adipocyte-differentiation progression live in single cells. Strikingly, we find a bursting behavior in the cell population whereby individual preadipocytes commit to differentiate primarily during a 12-h window each day, corresponding to the time of rest. Daily gating occurs because cells irreversibly commit to differentiate within only a few hours, which is much faster than the rest phase and the overall multiday differentiation process. The daily bursts in differentiation commitment result from a differentiation-stimulus driven variable and slow increase in expression of PPARG, the master regulator of adipogenesis, overlaid with circadian boosts in PPARG expression driven by fast, clock-driven PPARG regulators such as CEBPA. Our finding of daily bursts in cell differentiation only during the circadian cycle phase corresponding to evening in humans is broadly relevant, given that most differentiating somatic cells are regulated by the circadian clock. Having a restricted time each day when differentiation occurs may open therapeutic strategies to use timed treatment relative to the clock to promote tissue regeneration.
View details for DOI 10.1073/pnas.2204470119
View details for PubMedID 35939672
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Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells.
eLife
2022; 11
Abstract
In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation circuits. Here, we systematically dissect this process in single cells by recruiting and releasing repressive chromatin regulators at dual-gene synthetic reporters, and measuring how fast gene silencing and reactivation spread as a function of intergenic distance and configuration of insulator elements. We find that silencing by KRAB, associated with histone methylation, spreads between two genes within hours, with a time delay that increases with distance. This fast KRAB-mediated spreading is not blocked by the classical cHS4 insulators. Silencing by histone deacetylase HDAC4 of the upstream gene can also facilitate background silencing of the downstream gene by PRC2, but with a days-long delay that does not change with distance. This slower silencing can sometimes be stopped by insulators. Gene reactivation of neighboring genes is also coupled, with strong promoters and insulators determining the order of reactivation. Our data can be described by a model of multi-gene regulation that builds upon previous knowledge of heterochromatin spreading, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment.
View details for DOI 10.7554/eLife.75115
View details for PubMedID 35678392