Protecting heterochromatin from the remodeling machines: a fundamental mechanism of gene silencing
Research Summary: Our research finds that shielding heterochromatin, which typically contains repetitive DNA elements and transcriptionally inactive genes, from chromatin remodeling enzymes is crucial for maintaining its silent nature.
Author interview: Dr. Rakesh K. Sahu is a visiting postdoctoral fellow at the National Institutes of Health (NIH), USA. His research focuses on understanding the fundamental mechanisms of gene silencing and its implications for human diseases.
Lab: Dr. Shiv I.S. Grewal, National Institutes of Health (NIH) – National Cancer Institute (NCI), Bethesda, MD, USA.
What was the core problem you aimed to solve with this research?
Gene expression is precisely regulated to activate only specific genes in each cell type, while keeping others inactive. Errors in this regulation can lead to severe diseases, including cancer. To maintain proper gene expression patterns, DNA is organized into chromatin compartments with histone proteins. Euchromatin represents the open, accessible regions that facilitate gene expression, whereas heterochromatin is the condensed, closed form that inhibits it.
Heterochromatin typically features methylated histones, such as H3K9me3 and H3K27me3, which recruit silencing effector proteins like heterochromatin protein 1 (HP1). These proteins promote chromatin compaction and transcriptional silencing. This form of repression effectively silences repetitive DNA, transposons, and prevents inappropriate gene expression during development. Disruption of heterochromatin assembly can lead to detrimental genomic consequences, although the exact mechanisms by which heterochromatin induces transcriptional repression are not fully understood.
Some reports suggest that HP1 proteins form specialized structures, such as liquid condensates, that exclude transcription machinery, thereby silencing genes. However, recent studies question whether physical condensation alone is sufficient for gene silencing. We believe that HP1 proteins promote gene silencing by bringing various silencing effector proteins to heterochromatin, which alters the inherent properties of these domains, making them resistant to transcription. One such effector brought to heterochromatin by HP1 proteins is the histone deacetylase enzyme (HDAC). HDACs help maintain heterochromatic histones in a deacetylated state, which appears crucial for heterochromatin stability and gene repression. The precise way in which HDAC-mediated removal of acetyl marks promotes transcriptional silencing remains unknown, and we sought to address this in our study.
How did you go about solving this problem?
We utilized the powerful genetics of fission yeast to elucidate the mechanism of heterochromatic gene silencing. First, we conducted genetic interaction studies in cells lacking histone deacetylation activity to identify the factors involved in the loss of gene silencing and the inheritance of the silent state. This part of the study identified chromatin remodeling complexes as factors responsible for causing gene silencing defects. We observed the biological consequences of these genetic interactions on gene silencing using live-cell fluorescence microscopy. Next, we employed high-throughput sequencing techniques to examine their effects on a genome-wide scale. Additionally, to demonstrate the mechanism, we created a system that expresses fusion proteins—chromatin remodeler or histone acetyl transferase (HAT) fused with chromodomains—which can be targeted to heterochromatin domains, allowing us to assess their effects on gene silencing.
“Our work uncovers how chromatin modification, in particular, histone deacetylation, enforces gene silencing and has major implications for understanding developmental gene regulation in mammals.”
How would you explain your research outcomes (Key findings) to the non-scientific community?
Our study reveals that a key feature of heterochromatin—the absence of acetylation on histone proteins—protects these regions from being affected by enzyme complexes called chromatin remodelers, especially SWI/SNF remodelers. These remodelers generally promote gene expression by facilitating the assembly of transcription machinery over active genes. Here, HDAC enzymes help shield heterochromatin from SWI/SNF activity by actively removing acetyl modifications on histone proteins. This protection keeps the genes in these regions silent and allows this silenced state to be inherited by the next generation. To provide definitive proof of principle, when we artificially increased histone acetylation or crowded SWI/SNF complexes at heterochromatin regions, it resulted in the loss of gene silencing and the inheritance of the silenced state. We demonstrate this mechanism using a single-celled organism, but our findings may help explain how gene silencing problems can lead to cancer and have important implications for understanding gene regulation during development.
What are the potential implications of your findings for the field and society?
The implications of fundamental research can be wide-ranging. Our findings suggest that excluding SWI/SNF remodelers from heterochromatin is a mechanism for achieving sustained gene silencing. This has significant implications for human diseases, such as cancer. In certain types of cancer, mutations in SWI/SNF complexes or their misexpression can cause these complexes to activate previously silent oncogenes, contributing to cancer progression. Our study may explain how these remodelers are prevented from accessing the silent oncogenes and how alterations in this process can lead to cancer.
What was the exciting moment during your research?
It was quite rewarding to see our hypothesis validated when I targeted the remodeling complex to heterochromatin, and that could cause a silencing defect despite the presence of HDAC enzymes and other silencing factors in the cell. Excited by this result, when I further targeted this protein complex to ectopic heterochromatin, it hampered the epigenetic inheritance of this silenced domain. Thus, these experiments conclusively stated that preventing remodeling activities is critical for maintaining and propagating the silenced chromatin state of heterochromatin.
Figure Caption: Schematic illustrating how heterochromatic gene silencing occurs through HDAC-mediated exclusion of SWI/SNF chromatin remodelers. The high concentration of HDACs in heterochromatic regions promotes the removal of histone acetylation marks. This limits the ability of SWI/SNF remodelers to engage with heterochromatic regions, thereby preventing the turnover of nucleosomes with methyl marks. Suppressing nucleosome turnover helps maintain high levels of H3K9me3 necessary for heterochromatin propagation, and these stable nucleosomes support transcriptional gene silencing.
Paper reference: Sahu, R. K., Dhakshnamoorthy, J., Jain, S., Folco, H. D., Wheeler, D., & Grewal, S. I. S. Nucleosome remodeler exclusion by histone deacetylation enforces heterochromatic silencing and epigenetic inheritance. Molecular cell, (2024), 84(17), 3175–3191.e8. https://doi.org/10.1016/j.molcel.2024.07.006
