Shiv Grewal, Ph.D.

It is fascinating to consider how a multitude of cell types originate during development from a single genome. Epigenetic mechanisms allow specific gene expression outputs and involve DNA and histone modifications, including those associated with heterochromatin, a repressive form of chromatin. Heterochromatin maintains global patterns of gene expression and protects genomic integrity by prohibiting recombination between repetitive DNA elements and promoting proper segregation of chromosomes. Defects in heterochromatin mechanisms can lead to chromosome abnormalities that underlie many diseases, including cancer.

Over more than two decades, our lab has been delineating highly conserved heterochromatin assembly pathways. We were the first to define the epigenetic profile of an entire eukaryotic genome, using fission yeast Schizosaccharomyces pombe. Our comprehensive analyses have revealed that methylation of histone H3 lysine 9 (H3K9me) is strictly localized across heterochromatin domains, whereas H3K4me is specific to euchromatic regions. We have described the establishment of heterochromatin domains through nucleation and spreading activities catalyzed by the read/write activity of the histone methyltransferase Clr4/Suv39h, and we uncovered its role as a versatile recruiting platform for orchestrating diverse genome functions. Importantly, we have discovered that heterochromatin can be inherited epigenetically in a self-templating manner and described mechanisms that promote epigenetic inheritance. Our work also led to the discovery of RNA-based mechanisms of heterochromatin assembly, including RNAi that utilizes small interfering RNAs (siRNAs) to target heterochromatin. RNA-based mechanisms of heterochromatin assembly are conserved in higher eukaryotes and have broad implications for human biology and disease.

Excitingly, our current research has revealed that multiple pathways converge to enforce a single key feature crucial for epigenetic inheritance of heterochromatin, namely the critical density of tri-methylated histone H3 lysine 9 (H3K9me3) that is required for self-propagation. Our work suggests a model in which tethering of heterochromatin domains at the nuclear periphery provides an ideal microenvironment to maintain a high density of H3K9me3, thus facilitating self-templated inheritance of silenced domains through read/write histone methyltransferase activity. However, despite significant progress, much still remains to be discovered. Below are our current research areas.

Current Research Areas

RNA-mediated Heterochromatin Assembly: Our lab has discovered two distinct RNA-based mechanisms that direct the assembly of heterochromatin. We have described the role of the RNAi machinery and small RNAs in targeting heterochromatin (named “Breakthrough of the Year 2002” by Science magazine). We have also uncovered an RNAi-independent mechanism in which a nuclear RNA processing complex (named MTREC) cooperates with RNA polymerase II termination factors to direct the assembly of facultative heterochromatin domains under specific growth conditions. We are continuing to investigate mechanisms of heterochromatin assembly that are expected to have major implications for understanding gene regulatory processes in higher eukaryotes.

Signaling to Chromatin and Adaptive Genome Control: The complexity of eukaryotic genome regulation is evidenced by the ability to reprogram gene expression to adapt to environmental changes or respond to developmental signals. We are investigating how facultative heterochromatin domains can be modulated by signaling pathways. Our work has led to the finding that heterochromatin assembly factors are part of a rheostat-like buffering mechanism that limits the uncontrolled expression of a wide variety of genes in response to environmental change. Recently, we have found that the TOR signaling pathway, a key regulator of cell growth in all eukaryotes, controls gene expression by targeting MTREC nuclear RNA elimination complex that promotes facultative heterochromatin and RNA decay to regulate gene expression. Two key findings are: (1) the RNA degradation/facultative heterochromatin assembly machinery is a central regulator of TOR-mediated control of cell proliferation and (2) TORC1 dynamically controls RNA elimination machinery to modulate facultative heterochromatin and coordinate developmental gene expression during gametogenesis, an extremely important process that ensures the genomic integrity of future generations and lies at the heart of many heritable human disorders. We are continuing to explore how heterochromatin machinery intersects with signaling pathways to control gene expression programs.

Epigenetic Inheritance of Silenced Chromatin Domains: How are silenced chromatin domains heritably maintained? The assembly and maintenance of repressive heterochromatin domains is achieved through modification of histones, particularly H3K9me3 that is recognized and deposited by Clr4/SUV39h via its ability to both “read” and “write”. Using a carefully designed genetic system, we have identified several factors that ensure faithful inheritance of heterochromatin by suppressing histone turnover. We have also provided direct evidence that histones are carriers of epigenetic information and discovered that a critical density of H3K9me3 is required to provide an epigenetic template for the spreading and epigenetic inheritance of heterochromatin. This work elucidated a fundamental theme in which the activities of multiple silencing factors converge to maintain H3K9me3 density, which in turn promotes epigenetic inheritance of heterochromatin through both mitosis and meiosis.

Although silenced chromatin domains are often observed at the nuclear periphery, the purpose of this genome organization had remained unclear. We have discovered that the evolutionarily conserved process of peripheral tethering of heterochromatin promotes stable propagation by creating a microenvironment for loading of silencing effectors that maintain the threshold density of methylated histones required for self-templated heterochromatin propagation. We are further exploring how heterochromatin factors are coordinated to enforce silenced domains and support their stable propagation.

Biological Implications of Defects in RNA Elimination and Heterochromatin Assembly: Our work has revealed that heterochromatin shapes the three-dimensional (3D) architecture of the genome. In particular, heterochromatin formed at centromeres and telomeres provides structural constraints that are crucial for proper genome organization. Indeed, we have found that loss of heterochromatin relaxes constraints on chromosomes. Moreover, our analyses revealed that the arms of chromosomes are organized into ~50-100kb locally crumpled self-interacting chromatin domains called globules, which are dependent on cohesin. Together, heterochromatin- and cohesin-mediated organization promotes chromosome territoriality and facilitates functional genomic annotation. Our work has uncovered fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions. We are continuing to explore the importance and biological implications of 3D genome organization as cells respond to various signals including replication stress.

Defects that result in inappropriate gene expression can cause devastating consequences. We have found that untimely activation of the silenced gametogenic program (such as in cells lacking RNAi machinery or the RNA elimination factor Mmi1) triggers uniparental disomy (UPD), in which cells contain two copies of a chromosome originating from only one parent. UPD is frequently associated with congenital disorders and cancer. Our analyses revealed that UPD is linked to the misexpression of Rec8, a meiotic cohesin that is only expressed in cells undergoing sexual differentiation and promotes reductional chromosome segregation. Rec8 misexpression in mitotic diploid cells causes aberrant segregation and UPD. This work has important implications for understanding the UPD phenomenon in humans and provides a solid foundation for the development of therapies to treat diseases, including various cancers linked to the aberrant expression of germline genes in somatic cells.

Dr. Grewal began his scientific career at the University of Cambridge, UK, where he held the prestigious Cambridge-Nehru scholarship. In 1993, he joined National Cancer Institute as a postdoctoral fellow to pursue his interests in the epigenetic control of gene expression. Apart from his pioneering work on the role of centromeric repeats in heterochromatin assembly, he showed that epigenetic imprints can be stably propagated through meiosis and in some instances inherited in cis. Dr. Grewal joined Cold Spring Harbor Laboratory as an Assistant Professor in 1998, and was promoted to Associate Professor position. In 2003, he joined National Cancer Institute, Bethesda as a Senior Investigator. He has been named an NIH Distinguished Investigator and is currently serving as the Chief of the Laboratory of Biochemistry and Molecular Biology and the Head of the Chromosome Biology Section of the Center for Cancer Research, National Cancer Institute. Dr. Grewal and colleagues discovered a highly conserved connection between RNAi and heterochromatin assembly. This important contribution was selected as Breakthrough of the Year 2002 by Science magazine. Three papers from Dr. Grewal's laboratory are cited for historic discoveries over the past 50 years by Nature. He is recipient of the prestigious Newcomb-Cleveland Prize, NIH Merit Award, and the NIH Directors’ award. Dr. Grewal has been elected to the US National Academy of Sciences and the American Academy of Arts and Sciences.