Our laboratory focuses on the following areas:
EPIGENETIC MAINTENANCE OF HETROCHROMATIN
To investigate how heterochromatin is stably maintained, we used systems that allow us to separate the establishment and epigenetic inheritance phases of heterochromatin in yeast and mammalian cells. Using these systems, we have demonstrated that a histone modification read-write positive feedback loop can maintain heterochromatin independently of DNA sequence. However, sequence-independent inheritance is metastable. Contrary to prevailing models, we have demonstrated that robust epigenetic inheritance of heterochromatin requires input from either a small RNA positive feedback loop or specific DNA sequences, termed maintainers. By a combination of genetic and biochemical approaches, we have identified additional pathways that are critical for heterochromatin maintenance, including a conserved RNA decay complex that is recruited to heterochromatin by HP1, proteins that localize heterochromatin to the nuclear periphery, and components of the DNA replication machinery that may help transfer parental histones to newly synthesized DNA. We can now investigate how these pathways contribute to heterochromatin maintenance in fission yeast and mammalian cells.
Selected publications: Ragunathan et al., Science 2015; Wang et al., Science 2017; Jih et al., Nature 2017; Shipkovenska et al., eLife 2020; Iglesias, Paulo et al., Mol Cell 2020; Wang et al., Mol Cell 2021
Our group has elucidated the mechanism of RNAi-mediated heterochromatin assembly in the fission yeast S. pombe. The key steps in the assembly process involve (1) trigger primary small RNAs (siRNAs) are generated from noncoding RNAs that are transcribed from pericentromeric and other DNA repeats, (2, 3) the resulting primary siRNAs load onto the conserved Argonaute (Ago1) protein, first as a component of the Argonaute Chaperone (ARC) complex and then in the effector RNA-Induced Transcriptional Silencing (RITS) complex, (4, 5) the RITS complex binds to nascent noncoding RNAs via siRNA-dependent base pairing and recruits the Clr4 methyltransferase to initiate histone H3 lysine 9 methylation (H3K9me), (6,7) RITS binding is stabilizes via its recognition of H3K9me and leads to recruitment of the RNA-dependent RNA polymerase which synthesizes double strand RNA, (8) the Dicer ribonuclease cleaves the double strand RNA to generate secondary siRNAs. The cycle then starts over by the loading of secondary siRNAs onto the ARC and RITS complexes and forms a positive feedback loop that amplifies siRNAs and can maintain H3K9 methylation and heterochromatin. We are now focused on understanding how certain RNAs are recognized as foreign or non-self, triggering RNAi and heterochromatin formation, and exploring how nuclear RNAi pathways may contribute to gene and genome regulation in mammalian cells.
Selected publications: Verdel et al., Science 2004; Motamedi et al., Cell 2004; Buhler et al., Cell 2006; Buhler et al., Cell 2007; Colmenares et al., Mol Cell 2007; Iida et al., Mol Cell 2008; Halic and Moazed, Cell 2010; Gerace et al., Mol Cell 2010; Yu et al., Mol Cell 2014; Jain, Iglesias et al., Mol Cell 2016; Yu et al., Nature 2018
HP1- AND POLYCOMB-ASSOCIATED RNA DECAY
An unexpected outcome of our studies on the role of RNAi in heterochromatin assembly was the discovery that, in addition to its role in heterochromatin assembly, RNAi-dependent RNA decay contributes to heterochromatic gene silencing. This mechanism, which we call co-transcriptional gene silencing (CTGS), can mediate a relatively large reduction in transcriptional output, about 10- to 20-fold reduction in RNA levels, and may be critical for achieving full silencing. In addition, it removes RNA polymerase II/nascent RNA, which might interfere with heterochromatin formation. In fact, more recently, we discovered that a conserved RNA processing complex, which we call the rixosome, is recruited to heterochromatin via association with HP1 in fission yeast. The RNA degradation activities of the rixosome are required for heterochromatin maintenance and spreading in regions where RNAi is absent, suggesting redundant roles for different RNA decay pathways in heterochromatin. In human cells, we have shown that the rixosome is recruited to Polycomb target genes and contributes to Polycomb-mediated silencing via its RNA degradation activities. We can now study why RNA decay is critical for heterochromatin maintenance in fission yeast and human cells.
Buhler et al., Cell 2007; Motamedi et al., Mol Cell 2008; Jih et al., Nature 2017; Shipkovenska et al., eLife 2020; Iglesias, Paulo et al., Mol Cell, 2020; Zhou et al., bioRxiv 2021
STRUCTURAL BIOLOGY OF HETEROCHROMATIN
Our efforts on structural biology of heterochromatin proteins are ultimately directed toward gaining insight into how these proteins bind to nucleosomes, how this binding impedes transcription and access to DNA, and how histone methylation is accurately targeted to specific chromosome regions. By solving the X ray crystal structure of Clr4, the fission yeast homolog of the human SUV39H1/2 histone H3K9 methyltransferase, combined with biochemical and genetic analyses, we discovered an autoinhibited conformation of Clr4. In this conformation, a lysine in an internal autoinhibitory loop in the enzyme occludes the active site and inhibits methylation of the histone H3 substrate. Automethylation of the inhibitory lysine leads to a conformational switch that activates Clr4. Our findings suggest that autoinhibition is relieved upon the recruitment of Clr4 to cognate chromatin regions and acts to prevent inappropriate heterochromatin formation. The Clr4 enzyme is a component of a large complex that also contains a Cul4 E3 ubiquitin ligase, which has been shown to ubiquitinate histone H3K14. Our current efforts are focused on using cryoEM to solve the structure of the Clr4 complex bound to methylated and ubiquitinated nucleosome, and the structures of nucleosome-bound bridging proteins such as HP1 and Sir3.
Selected publications: Onishi et al., Mol Cell 2007; Wang et al., PNAS 2013; Behrouzi et al., eLife 2016; Iglesias, Currie et al., Nature 2018