Department of Cell Biology, Harvard Medical School
Investigator, Howard Hughes Medical Institute
We are interested in understanding how epigenetic chromatin domains are assembled and stably propagated.
We use silent chromatin domains, also called heterochromatin, as model systems to study this problem. In order to understand this process at a molecular and mechanistic level, we are working toward establishing defined biochemical systems to reconstitute and ultimately replicate silent chromatin domains in vitro. In addition, we are developing chromatin templates that are suitable for specific association with silencing complexes so that we could use X-ray crystallography and electron microscopy to determine the structure of silent chromatin. Yeast silent chromatin domains regulate gene expression and chromosome stability and share central features with epigenetically heritable chromatin domains in multicellular eukaryotes. Our studies are focused on yeast (both budding and fission yeasts) because they represent the most experimentally amenable systems to attack this problem. Three major types of budding or fission yeast silent chromatin, which require distinct but overlapping factors, are under investigation in our laboratory, and are described below.
The SIR Silencing System. We have defined the composition of the silencing complex that mediates heterochromatin assembly at the mating type regions and telomeres in the budding yeast Saccharomyces cerevisiae. This silencing complex, called the SIR complex, contains the Sir2, Sir3 and Sir4 proteins, and brings together two types of biochemical activities: Sir2 is an NAD-dependent histone deacetylase, and the Sir3 and Sir4 subunits are histone-binding proteins. We have shown that deacetylation by Sir2 is coupled to NAD hydrolysis and the synthesis of O-acetyl-ADP-ribose (OAADPR or AAR). We have discovered that both histone deacetylation and AAR synthesis are critical for SIR complex assembly in vitro. Moreover, using purified Sir proteins, defined chromatin templates, and transcription extracts, we have reconstituted Sir-dependent transcriptional gene silencing. Our reconstituted system recapitulates some of the main features of in vivo silencing, including requirement for all three Sir proteins and NAD-dependent deacetylation, and sensitivity to specific histone mutations. Moreover, we have established a system for structural analysis of Sir-nucleosome complexes and successfully solved the structure of the Sir3-BAH domain bound to the yeast mono-nucleosome. The structural data suggests that Sir3-BAH induces a conformational change in the N terminus of histone H4 that clamps two conserved H4 arginines onto nucleosomal DNA. We will test this arginine clamp model using optical tweezer experiments to test whether the force required to unwrap DNA from histones increases when Sir3-BAH is bound to the nucleosome.
Stabilization of DNA repeats by silencing mechanisms. The silencing protein, Sir2, also regulates ribosomal DNA (rDNA) chromatin structure and is important for maintaining the stability of the highly repeated rDNA genes (100-200 copies in most eukaryotes). We identified a second silencing complex, called RENT (Regulator of nucleolar silencing and telophase exit), which contains Sir2, Net1 (also called Cfi1), and the protein phosphatase Cdc14, and mediates rDNA silencing. Our findings suggest a new mechanism for control of rDNA recombination in which RENT recruits monopolin, a complex previously identified for its role as a sister kinetochore clamp during meiosis, to specific sites in rDNA and assembles a mitotic rDNA sister chromatid clamp that prevents unequal crossover between rDNA repeat units. We have also uncovered a network of proteins that tethers the rDNA to the nuclear periphery through interactions with conserved inner nuclear membrane (INM) proteins. This network plays a major role in stabilizing the rDNA repeats by preventing inappropriate recombination. Several components of this network, including the INM proteins, are conserved in fission yeast and other eukaryotes. Our future goals include understanding how INM proteins participate in genome organization in the nucleus.
RNAi and heterochromatin. The RNAi machinery is required for heterochromatin formation in the fission yeast Schizosaccharomyces pombe, plants, Drosophila, and possibly other eukaryotes, but is absent in S. cerevisiae. We purified the RNA-Induced Transcriptional Silencing (RITS) complex, which directly links the RNAi pathway to heterochromatin assembly in fission yeast. We have identified a second RNAi complex, termed RDRC (RNA-directed RNA polymerase complex), which is recruited via interactions with RITS and is responsible for the generation of double stranded RNA. Furthermore, we have shown that (1) RITS/RDRC can be crosslinked to noncoding centromeric RNAs and (2) tethering the RITS complex to an RNA transcript can induce heterochromatin assembly at the corresponding coding DNA. Our results suggest that RNAi complexes can initiate heterochromatin formation by using nascent noncoding transcripts as anchors. Moreover, cotranscriptional degradation of such nascent transcripts is a primary feature of silencing in heterochromatin. Our future efforts will be focused on in vitro reconstitution of RNAi-mediated silencing and on further in vivo tests of the nascent transcript model for heterochromatin assembly. In addition, we are trying to understand how RNAi is first triggered, how another Argonaute containing complex, called ARC (Argonaute siRNA Chaperone), mediates the loading of siRNAs onto Argonaute, and how heterochromatin is epigenetically maintained during cell division.
Yu, R., Jih, G., Iglesias, N., and Moazed, D. (2014) Determinants of Heterochromatic siRNA Biogenesis and Function. Mol Cell. 53(2):262-76.
Johnson, A., Wu, R., Peetz, M., Gygi, S.P., and Moazed, D. (2013) Heterochromatic gene silencing by activator interference and a transcription elongation barrier. J Biol Chem. 288(40):28771-82.
Heard, E., and Moazed, D. (2013) Cell nucleus. Curr Opin Cell Biol. 25(3):279-80.
Moazed, D. (2013) Chromatin: a tail of repression. Curr Biol. 23(10):R456-9.
Wang, F., Li, G., Altaf, M., Lu, C., Currie, M.A., Johnson, A., and Moazed, D. (2013) Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA. PNAS USA. 110(21):8495-500.
Moazed, D. (2012) A piRNA to remember. Cell. 149(3):512-4.
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