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Danesh Moazed

Publications and source records attributed to Danesh Moazed.

26 records · Page 2Linked to original sources
PubMed2003

Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing.

Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span. rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit). We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region. Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities. In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased. We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I. Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex. Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging.

Cell Cycle Proteins↗
PubMed2003

Heterochromatin and epigenetic control of gene expression.

Eukaryotic DNA is organized into structurally distinct domains that regulate gene expression and chromosome behavior. Epigenetically heritable domains of heterochromatin control the structure and expression of large chromosome domains and are required for proper chromosome segregation. Recent studies have identified many of the enzymes and structural proteins that work together to assemble heterochromatin. The assembly process appears to occur in a stepwise manner involving sequential rounds of histone modification by silencing complexes that spread along the chromatin fiber by self-oligomerization, as well as by association with specifically modified histone amino-terminal tails. Finally, an unexpected role for noncoding RNAs and RNA interference in the formation of epigenetic chromatin domains has been uncovered.

Animals↗
PubMed2003

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast.

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.

Acetylation↗
PubMed2003

Structure of the coiled-coil dimerization motif of Sir4 and its interaction with Sir3.

The yeast silent information regulators Sir2, Sir3, and Sir4 physically interact with one another to establish a transcriptionally silent state by forming repressive chromatin structures. The Sir4 protein contains binding sites for both Sir2 and Sir3, and these protein-protein interactions are required for gene silencing. Here, we report the X-ray structure of the coiled-coil dimerization motif within the C-terminus of Sir4 and show that it forms a stable 1:1 complex with a dimeric fragment of Sir3 (residues 464-978). We have identified a cluster of residues on the surface of the Sir4 coiled coil required for specific interactions with Sir3. The histone deacetylase Sir2 can also bind to this complex, forming a ternary complex with the truncated Sir3 and Sir4 proteins. The dual interactions of Sir4 with Sir3 and Sir2 suggest a physical basis for recruiting Sir3 to chromatin by virtue of its interactions with Sir4 and with deacetylated histones in chromatin.

Amino Acid Sequence↗
PubMed2002

Association of the histone methyltransferase Set2 with RNA polymerase II plays a role in transcription elongation.

The Saccharomyces cerevisiae protein, Set2, has recently been shown to be a histone methyltransferase. To elucidate the function of Set2, its associated proteins were identified using tandem affinity purification and mass spectrometry. We found that Set2 associates with RNA polymerase II. The interaction between the Set2 protein and RNA polymerase II requires the WW domain in Set2 and phosphorylation of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Set2 directly binds to the carboxyl-terminal domain with phosphorylated Ser(2) in the heptapeptide repeats. set2 deletion mutant is sensitive to 6-azauracil, a property often associated with impaired transcription elongation. Together, our results suggest that Set2 through association with the elongating form of RNA polymerase II plays an important role in transcription elongation.

Amino Acid Sequence↗
PubMed2002

Steps in assembly of silent chromatin in yeast: Sir3-independent binding of a Sir2/Sir4 complex to silencers and role for Sir2-dependent deacetylation.

Transcriptional silencing at the budding yeast silent mating type (HM) loci and telomeric DNA regions requires Sir2, a conserved NAD-dependent histone deacetylase, Sir3, Sir4, histones H3 and H4, and several DNA-binding proteins. Silencing at the yeast ribosomal DNA (rDNA) repeats requires a complex containing Sir2, Net1, and Cdc14. Here we show that the native Sir2/Sir4 complex is composed solely of Sir2 and Sir4 and that native Sir3 is not associated with other proteins. We further show that the initial binding of the Sir2/Sir4 complex to DNA sites that nucleate silencing, accompanied by partial Sir2-dependent histone deacetylation, occurs independently of Sir3 and is likely to be the first step in assembly of silent chromatin at the HM loci and telomeres. The enzymatic activity of Sir2 is not required for this initial binding, but is required for the association of silencing proteins with regions distal from nucleation sites. At the rDNA repeats, we show that histone H3 and H4 tails are required for silencing and rDNA-associated H4 is hypoacetylated in a Sir2-dependent manner. However, the binding of Sir2 to rDNA is independent of its histone deacetylase activity. Together, these results support a stepwise model for the assembly of silent chromatin domains in Saccharomyces cerevisiae.

Acetylation↗