Project description:H3K4me3 is catalyzed by the Set1/MLL family of methyltransferases, whose function in catalyzing H3K4me3 is unique. Impaired function of Set1/MLL family members can lead to many abnormalities, such as bone and nerve defects, leukemia, and even death. Although the Set1 family plays an important regulatory role in various biological processes, it is still unclear how the Set1 protein itself is regulated and how protein levels are maintained. Due to the numerous homologues, complex composition, and high molecular weight of Set1 in higher organisms, especially humans, related research is greatly limited. In brewing yeast, Set1 is the only methyltransferase that catalyzes H3K4me3 and is highly conserved between species. Therefore, yeast is an ideal model for studying the functions and mechanisms of the Set1 family. In addition, Set1 protein plays an important role in regulating gene transcription, promoting telomere silencing, and maintaining cell lifespan. The Set1 family also plays an important regulatory role in the occurrence and development of various cancers.
Project description:The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact action mechanism of H3pT11 is poorly understood. Here, we report that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they work together to promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM36 taining Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a novel histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.
Project description:The actin-related proteins (ARPs) comprise a conserved protein family. Arp4p is found in large multisubunits of the INO80 and SWR1 chromatin remodeling complexes and in the NuA4 histone acetyltransferase complex. Here we show that arp4 (arp4S23AD159A) temperature-sensitive cells are defective in G2/M phase function. arp4 mutants are sensitive to the microtubule depolymering agent benomyl and arrest at G2/M phase at restrictive temperature. Arp4p is associated with centromeric and telomeric regions throughout cell cycle. Ino80p, Esa1p, and Swr1p, components of the INO80, NuA4, and SWR1 complexes, respectively, also associate with centromeres. The association of many kinetochore components including Cse4p, a component of the centromere nucleosome, Mtw1p, and Ctf3p is partially impaired in arp4 cells, suggesting that the G2/M arrest of arp4 mutant cells is due to a defect in formation of the chromosomal segregation apparatus. Keywords: ChIP-chip ⢠The goal of the experiment Genome-wide localization of Arp4 binding sites in Saccharomyces cerevisiae ⢠Experimental factors Distribution of Arp4 in WT in G2/M phase in the presence of nocodazole (Saccharomyces cerevisiae). ⢠Experimental design ChIP analysis: Hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Chromosome III, IV,V,VI S. cerevisiae: SC3456a520015F, P/N# 520015, affymetrix tiling array were used.
Project description:ATP-dependent chromatin remodeling complexes have been shown to participate in DNA replication in addition to transcription and DNA repair. However, the mechanisms of their involvement in DNA replication remain unclear. Here, we reveal a specific function of the yeast INO80 chromatin remodeling complex in the DNA damage tolerance pathways. Whereas INO80 is necessary for the resumption of replication at forks stalled by methyl methane sulfonate (MMS), it is not required for replication fork collapse after treatment with hydroxyurea (HU). Mechanistically, INO80 regulates DNA damage tolerance during replication through modulation of PCNA (proliferating cell nuclear antigen) ubiquitination and Rad51-mediated processing of recombination intermediates at impeded replication forks. Our findings establish a mechanistic link between INO80 and DNA damage tolerance pathways, indicating that chromatin remodeling is important for accurate DNA replication. INO80 distribution in WT cells was measured.
Project description:ChIP on CHIP analysis were performed to analyse the direct involvement of Ino80 in the regulation of the phosphate responsive and nucleotide metabolism genes upon phosphate starvation: We immunoprecipitated Ino80-bound chromatin fragments after formaldehyde crosslinking from cells grown in minimal medium (EMM) or minimal medium lacking phosphate and purified, amplified and probed the Ino80-associated DNA using oligonucleotide tiling arrays.
Project description:The project aimed to investigate the composition/interactome of the human INO80 complex. To this end, INO80B - a core INO80 complex subunit - was endogenously tagged with V5 epitope tag at its C terminus in MCF-7 cells, and co-immunoprecipitation followed by LC-MS/MS analysis was performed using anti-V5 Abs.