{"id":485,"date":"2023-07-18T13:12:55","date_gmt":"2023-07-18T20:12:55","guid":{"rendered":"https:\/\/live-usc-dornsife.pantheonsite.io\/pombenet\/?page_id=485"},"modified":"2023-08-18T10:59:06","modified_gmt":"2023-08-18T17:59:06","slug":"histones","status":"publish","type":"page","link":"https:\/\/dornsife.usc.edu\/pombenet\/histones\/","title":{"rendered":"Forsburg Lab Research: Histones and heterochromatin"},"content":{"rendered":"\n\n \n \n\n\n\n\n\n\n\n
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We have several projects related to epigenetics and heterochromatin relevant to the broad questions of DNA replication and genome stability. Some background on heterochromatin can be found on the\u00a0Chromosomes Page<\/a>.<\/p>\n<\/div>\n<\/div>\n

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DNA Replication and the Heterochromatin<\/h3>\n
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As described on the DDK page, we identified an interaction between Swi6 and the\u00a0DDK kinase<\/a>. Swi6 is heterochromatin protein 1 (HP1), a conserved protein that binds methylated histones and is important for the establishment of transcriptionally silent heterochromatin (more\u00a0background on the our chromosomes page<\/a>). In a collaboration with Robin Allshire, Julie Bailis showed that\u00a0hsk1<\/em>\u00a0and\u00a0dfp1<\/em>\u00a0mutants have defective chromosome segregation and centromere cohesin. Hisao Masukata’s lab went on to show that DDK interaction with Swi6 is also important for the early timing of centromere replication.<\/p>\n

This movie shows the effect of a\u00a0swi6\u00a0<\/em>mutation in mutant cells labeled with histone-GFP. Under these conditions, the chromosomes are at risk for inappropriate attachments to the spindle apparatus; some chromosomes may get attached to both poles of the spindle. You can see the cell in the center has this merotelic association and undergoes abnormal segregation and chromosome loss. However, most of the cells manage to divide safely.<\/p>\n<\/div>\n

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Another link between Swi6 and replication was studied by Pao-Chen Li in our lab, in a collaboration with Tom Kelly. Swi6 interacts with the Cdc18 (CDC6) replication protein, which is an MCM loading factor important for formation of the preRC (see\u00a0replication page for background<\/a>). The domain of Swi6 responsible is the chromo-shadow domain, a protein-association module that is responsible for most of Swi6’s molecular interactions. A point mutation in Cdc18 that disrupts this interaction is fully intact for replication; its only defect is an increased rate of replication in the centromere! Pao-Chen also showed that replication in the centromere is differently affected by heterochromatin mutants: while\u00a0swi6\u2206<\/em>\u00a0is delayed in replication, loss of\u00a0clr4<\/em>\u00a0or\u00a0chp1<\/em>\u00a0restores timely replication. RNAi mutants have delayed timing. We conclude that the unopposed presence of Chp1 leads to delayed replication.<\/div>\n
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Heterochromatin and Genome Stability<\/h3>\n
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See the\u00a0genome stability page<\/a><\/p>\n<\/div>\n<\/div>\n

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The Myst family of Histone Acetyltransferases<\/h3>\n
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\"Hbo1\"In metazoans, the MCMs have been shown to interact with a protein called Hbo1 which is a member of the MYST family of histone acetyltransferases. This family has numerous members, and is defined mainly by characteristics of the MYST-acetyltransferase domain. The different members of the family have different additional domains, indicated in the figure at right.<\/p>\n

Although there is no obvious Hbo1 orthologue in fission yeast, there are two members of this family: Mst1, which is the highly conserved Kat5 orthologue corresponding to Tip60\/Esa1, and Mst2., which is less well conserved but has features in common with\u00a0S. cerevisiae<\/em>\u00a0HATs called Sas2 and Sas3. Eliana G\u00f3mez cloned both\u00a0mst1+\u00a0<\/em>and\u00a0mst2+<\/em>\u00a0and performed their initial characterization.<\/p>\n

mst1+<\/em><\/strong>\u00a0<\/strong>is an essential gene. Eliana constructed a temperature sensitive allele and showed that the cells have significant defects in chromosome segregation. Rebecca Nugent followed up on Eliana’s observations. Interestingly,\u00a0mst1ts<\/em>\u00a0mutants have strong genetic interactions with heterochromatin mutants including\u00a0swi6<\/em>\u00a0and\u00a0clr4.\u00a0<\/em>However, 2-hybrid analysis showed that Mst1 interacts physically with proteins that influence the central core of the centromere, including Msc1and CenpB. Rebecca showed that\u00a0mst1ts<\/em>\u00a0disrupts the kinetochore but not Cnp1 binding in the central core.<\/p>\n

Ruben Petreaca has followed up data from Eliana showing that Mst1 interacts with the Rad22 (Rad52) homologous recombination protein. He has dissected the domains of the proteins required for this and assessed the effect on double strand break repair.<\/p>\n

mst2+<\/em><\/strong>\u00a0<\/em>is not essential. Eliana showed that\u00a0Mst2<\/strong>\u00a0antagonizes the\u00a0Sir2<\/strong>\u00a0histone deacetylase in silencing of the telomere. Working with Anthony Wright’s lab, Rebecca showed that\u00a0mst2<\/em>\u00a0overlaps with\u00a0gcn5\u00a0<\/em>in the acetylation of H3K14, and the double mutant is quite damage sensitive. We continue to work on this family of proteins.<\/p>\n<\/div>\n<\/div>\n<\/div>\n

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\"ade6+<\/p>\n

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Silencing of an\u00a0ade6+\u00a0<\/em>gene in the telomere in\u00a0mst2\u00a0<\/em>mutants. In the absence of Mst2, there is increased silencing, and this depends on both Swi6 and Sir2. From Gomez (2004).<\/p>\n

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Chromosome Passenger Proteins<\/h3>\n
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In collaboration with Tony Hunter’s lab, we also characterized components of the Chromosome Passenger Complex, or CPC. This complex, made up of Ark1, the Aurora kinase, Bir1\/Survivin, and Pic1\/INCENP, is important for proper attachment of the kinetochore to the centromere in mitosis. Interestingly, we found that a member of the GINS complex, Psf2, interacts with this complex, suggesting that it may be coordinated with the events of S phase.<\/p>\n<\/div>\n<\/div>\n

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Reviews<\/h3>\n
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