Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes

Abstract

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I-and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1-and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.To regulate expression of a gene epigenetically, cells must efficiently assemble specialized chromatin at those genes during or shortly after DNA replication each cell cycle. This process ensures that the gene expression state is inherited in future generations. In the budding yeast Saccharomyces cerevisiae, an epigenetic process called silencing prevents transcription of the mating-type genes at the silent mating-type loci HMR and HML (1). When silent chromatin is first formed, the silent information regulator proteins Sir1p, Sir2p, Sir3p, and Sir4p are recruited to the HM loci through their physical interactions with proteins bound to DNA elements called silencers adjacent to the HM loci. At HMR, the four Sir proteins interact with each other and with the origin recognition complex (ORC), 2 Rap1p and Abf1p bound to the E silencer (2-9). Sir2p, Sir3p, and Sir4p then spread along the chromosome and across the mating-type genes a2 and a1 at HMR as the deacetylase Sir2p removes acetyl groups from histones H3 and H4 and creates binding sites for Sir3p and Sir4p on nucleosomes (7, 9) (see also Ref. 5). Once silent chromatin is formed upon passage through S phase, the a2 and a1 genes at HMR are inactivated and their silenced state will be inherited as the genome is duplicated in subsequent generations (1, 10 -14).Although DNA replication itself is not required to establish silent chromatin in S phase (10 -12), a link between DNA replication and silencing in S. cerevisiae has been reinforced by numerous studies. In y...

Expressão de metaloproteinases de matriz e PCNA em úlceras de córnea profundas, induzidas em coelhos, tratadas com plasma rico em plaquetas

“…A ação imediata desses fatores pôde ser observada no centro da lesão dos animais do GR, em que se verificou maior expressão de PCNA no M4 do que nos demais momentos. O PCNA é uma proteína expressa durante a mitose celular (Miller et al, 2010), sendo um valioso marcador de proliferação celular em córneas humanas e de animais (Gan et al, 1999).…”

Propagating Epigenetic States During DNA Replication

“…Misregulation of H3 K56ac leads to silencing defects (Hyland et al, 2005, Miller et al, 2008, Sharp et al, 2001) and SAS-I and rtt109 mutants have silencing phenotypes similar to those of cac1, asf1 and pol30 mutants (Ehrenhofer-Murray et al, 1999, Meijsing & Ehrenhofer-Murray, 2001, Miller et al, 2010, Miller et al, 2008, Osada et al, 2001. PCNA interacts with Rtt109p and SAS-I in vivo, but this interaction is lost in pol30 mutants with defects in CAF-1-and Asf1p-dependent pathways (Miller et al, 2010), implying that acetylation of H3 K56 and H4 K16 are coupled to DNA replication. In addition, Asf1p binds the histone chaperone complex Hif1p/Hat1p/Hat2p in a Hat2p-dependent manner (Fillingham et al, 2008).…”

“…And, in gcn5 or H3 K5R mutants, reduced levels of H3 containing modifications of newly synthesized histones, K9ac, K27ac and K56ac, are incorporated into chromatin adjacent to an early firing replication origin in cells arrested in early S phase (Burgess et al, 2010). RTT109 and GCN5-dependent H3 K9ac (Fillingham et al, 2008, Adkins et al, 2007, Berndsen et al, 2008, Kuo et al, 1996 is also defective in po30 mutants (Miller et al, 2010). This loss of H3 K9ac is consistent with loss of interactions between pol30p mutants and Rtt109p (Miller et al, 2010), but could also indicate the activity of Gcn5p during chromatin assembly was compromised in pol30 mutants.…”

“…Asf1p also binds the H3 K56 acetyltransferase Rtt109p, stimulates H3 K56ac in vitro and is required, along with RTT109, for H3 K56ac in S phase in vivo (Driscoll et al, 2007, Recht et al, 2006, Schneider et al, 2006, Tsubota et al, 2007, Han et al, 2007. Misregulation of H3 K56ac leads to silencing defects (Hyland et al, 2005, Miller et al, 2008, Sharp et al, 2001) and SAS-I and rtt109 mutants have silencing phenotypes similar to those of cac1, asf1 and pol30 mutants (Ehrenhofer-Murray et al, 1999, Meijsing & Ehrenhofer-Murray, 2001, Miller et al, 2010, Miller et al, 2008, Osada et al, 2001. PCNA interacts with Rtt109p and SAS-I in vivo, but this interaction is lost in pol30 mutants with defects in CAF-1-and Asf1p-dependent pathways (Miller et al, 2010), implying that acetylation of H3 K56 and H4 K16 are coupled to DNA replication.…”

“…Yeast pol30 mutants with silencing defects have defects in CAF-1 and ASF1-dependent pathways (Zhang et al, 2000, Sharp et al, 2001). The silencing defects in pol30, cac1, asf1 and rfc1-1 mutants reflect, in part, misregulation of histone acetylation by SAS-I, Rtt109p and/or Gcn5p leading to hypoacetylation of at least H3 K9, H3 K56 and H4 K16 throughout the genome (Miller et al, 2010, Miller et al, 2008. CAF-1 and Asf1p both bind to the H4 K16 acetyltransferase complex SAS-I (Meijsing & Ehrenhofer-Murray, 2001, Osada et al, 2001) and loss of the catalytic subunit of SAS-I, Sas2p, alters the chromosomal distribution of Sirs and results in silencing defects (Kimura et al, 2002, Meijsing and Ehrenhofer-Murray, 2001, Osada et al, 2001, Reifsnyder et al, 1996, Suka et al, 2002.…”