Histone H3 lysine 4 trimethylation (H3K4me3) is a hallmark of transcription initiation, but how H3K4me3 is demethylated during gene repression is poorly understood. Jhd2, a JmjC domain protein, was recently identified as the major H3K4me3 histone demethylase (HDM) in Saccharomyces cerevisiae. Although JHD2 is required for removal of methylation upon gene repression, deletion of JHD2 does not result in increased levels of H3K4me3 in bulk histones, indicating that this HDM is unable to demethylate histones during steady-state conditions. In this study, we showed that this was due to the negative regulation of Jhd2 activity by histone H3 lysine 14 acetylation (H3K14ac), which colocalizes with H3K4me3 across the yeast genome. We demonstrated that loss of the histone H3-specific acetyltransferases (HATs) resulted in genome-wide depletion of H3K4me3, and this was not due to a transcription defect. Moreover, H3K4me3 levels were reestablished in HAT mutants following loss of JHD2, which suggested that H3-specific HATs and Jhd2 serve opposing functions in regulating H3K4me3 levels. We revealed the molecular basis for this suppression by demonstrating that H3K14ac negatively regulated Jhd2 demethylase activity on an acetylated peptide in vitro. These results revealed the existence of a general mechanism for removal of H3K4me3 following gene repression.Gcn5 | histone acetylation | histone methylation | Sas3

Histone post-translational modifications (PTMs) alter chromatin structure by promoting the interaction of chromatinmodifying complexes with nucleosomes. The majority of chromatin-modifying complexes contain multiple domains that preferentially interact with modified histones, leading to speculation that these domains function in concert to target nucleosomes with distinct combinations of histone PTMs. In Saccharomyces cerevisiae, the NuA3 histone acetyltransferase complex contains three domains, the PHD finger in Yng1, the PWWP domain in Pdp3, and the YEATS domain in Taf14; which in vitro bind to H3K4 methylation, H3K36 methylation, and acetylated and crotonylated H3K9, respectively. While the in vitro binding has been well characterized, the relative in vivo contributions of these histone PTMs in targeting NuA3 is unknown. Here, through genome-wide colocalization and by mutational interrogation, we demonstrate that the PHD finger of Yng1, and the PWWP domain of Pdp3 independently target NuA3 to H3K4 and H3K36 methylated chromatin, respectively. In contrast, we find no evidence to support the YEATS domain of Taf14 functioning in NuA3 recruitment. Collectively our results suggest that the presence of multiple histone PTM binding domains within NuA3, rather than restricting it to nucleosomes containing distinct combinations of histone PTMs, can serve to increase the range of nucleosomes bound by the complex. Interestingly, however, the simple presence of NuA3 is insufficient to ensure acetylation of the associated nucleosomes, suggesting a secondary level of acetylation regulation that does not involve control of HAT-nucleosome interactions.

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ␤-galactosidase levels reflecting autoregulation and apparent K D values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. Polynucleotide phosphorylase (PNPase) is a conserved, widely distributed phosphorolytic 3=-5= exoribonuclease that may also function under some circumstances as a template-independent RNA polymerase (1; reviewed in reference 2). Although it is not essential, deletions of its gene (pnp) are synthetically lethal when either RNase II or RNase R, both of which are hydrolytic 3=-5= exoribonucleases, is deficient (3-5). Strains deficient in PNPase are also sensitive to cold shock and other stresses (6-9). Thus, PNPase is believed to play significant roles in mRNA turnover and other aspects of RNA processing and metabolism (4, 10-12). Partial or full structures of PNPase from several microorganisms (13-16), as well as structures of the related archaeal exosome (17, 18), have shed considerable light on its mechanism of action. Bacterial PNPase is composed of three identical subunits. Each subunit consists of two tandem globular domains (residues 8 to 210 and 312 to 541; see Fig. 1a) derived from RNase PH that form a core whose central channel is accessible from both the upper and lower surfaces of the core (14,19). Each subunit also contains a C-terminal extension that consists of two additional small domains, KH and S1 (residues 551 to 591 and 622 to 691, respectively; Fig. 1), which are positioned on the upper surface of the core. The KH and S1 domains have been implicated in autoregulation (20), in resistance to cold shock (7), in pathogenesis (21), and in substrate binding (22). Although a solution structure of the S1 domain from Escherichia coli PNPase has been available (13), the positions of the S1 and KH domains relative to the core have been elucidated only recently from the structure of PNPase from Caulobacter crescentus (16). The S1 and KH domains do not appear to be required for the enzymatic activity of PNPase (20,22,23). Nonetheless, deletion of the S1, the KH or both domains results in significant loss of RNA binding and inefficient enzymatic turnover (22).PNPase from E. coli and other organisms exhibits strong autoregulation...

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 a3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 a3 helix play organismal roles in defining chromatin structure. KEYWORDS histone; H3; S. cerevisiae; nucleosome positioning I N eukaryotes, DNA is packaged into a nucleoprotein structure known as chromatin, which consists of DNA, histones, and nonhistone proteins. The basic unit of chromatin is the nucleosome core particle, which is made up of an octamer of the four core histones, H2A, H2B, H3, and H4, wrapped with 147 bp of DNA (Luger et al. 1997;Kornberg and Lorch 1999). Although nucleosomes will form on most sequences in vitro, they are not randomly positioned in vivo. Genomewide mapping studies have shown that gene promoters and other regulatory regions tend to be nucleosome depleted and two general mechanisms have been proposed to explain this (Hughes and Rando 2014). First, certain DNA sequences, such as AT-rich regions, are refractory to nucleosome formation. Second, trans-acting factors, such as transcriptional activators, RNA polymerases, and ATP-dependent chromatin remodelers can either evict or reposition nucleosomes. Nucleosomes immediately adjacent to nucleosome-depleted regions (NDRs) are generally well positioned, but nucleosome position shows more cell-to-cell variability with increasing distance from NDRs (Yuan et al. 2005;Mavrich et al. 2008). This has led to proposal of the statistical positioning model, which suggests that strongly positioned nucleosomes create barriers against which other nucleosomes are packed into positioned and phased arrays (Kornberg and Stryer 1988;Zhang et al. 2011).Nucleosomes block the access of proteins to DNA and thus chromatin structure can modulate DNA-dependent processes such as transcription, replication, recombination, and DNA repair. Much of our insight into the role of chromatin in regulating the access of cellular machinery to DNA was driven by work with the budding yeast, Saccharomyces cerevisiae. Genetic analyses in this organism have revealed the roles played by histones and multiprotein complexes in regulating DNA-dependent processes (Rando and Winston 2012). However, although histones are among the most well-conserved proteins known, noted differences do exist between yeast and metazoan histones. First, although histone H4 is 92% conserved between S. cerevisiae and humans, H2A and H2B are less so (77 and 73% identity, respectively), which is su...