Biomedical subjects

M Mitchell Smith

Publications and source records attributed to M Mitchell Smith.

10 recordsLinked to original sources

PubMed2006

Regulation of replication licensing by acetyltransferase Hbo1.

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclin-dependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.

Acetylation↗

PubMed2005

Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae.

Apoptosis is a highly coordinated cell suicide mechanism in vertebrates. Phosphorylation of serine 14 of histone H2B, catalyzed by Mst1 kinase, has been linked to chromatin compaction during apoptosis. We extend these results to unicellular eukaryotes by demonstrating that H2B is specifically phosphorylated at serine 10 (S10) in a hydrogen peroxide-induced cell death pathway in S. cerevisiae. H2B S10A mutants are resistant to cell death elicited by H(2)O(2) while H2B S10E phospho-site mimics promote cell death and induce the "constitutive" formation of condensed chromatin. Ste20 kinase, a yeast homolog of mammalian Mst1 kinase, translocates into the nucleus in a caspase-independent fashion and directly phosphorylates H2B at S10. Conservation of targeted H2B phosphorylation and the enzyme system responsible for the process point to an ancient mechanism of chromatin remodeling that likely plays an important role in governing cellular homeostasis in a wide range of organisms.

Apoptosis↗

PubMed2003

Global and specific transcriptional repression by the histone H3 amino terminus in yeast.

The yeast CHA1 promoter is activated in the presence of serine or threonine. Activation requires the Cha4p activator, and it results in perturbation of a nucleosome that incorporates the TATA element under noninducing conditions. We show that in yeast lacking the amino terminus of histone H3, the promoter is constitutively active and the chromatin is concomitantly perturbed. This derepression occurs in the absence of elevated intracellular levels of serine or threonine and is not observed in cells lacking Rpd3p, Tup1p, or the amino terminus of histone H4. Furthermore, derepression in the absence of the H3 amino terminus requires the primary activator of this promoter, Cha4p, which we show by chromatin immunoprecipitation to be constitutively bound to the CHA1 promoter in WT yeast. Thus, the H3 amino terminus is required to prevent Cha4p from activating CHA1 in the absence of inducer. We also present results of a microarray experiment showing that the H3 amino terminus has a substantial repressive effect on a genome-wide scale.

Amino Acids↗

PubMed2003

Functional consequences of histone modifications.

Covalent modifications of the histone proteins have well-known roles in gene expression. Experiments reported during the past year have extended this paradigm to include roles for histone acetylation and phosphorylation in DNA double-strand break repair. In addition, new results now provide a definitive example of an acetylation histone code, whereas others reveal the workings of a charge patch mechanism. Finally, exciting research has identified new modifications, complex modification cascades, and functional links to DNA methylation and RNA interference pathways.

Acetylation↗

PubMed2003

Yaf9, a novel NuA4 histone acetyltransferase subunit, is required for the cellular response to spindle stress in yeast.

Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.

Acetylation↗

PubMed2002

Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair.

Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.

Acetylation↗

PubMed2002

Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1.

The budding yeast Saccharomyces cerevisiae was used as a model system to study the function of the adenovirus E1A oncoprotein. Previously we demonstrated that expression of the N-terminal 82 amino acids of E1A in yeast causes pronounced growth inhibition and specifically interferes with SWI/SNF-dependent transcriptional activation. Further genetic analysis identified the yeast transcription factor Adr1 as a high copy suppressor of E1A function. Transcriptional activation by Adr1 requires interaction with co-activator proteins Ada2 and Gcn5, components of histone acetyltransferase complexes including ADA and SAGA. Analysis of mutant alleles revealed that several components of the SAGA complex, including proteins from the Ada, Spt, and Taf classes were required for E1A-induced growth inhibition. Growth inhibition also depended on the Gcn5 histone acetyltransferase, and point mutations within the Gcn5 HAT domain rendered cells E1A-resistant. Also required was SAGA component Tra1, a homologue of the mammalian TRRAP protein which is required for c-myc and E1A induced cellular transformation. Additionally, Gcn5 protein could associate with E1A in vitro in a manner that depended on the N-terminal domain of E1A, and Tra1 protein was co-immunoprecipitated with E1A in vivo. These results indicate a strong requirement for intact SAGA complex for E1A to function in yeast, and suggest a role for SAGA-like complexes in mammalian cell transformation.

Acetyltransferases↗

PubMed2002

Centromeres and variant histones: what, where, when and why?

The CENP-A histone H3-like variants are centromere-specific histones found in all eukaryotes examined to date, from budding yeast to man. New experiments using antibodies, green fluorescent protein fusions, and epitope tags show that CENP-A replaces the major histone H3 subunits in a specialized histone octamer and that it does so with histones H4, and probably H2A and H2B. One of the classic hallmarks of chromatin molecular biology is that nucleosomes are deposited on DNA during replication in S phase. However, dramatic new results in mammalian and Drosophila cells show that CENP-A deposition is uncoupled from the replication of centromere DNA. Furthermore, genetic and phenotypic knockout experiments over the past year have demonstrated that the deposition of CENP-A at newly duplicated sister centromeres is an early step in the biogenesis of new centromeres and is required for the recruitment of other proteins to the centromere and kinetochore. In organisms with complex regional or holocentric centromeres, centromere identity was thought to be defined by the epigenetic state of centromere chromatin. Now, new experiments solidify this model and show that the epigenetic state can be spread in cis experimentally, creating a neocentromere, in a mechanism reminiscent of chromatin transcriptional silencing. Finally, a new report provides a glimpse into the potential regulation of CENP-A through specific post-translational phosphorylation, suggesting a broad level of control through histone tail modifications.

Amino Acid Sequence↗

PubMed2002

Histone variants and nucleosome deposition pathways.

In this issue of Molecular Cell, Ahmad and Henikoff show that the replication-independent pathway of chromatin assembly in vivo can discriminate between different histone variants on the basis of their primary amino acid sequences. These results have important implications for chromatin remodeling and epigenetic imprinting.

Animals↗

PubMed2001

Replication of minichromosomes in Saccharomyces cerevisiae is sensitive to histone gene copy number and strain ploidy.

We have characterized a defect in the mitotic transmission of plasmid minichromosomes in yeast strains deleted for the more highly expressed pair of histone H3 and H4 genes. Several observations indicate that an impairment in DNA replication contributes to the decrease in minichromosome stability. First, the maintenance of ARS plasmids that lack centromeres was also defective. Second, the addition of multiple ARS elements suppressed the defect in plasmid maintenance. Third, a synergistic increase in plasmid loss rate was seen when a plasmid containing an inefficient mutated ARS was tested in a strain deleted for histone genes, implying an interaction between ARS activity and the histone gene deletion. These results support the existence of a histone-dependent step in the initiation of DNA replication. We find that the stability of native chromosomes is not affected in strains deleted for histone genes. We propose that reduced histone H3 and H4 protein decreases the efficiency of initiation at ARS elements on plasmids and chromosomes, but that the presence of multiple origins on chromosomes compensates for the reduced efficiency. We find that decreased minichromosome stability is suppressed by increases in strain ploidy. The greater stability due to ploidy increases is not due to a relative increase in the expression of histone genes. We discuss models for the effect of strain ploidy on minichromosome maintenance.

Chromosome Deletion↗