tRNA genes as chromatin barriers.
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Chromatin↗
Biomedical subjects
Rohinton T Kamakaka
Publications and source records attributed to Rohinton T Kamakaka.
H2A.Z functions to regulate progression through the cell cycle.
Histone H2A variants are highly conserved proteins found ubiquitously in nature and thought to perform specialized functions in the cell. Studies in yeast on the histone H2A variant H2A.Z have shown a role for this protein in transcription as well as chromosome segregation. Our studies have focused on understanding the role of H2A.Z during cell cycle progression. We found that htz1delta cells were delayed in DNA replication and progression through the cell cycle. Furthermore, cells lacking H2A.Z required the S-phase checkpoint pathway for survival. We also found that H2A.Z localized to the promoters of cyclin genes, and cells lacking H2A.Z were delayed in the induction of these cyclin genes. Several different models are proposed to explain these observations.
Cell Cycle↗
Chromatin insulators.
Active and silenced chromatin domains are often in close juxtaposition to one another, and enhancer and silencer elements operate over large distances to regulate the genes in these domains. The lack of promiscuity in the function of these elements suggests that active mechanisms exist to restrict their activity. Insulators are DNA elements that restrict the effects of long-range regulatory elements. Studies on different insulators from different organisms have identified common themes in their mode of action. Numerous insulators map to promoters of genes or have binding sites for transcription factors and like active chromatin hubs and silenced loci, insulators also cluster in the nucleus. These results bring into focus potential conserved mechanisms by which these elements might function in the nucleus.
Animals↗
Analyses of SUM1-1-mediated long-range repression.
In Saccharomyces cerevisiae, local repression is promoter specific and localized to a small region on the DNA, while silencing is promoter nonspecific, encompasses large domains of chromatin, and is stably inherited for multiple generations. Sum1p is a local repressor protein that mediates repression of meiosis-specific genes in mitotic cells while the Sir proteins are long-range repressors that stably silence genes at HML, HMR, and telomeres. The SUM1-1 mutation is a dominant neomorphic mutation that enables the mutant protein to be recruited to the HMR locus and repress genes, even in the absence of the Sir proteins. In this study we show that the mutation in Sum1-1p enabled it to spread, and the native HMR barrier blocked it from spreading. Thus, like the Sir proteins, Sum1-1p was a long-range repressor, but unlike the Sir proteins, Sum1-1p-mediated repression was more promoter specific, repressing certain genes better than others. Furthermore, repression mediated by Sum1-1p was not stably maintained or inherited and we therefore propose that Sum1-1p-mediated long-range repression is related but distinct from silencing.
Binding Sites↗
Barrier function at HMR.
The silenced HMR domain is restricted from spreading by barrier elements, and the right barrier is a unique t-RNA(THR) gene. We show that sequences immediately flanking the silenced domain were enriched in acetylated, but not methylated, histones, whereas the barrier element was associated with a nucleosome-free region. Surprisingly, the SAGA acetyltransferase resided across the entire region. We further demonstrate that a mutation in the barrier eliminated the nucleosome-free gap but only subtly altered the distribution of SAGA. Interestingly, neither reformation of the nucleosome nor mutations in chromatin-modifying enzymes alone led to an unrestricted spread of silenced chromatin. Double mutations in the t-RNA barrier and these complexes, on the other hand, led to a significant spread of Sir proteins. These results suggest two overlapping mechanisms function to restrict the spread of silencing: one of which involves a DNA binding element, whereas the other mechanism involves specific chromatin-modifying activities.
Chromatin↗
Histone variants: deviants?
Histones are a major component of chromatin, the protein-DNA complex fundamental to genome packaging, function, and regulation. A fraction of histones are nonallelic variants that have specific expression, localization, and species-distribution patterns. Here we discuss recent progress in understanding how histone variants lead to changes in chromatin structure and dynamics to carry out specific functions. In addition, we review histone variant assembly into chromatin, the structure of the variant chromatin, and post-translational modifications that occur on the variants.
Animals↗
Purification of Sir2 proteins from yeast.
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Chromatography, Affinity↗
Barrier proteins remodel and modify chromatin to restrict silenced domains.
Transcriptionally active and inactive domains are frequently found adjacent to one another in the eukaryotic nucleus. To better understand the underlying mechanisms by which domains maintain opposing transcription patterns, we performed a systematic genomewide screen for proteins that may block the spread of silencing in yeast. This analysis identified numerous proteins with efficient silencing blocking activities, and some of these have previously been shown to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID, as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes, as barrier proteins. We demonstrate that histone acetylation and chromatin remodeling occurred at the barrier and correlated with a block to the spread of silencing. Our data suggest that multiple overlapping mechanisms were involved in delimiting silenced and active domains in vivo.
Acetylation↗
Heterochromatin: proteins in flux lead to stable repression.
Heterochromatin is a phenotypically stable entity, but recent studies on the binding of HP1 protein in heterochromatin indicate that the individual components within these domains are not stably bound but in constant flux. These results force us to reexamine previous models of heterochromatin.
Chromosomal Proteins, Non-Histone↗
Chromatin: a connection between loops and barriers?
A genetic screen for proteins that can block the spread of silenced heterochromatin has identified components of the nuclear pores with potential barrier activity. These results suggest that formation of loops of chromatin anchored to the pore could be one mechanism of barrier function.
Chromatin↗
Braking the silence: how heterochromatic gene repression is stopped in its tracks.
Eukaryotic DNA is assembled into nucleosomes, which are further packaged into higher order chromatin structures containing many non-histone chromosomal proteins. The details of this packaging have profound effects on gene expression and other cellular processes involving the genetic material. Heterochromatic domains of the genome are usually transcriptionally repressed, while euchromatic regions are transcriptionally competent. Current models of gene activation postulate the existence of boundary elements that either prevent inappropriate activation of genes by distal enhancers (enhancer blockers), or sequences that block the propagation of heterochromatin into euchromatic regions (barriers). While numerous boundary sequences have been identified, little is known with regard to the molecular mechanisms used to punctuate the genome. This review will focus on recent data that provide insight into the mode of action of barrier elements.
Animals↗
Blockers and barriers to transcription: competing activities?
In the eukaryotic cell active and inactive genes reside adjacent to one another and are modulated by numerous regulatory elements. Insulator elements prevent the misregulation of adjacent genes by restricting the effects of the regulatory elements to specific domains. Enhancer blockers prevent enhancers from inadvertently activating neighboring genes, and recent results suggest that they might function by a conserved mechanism across species. These elements appear to disrupt enhancer-promoter "communications" by interacting with the regulatory elements and sequestering these elements into specific regions of the nucleus thus rendering them non-functional. Barrier elements insulate active genes from neighboring heterochromatin and recent results suggest that they function by specific localized recruitment of acetyltransferases that antagonize the spread of heterochromatin-associated deacetylases, thus preventing the propagation of heterochromatin.
Animals↗
Breaking through to the other side: silencers and barriers.
The establishment and restriction of transcriptionally inactive regions in the nucleus is mediated by silencer and barrier elements. Silencer-bound proteins recruit additional factors to establish the silenced domain during the S-phase of the cell cycle but, contrary to previous models, DNA replication is not a pre-requisite for the establishment. Characteristically, silenced domains contain hypoacetylated histones and recent data have identified residue-specific methylation of histone H3 as an additional signature that distinguishes active regions from inactive ones. Peaks of acetylated histones demarcate the boundaries between these regions and recruitment of HAT activities provides a mechanism to restrict the spread of heterochromatin.
Gene Silencing↗