Human tRNA genes function as chromatin insulators

Insulators help separate active chromatin domains from silenced ones. In yeast, gene promoters act as insulators to block the spread of Sir and HP1 mediated silencing while in metazoans most insulators are multipartite autonomous entities. tDNAs are repetitive sequences dispersed throughout the human genome and we now show that some of these tDNAs can function as insulators in human cells. Using computational methods, we identified putative human tDNA insulators. Using silencer blocking, transgene protection and repressor blocking assays we show that some of these tDNA-containing fragments can function as barrier insulators in human cells. We find that these elements also have the ability to block enhancers from activating RNA pol II transcribed promoters. Characterization of a putative tDNA insulator in human cells reveals that the site possesses chromatin signatures similar to those observed at other better-characterized eukaryotic insulators. Enhanced 4C analysis demonstrates that the tDNA insulator makes long-range chromatin contacts with other tDNAs and ETC sites but not with intervening or flanking RNA pol II transcribed genes.     

Eaf1 is the platform for NuA4 molecular assembly that evolutionarily links chromatin acetylation to ATP-dependent exchange of histone H2A variants

Eaf1 (for Esa1-associated factor 1) and Eaf2 have been identified as stable subunits of NuA4, a yeast histone H4/H2A acetyltransferase complex implicated in gene regulation and DNA repair. While both SWI3-ADA2-N-CoR-TF IIIB domain-containing proteins are required for normal cell cycle progression, their depletion does not affect the global Esa1-dependent acetylation of histones. In contrast to all other subunits, Eaf1 is found exclusively associated with the NuA4 complex in vivo. It serves as a platform that coordinates the assembly of functional groups of subunits into the native NuA4 complex. Eaf1 shows structural similarities with human p400/Domino, a subunit of the NuA4-related TIP60 complex. On the other hand, p400 also possesses an SWI2/SNF2 family ATPase domain that is absent from the yeast NuA4 complex. This domain is highly related to the yeast Swr1 protein, which is responsible for the incorporation of histone variant H2AZ in chromatin. Since all of the components of the TIP60 complex are homologous to SWR1 or NuA4 subunits, we proposed that the human complex corresponds to a physical merge of two yeast complexes. p400 function in TIP60 then would be accomplished in yeast by cooperation between SWR1 and NuA4. In agreement with such a model, NuA4 and SWR1 mutants show strong genetic interactions, NuA4 affects histone H2AZ incorporation/acetylation in vivo, and both preset the PHO5 promoter for activation. Interestingly, the expression of a chimeric Eaf1-Swr1 protein recreates a single human-like complex in yeast cells. Our results identified the key central subunit for the structure and functions of the NuA4 histone acetyltransferase complex and functionally linked this activity with the histone variant H2AZ from yeast to human cells.     

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.     

Ordered nucleation and spreading of silenced chromatin in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, silencing at the HM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region at HMR. Sir1p, in contrast, associated only with the HMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading across HMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.     

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.     

Ten members of the Arabidopsis gene family encoding methyl-CpG-binding domain proteins are transcriptionally active and at least one,AtMBD11, is crucial for normal development

Animal proteins that contain a methyl-CpG-binding domain (MBD) are suggested to provide a link between DNA methylation, chromatin remodelling and gene silencing. However, some MBD proteins reside in chromatin remodelling complexes, but do not have specific affinity for methylated DNA. It has recently been shown that the Arabidopsis genome contains 12 putative genes encoding proteins with domains similar to MBD, of which at least three bind symmetrically methylated DNA. Using a bioinformatics approach, we have identified additional domains in a number of these proteins and, on this basis and extended sequence similarity, divided the proteins into subgroups. Using RT-PCR we show that 10 of the AtMBD genes are active and differentially expressed in diverse tissues. To investigate the biological significance of AtMBD proteins, we have transformed Arabidopsis with a construct aimed at RNA interference with expression of the AtMBD11 gene, normally active in most tissues. The resulting 35S::AtMBD11-RNAi plants displayed a variety of phenotypic effects, including aerial rosettes, serrated leaves, abnormal position of flowers, fertility problems and late flowering. Arabidopsis lines with reduced expression of genes involved in chromatin remodelling and transgene silencing show similar phenotypes. Our results suggest an important role for AtMBD proteins in plant development.     

Insulators: exploiting transcriptional and epigenetic mechanisms

Insulators are DNA sequence elements that prevent inappropriate interactions between adjacent chromatin domains. One type of insulator establishes domains that separate enhancers and promoters to block their interaction, whereas a second type creates a barrier against the spread of heterochromatin. Recent studies have provided important advances in our understanding of the modes of action of both types of insulator. These new insights also suggest that the mechanisms of action of both enhancer blockers and barriers might not be unique to these types of element, but instead are adaptations of other gene-regulatory mechanisms.     

PRE-mediated bypass of two Su(Hw) insulators targets PcG proteins to a downstream promoter

Drosophila Polycomb group response elements (PRE) silence neighboring genes, but silencing can be blocked by one copy of the Su(Hw) insulator element. We show here that Polycomb group (PcG) proteins can spread from a PRE in the flanking chromatin region and that PRE blocking depends on a physical barrier established by the insulator to PcG protein spreading. On the other hand, PRE-mediated silencing can bypass two Su(Hw) insulators to repress a downstream reporter gene. Strikingly, insulator bypass involves targeting of PcG proteins to the downstream promoter, while they are completely excluded from the intervening insulated domain. This shows that PRE-dependent silencing is compatible with looping of the PRE in order to bring PcG proteins in contact with the promoter and does not require the coating of the whole chromatin domain between PRE and promoter.