Chromatin insulator elements: establishing barriers to set heterochromatin boundaries

Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates

Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper

Programmed cell death, or apoptosis, is a highly conserved cellular process that is crucial for tissue homeostasis under normal development as well as environmental stress. Misregulation of apoptosis is linked to many developmental defects and diseases such as tumour formation, autoimmune diseases and neurological disorders. In this paper, we show a novel role for the exoribonuclease Pacman/Xrn1 in regulating apoptosis. Using Drosophila wing imaginal discs as a model system, we demonstrate that a null mutation in pacman results in small imaginal discs as well as lethality during pupation. Mutant wing discs show an increase in the number of cells undergoing apoptosis, especially in the wing pouch area. Compensatory proliferation also occurs in these mutant discs, but this is insufficient to compensate for the concurrent increase in apoptosis. The phenotypic effects of the pacman null mutation are rescued by a deletion that removes one copy of each of the pro-apoptotic genes reaper, hid and grim, demonstrating that pacman acts through this pathway. The null pacman mutation also results in a significant increase in the expression of the pro-apoptotic mRNAs, hid and reaper, with this increase mostly occurring at the post-transcriptional level, suggesting that Pacman normally targets these mRNAs for degradation. Our results uncover a novel function for the conserved exoribonuclease Pacman and suggest that this exoribonuclease is important in the regulation of apoptosis in other organisms

Transcription-coupled eviction of histones H2A/H2B governs V(D)J recombination.

Initiation of V(D)J recombination critically relies on the formation of an accessible chromatin structure at recombination signal sequences (RSSs) but how this accessibility is generated is poorly understood. Immunoglobulin light-chain loci normally undergo recombination in pre-B cells. We show here that equipping (earlier) pro-B cells with the increased pre-B-cell levels of just one transcription factor, IRF4, triggers the entire cascade of events leading to premature light-chain recombination. We then used this finding to dissect the critical events that generate RSS accessibility and show that the chromatin modifications previously associated with recombination are insufficient. Instead, we establish that non-coding transcription triggers IgL RSS accessibility and find that the accessibility is transient. Transcription transiently evicts H2A/H2B dimers, releasing 35-40 bp of nucleosomal DNA, and we demonstrate that H2A/H2B loss can explain the RSS accessibility observed in vivo. We therefore propose that the transcription-mediated eviction of H2A/H2B dimers is an important mechanism that makes RSSs accessible for the initiation of recombination

Differential range and activity of various forms of the Hedgehog protein

BACKGROUND: The Hedgehog (Hh) family of secreted proteins act as extracellular messengers to control and coordinate growth and differentiation. The mechanism by which Hh protein travels across a field of cells, and results in a range of specific effects relating to the distance from the source, has been the subject of much debate. It has been suggested that the range and activity of the pathway can be linked to modifications of the Hh protein, specifically the addition of lipid groups at N- and C-terminal sites. RESULTS: Here we have addressed the potency of different forms of Hh protein by expressing these in Drosophila, where we are able to precisely establish pathway activity and range in naïve but responsive tissues. As expected, a construct that can produce all forms of Hh recapitulates endogenous signaling potencies. In comparison, expression of a form that lacks the cholesterol moiety (HhN) leads to an extended range, but the product is less effective at inducing maximal Hh responses. Expression of a point mutant that lacks the N-terminal palmitate binding site shows that the palmitoylation of Hh is absolutely required for activity in this system. CONCLUSION: We conclude that the addition of the cholesterol moiety limits the range of the protein and is required for maximal activity, while addition of palmitate is required for all activity. These findings have implications for understanding how Hedgehog proteins move, and thus their potential at influencing distant sites, and concomitantly, how modifications of the signaling protein can affect the efficacy of the response in exposed cells

The HTLV-1-encoded protein HBZ directly inhibits the acetyl transferase activity of p300/CBP

The homologous cellular coactivators p300 and CBP contain intrinsic lysine acetyl transferase (termed HAT) activity. This activity is responsible for acetylation of several sites on the histones as well as modification of transcription factors. In a previous study, we found that HBZ, encoded by the Human T-cell Leukemia Virus type 1 (HTLV-1), binds to multiple domains of p300/CBP, including the HAT domain. In this study, we found that HBZ inhibits the HAT activity of p300/CBP through the bZIP domain of the viral protein. This effect correlated with a reduction of H3K18 acetylation, a specific target of p300/CBP, in cells expressing HBZ. Interestingly, lower levels of H3K18 acetylation were detected in HTLV-1 infected cells compared to non-infected cells. The inhibitory effect of HBZ was not limited to histones, as HBZ also inhibited acetylation of the NF-κB subunit, p65, and the tumor suppressor, p53. Recent studies reported that mutations in the HAT domain of p300/CBP that cause a defect in acetylation are found in certain types of leukemia. These observations suggest that inhibition of the HAT activity by HBZ is important for the development of adult T-cell leukemia associated with HTLV-1 infection

Changes in the Histone Acetylation Patterns during the Development of the Nervous System

Epigenetic modification such as DNA methylation and histone acetylation plays essential roles in many aspects of cellular function and development of animals. There is an increasing amounts of evidence for dynamic changes in the histone acetylation of specific gene segments, but little attempt was made to examine global pattern changes in the histone acetylation in developing nervous system. In this study, we found that acetylated histone H3 and H4 immunoreactivities were relatively weak in neuroepithelial cells in the ventricular zone of developing rat cerebral cortex or chick spinal cord, compared to the immature young neurons in the cortical plate of a rat embryo or lateral motor column in chick spinal cord. On the other hand, adult neural stem cells in the dentate gyrus (DG) of rat hippocampal formation did not exhibit such diminished histone acetylation, compared to neuroblasts and mature DG neurons. These results suggest that the level of histone acetylation is highly dynamic and tightly linked to the neuronal types and the differentiation stages

Coordinated allele-specific histone acetylation at the differentially methylated regions of imprinted genes

Genomic imprinting is an epigenetic inheritance system characterized by parental allele-specific gene expression. Allele-specific DNA methylation and chromatin composition are two epigenetic modification systems that control imprinted gene expression. To get a general assessment of histone lysine acetylation at imprinted genes we measured allele-specific acetylation of a wide range of lysine residues, H3K4, H3K18, H3K27, H3K36, H3K79, H3K64, H4K5, H4K8, H4K12, H2AK5, H2BK12, H2BK16 and H2BK46 at 11 differentially methylated regions (DMRs) in reciprocal mouse crosses using multiplex chromatin immunoprecipitation SNuPE assays. Histone acetylation marks generally distinguished the methylation-free alleles from methylated alleles at DMRs in mouse embryo fibroblasts and embryos. Acetylated lysines that are typically found at transcription start sites exhibited stronger allelic bias than acetylated histone residues in general. Maternally methylated DMRs, that usually overlap with promoters exhibited higher levels of acetylation and a 10% stronger allele-specific bias than paternally methylated DMRs that reside in intergenic regions. Along the H19/Igf2 imprinted domain, allele-specific acetylation at each lysine residue depended on functional CTCF binding sites in the imprinting control region. Our results suggest that many different histone acetyltransferase and histone deacetylase enzymes must act in concert in setting up and maintaining reciprocal parental allelic histone acetylation at DMRs

Inactive X chromosome-specific histone H3 modifications and CpG hypomethylation flank a chromatin boundary between an X-inactivated and an escape gene

In mammals, the dosage compensation of sex chromosomes between males and females is achieved by transcriptional inactivation of one of the two X chromosomes in females. However, a number of genes escape X-inactivation in humans. It remains poorly understood how the transcriptional activity of these ‘escape genes’ is maintained despite the chromosome-wide heterochromatin formation. To address this question, we analyzed a putative chromatin boundary between the inactivated RBM10 and an escape gene, UBA1/UBE1. Chromatin immunoprecipitation revealed that trimethylated histone H3 lysine 9 and H4 lysine 20 were enriched in the last exon through the proximal downstream region of RBM10, but were remarkably diminished at ∼2 kb upstream of the UBA1 transcription start site. Whereas RNA polymerase II was not loaded onto the intergenic region, CTCF (CCCTC binding factor) was enriched around the boundary, where some CpG sites were hypomethylated specifically on inactive X. These findings suggest that local DNA hypomethylation and CTCF binding are involved in the formation of a chromatin boundary, which protects the UBA1 escape gene against the chromosome-wide transcriptional silencing

Acetylation increases access of remodelling complexes to their nucleosome targets to enhance initiation of V(D)J recombination

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity

Single-Nucleosome Mapping of Histone Modifications in S. cerevisiae

Covalent modification of histone proteins plays a role in virtually every process on eukaryotic DNA, from transcription to DNA repair. Many different residues can be covalently modified, and it has been suggested that these modifications occur in a great number of independent, meaningful combinations. Published low-resolution microarray studies on the combinatorial complexity of histone modification patterns suffer from confounding effects caused by the averaging of modification levels over multiple nucleosomes. To overcome this problem, we used a high-resolution tiled microarray with single-nucleosome resolution to investigate the occurrence of combinations of 12 histone modifications on thousands of nucleosomes in actively growing S. cerevisiae. We found that histone modifications do not occur independently; there are roughly two groups of co-occurring modifications. One group of lysine acetylations shows a sharply defined domain of two hypo-acetylated nucleosomes, adjacent to the transcriptional start site, whose occurrence does not correlate with transcription levels. The other group consists of modifications occurring in gradients through the coding regions of genes in a pattern associated with transcription. We found no evidence for a deterministic code of many discrete states, but instead we saw blended, continuous patterns that distinguish nucleosomes at one location (e.g., promoter nucleosomes) from those at another location (e.g., over the 3′ ends of coding regions). These results are consistent with the idea of a simple, redundant histone code, in which multiple modifications share the same role