Complex exon-intron marking by histone modifications is not determined solely by nucleosome distribution

It has recently been shown that nucleosome distribution, histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (“exon-intron marking”), linking chromatin structure and function to co-transcriptional splicing in a variety of eukaryotes. Previous ChIP-sequencing studies suggested that these marking patterns reflect the nucleosomal landscape. By analyzing ChIP-chip datasets across the human genome in three cell types, we have found that this marking system is far more complex than previously observed. We show here that a range of histone modifications and Pol II are preferentially associated with exons. However, there is noticeable cell-type specificity in the degree of exon marking by histone modifications and, surprisingly, this is also reflected in some histone modifications patterns showing biases towards introns. Exon-intron marking is laid down in the absence of transcription on silent genes, with some marking biases changing or becoming reversed for genes expressed at different levels. Furthermore, the relationship of this marking system with splicing is not simple, with only some histone modifications reflecting exon usage/inclusion, while others mirror patterns of exon exclusion. By examining nucleosomal distributions in all three cell types, we demonstrate that these histone modification patterns cannot solely be accounted for by differences in nucleosome levels between exons and introns. In addition, because of inherent differences between ChIP-chip array and ChIP-sequencing approaches, these platforms report different nucleosome distribution patterns across the human genome. Our findings confound existing views and point to active cellular mechanisms which dynamically regulate histone modification levels and account for exon-intron marking. We believe that these histone modification patterns provide links between chromatin accessibility, Pol II movement and co-transcriptional splicing

Relationship between Gene Body DNA Methylation and Intragenic H3K9me3 and H3K36me3 Chromatin Marks

To elucidate the relationship between intragenic DNA methylation and chromatin marks, we performed epigenetic profiling of chromosome 19 in human bronchial epithelial cells (HBEC) and in the colorectal cancer cell line HCT116 as well as its counterpart with double knockout of DNMT1 and DNMT3B (HCT116-DKO). Analysis of H3K36me3 profiles indicated that this intragenic mark of active genes is associated with two categories of genes: (i) genes with low CpG density and H3K9me3 in the gene body or (ii) genes with high CpG density and DNA methylation in the gene body. We observed that a combination of low CpG density in gene bodies together with H3K9me3 and H3K36me3 occupancy is a specific epigenetic feature of zinc finger (ZNF) genes, which comprise 90% of all genes carrying both histone marks on chromosome 19. For genes with high intragenic CpG density, transcription and H3K36me3 occupancy were not changed in conditions of partial or intensive loss of DNA methylation in gene bodies. siRNA knockdown of SETD2, the major histone methyltransferase responsible for production of H3K36me3, did not reduce DNA methylation in gene bodies. Our study suggests that the H3K36me3 and DNA methylation marks in gene bodies are established largely independently of each other and points to similar functional roles of intragenic DNA methylation and intragenic H3K9me3 for CpG-rich and CpG-poor genes, respectively

ChromaSig: A Probabilistic Approach to Finding Common Chromatin Signatures in the Human Genome

Computational methods to identify functional genomic elements using genetic information have been very successful in determining gene structure and in identifying a handful of cis-regulatory elements. But the vast majority of regulatory elements have yet to be discovered, and it has become increasingly apparent that their discovery will not come from using genetic information alone. Recently, high-throughput technologies have enabled the creation of information-rich epigenetic maps, most notably for histone modifications. However, tools that search for functional elements using this epigenetic information have been lacking. Here, we describe an unsupervised learning method called ChromaSig to find, in an unbiased fashion, commonly occurring chromatin signatures in both tiling microarray and sequencing data. Applying this algorithm to nine chromatin marks across a 1% sampling of the human genome in HeLa cells, we recover eight clusters of distinct chromatin signatures, five of which correspond to known patterns associated with transcriptional promoters and enhancers. Interestingly, we observe that the distinct chromatin signatures found at enhancers mark distinct functional classes of enhancers in terms of transcription factor and coactivator binding. In addition, we identify three clusters of novel chromatin signatures that contain evolutionarily conserved sequences and potential cis-regulatory elements. Applying ChromaSig to a panel of 21 chromatin marks mapped genomewide by ChIP-Seq reveals 16 classes of genomic elements marked by distinct chromatin signatures. Interestingly, four classes containing enrichment for repressive histone modifications appear to be locally heterochromatic sites and are enriched in quickly evolving regions of the genome. The utility of this approach in uncovering novel, functionally significant genomic elements will aid future efforts of genome annotation via chromatin modifications

NOF1 Encodes an Arabidopsis Protein Involved in the Control of rRNA Expression

The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes

Genic and Global Functions for Paf1C in Chromatin Modification and Gene Expression in Arabidopsis

In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 3′ shift in the distribution of H3K4me3 and a 5′ shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory

DNA Methylation of the First Exon Is Tightly Linked to Transcriptional Silencing

Tissue specific patterns of methylated cytosine residues vary with age, can be altered by environmental factors, and are often abnormal in human disease yet the cellular consequences of DNA methylation are incompletely understood. Although the bodies of highly expressed genes are often extensively methylated in plants, the relationship between intragenic methylation and expression is less clear in mammalian cells. We performed genome-wide analyses of DNA methylation and gene expression to determine how the pattern of intragenic methylation correlates with transcription and to assess the relationship between methylation of exonic and intronic portions of the gene body. We found that dense exonic methylation is far more common than previously recognized or expected statistically, yet first exons are relatively spared compared to more downstream exons and introns. Dense methylation surrounding the transcription start site (TSS) is uncoupled from methylation within more downstream regions suggesting that there are at least two classes of intragenic methylation. Whereas methylation surrounding the TSS is tightly linked to transcriptional silencing, methylation of more downstream regions is unassociated with the magnitude of gene expression. Notably, we found that DNA methylation downstream of the TSS, in the region of the first exon, is much more tightly linked to transcriptional silencing than is methylation in the upstream promoter region. These data provide direct evidence that DNA methylation is interpreted dissimilarly in different regions of the gene body and suggest that first exon methylation blocks transcript initiation, or vice versa. Our data also show that once initiated, downstream methylation is not a significant impediment to polymerase extension. Thus, the consequences of most intragenic DNA methylation must extend beyond the modulation of transcription magnitude

Transcription and Chromatin Organization of a Housekeeping Gene Cluster Containing an Integrated β-Globin Locus Control Region

The activity of locus control regions (LCR) has been correlated with chromatin decondensation, spreading of active chromatin marks, locus repositioning away from its chromosome territory (CT), increased association with transcription factories, and long-range interactions via chromatin looping. To investigate the relative importance of these events in the regulation of gene expression, we targeted the human β-globin LCR in two opposite orientations to a gene-dense region in the mouse genome containing mostly housekeeping genes. We found that each oppositely oriented LCR influenced gene expression on both sides of the integration site and over a maximum distance of 150 kilobases. A subset of genes was transcriptionally enhanced, some of which in an LCR orientation-dependent manner. The locus resides mostly at the edge of its CT and integration of the LCR in either orientation caused a more frequent positioning of the locus away from its CT. Locus association with transcription factories increased moderately, both for loci at the edge and outside of the CT. These results show that nuclear repositioning is not sufficient to increase transcription of any given gene in this region. We identified long-range interactions between the LCR and two upregulated genes and propose that LCR-gene contacts via chromatin looping determine which genes are transcriptionally enhanced

SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State

SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs) and multipotent neural progenitor cells (NPCs); however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1) in ESCs, the related POU family member BRN2 (Pou3f2) co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome

Chromosomal organization at the level of gene complexes

Metazoan genomes primarily consist of non-coding DNA in comparison to coding regions. Non-coding fraction of the genome contains cis-regulatory elements, which ensure that the genetic code is read properly at the right time and space during development. Regulatory elements and their target genes define functional landscapes within the genome, and some developmentally important genes evolve by keeping the genes involved in specification of common organs/tissues in clusters and are termed gene complex. The clustering of genes involved in a common function may help in robust spatio-temporal gene expression. Gene complexes are often found to be evolutionarily conserved, and the classic example is the hox complex. The evolutionary constraints seen among gene complexes provide an ideal model system to understand cis and trans-regulation of gene function. This review will discuss the various characteristics of gene regulatory modules found within gene complexes and how they can be characterized