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

Detection and Removal of Biases in the Analysis of Next-Generation Sequencing Reads

Since the emergence of next-generation sequencing (NGS) technologies, great effort has been put into the development of tools for analysis of the short reads. In parallel, knowledge is increasing regarding biases inherent in these technologies. Here we discuss four different biases we encountered while analyzing various Illumina datasets. These biases are due to both biological and statistical effects that in particular affect comparisons between different genomic regions. Specifically, we encountered biases pertaining to the distributions of nucleotides across sequencing cycles, to mappability, to contamination of pre-mRNA with mRNA, and to non-uniform hydrolysis of RNA. Most of these biases are not specific to one analyzed dataset, but are present across a variety of datasets and within a variety of genomic contexts. Importantly, some of these biases correlated in a highly significant manner with biological features, including transcript length, gene expression levels, conservation levels, and exon-intron architecture, misleadingly increasing the credibility of results due to them. We also demonstrate the relevance of these biases in the context of analyzing an NGS dataset mapping transcriptionally engaged RNA polymerase II (RNAPII) in the context of exon-intron architecture, and show that elimination of these biases is crucial for avoiding erroneous interpretation of the data. Collectively, our results highlight several important pitfalls, challenges and approaches in the analysis of NGS reads

A High-Resolution Whole-Genome Map of Key Chromatin Modifications in the Adult Drosophila melanogaster

Epigenetic research has been focused on cell-type-specific regulation; less is known about common features of epigenetic programming shared by diverse cell types within an organism. Here, we report a modified method for chromatin immunoprecipitation and deep sequencing (ChIP–Seq) and its use to construct a high-resolution map of the Drosophila melanogaster key histone marks, heterochromatin protein 1a (HP1a) and RNA polymerase II (polII). These factors are mapped at 50-bp resolution genome-wide and at 5-bp resolution for regulatory sequences of genes, which reveals fundamental features of chromatin modification landscape shared by major adult Drosophila cell types: the enrichment of both heterochromatic and euchromatic marks in transposons and repetitive sequences, the accumulation of HP1a at transcription start sites with stalled polII, the signatures of histone code and polII level/position around the transcriptional start sites that predict both the mRNA level and functionality of genes, and the enrichment of elongating polII within exons at splicing junctions. These features, likely conserved among diverse epigenomes, reveal general strategies for chromatin modifications

Histone Deacetylase Activity Modulates Alternative Splicing

There is increasing evidence to suggest that splicing decisions are largely made when the nascent RNA is still associated with chromatin. Here we demonstrate that activity of histone deacetylases (HDACs) influences splice site selection. Using splicing-sensitive microarrays, we identified ∼700 genes whose splicing was altered after HDAC inhibition. We provided evidence that HDAC inhibition induced histone H4 acetylation and increased RNA Polymerase II (Pol II) processivity along an alternatively spliced element. In addition, HDAC inhibition reduced co-transcriptional association of the splicing regulator SRp40 with the target fibronectin exon. We further showed that the depletion of HDAC1 had similar effect on fibronectin alternative splicing as global HDAC inhibition. Importantly, this effect was reversed upon expression of mouse HDAC1 but not a catalytically inactive mutant. These results provide a molecular insight into a complex modulation of splicing by HDACs and chromatin modifications

Evidence for control of splicing by alternative RNA secondary structures in Dipteran homothorax pre-mRNA

In a recent study that identified highly evolutionary conserved sequences in three genomes of Diptera species we described an ultraconserved element found at an internal exon-intron junction of the Drosophila melanogaster homothorax (hth) gene that appeared to be involved in the control of hth pre-mRNA splicing. We also discussed a possible role of RNA secondary structure at this site in the regulation of hth pre-mRNA splicing. In this report we identify a shorter evolutionary conserved intronic element within the hth gene that is located downstream of the first element and has sequence complementarity to it. We demonstrate that intramolecular interactions between these two elements would give rise to alternative RNA secondary structures, which in turn may result in differential control of homothorax pre-mRNA splicing. We also provide additional comparative genomic data from several newly available insect genomes supporting our original conclusion that these conserved elements are important in the post-transcriptional regulation of homothorax gene expression in Diptera

Nucleosomes are preferentially positioned at exons in somatic and sperm cells

Nucleosome positioning is constrained at eukaryotic transcription start sites and implicated in transcriptional regulation. Moreover, recent observations indicate that chromatin structure, transcription and splicing are functionally intertwined, and that modified nucleosomes with trimethylation of lysine 36 in histone subunit 3 (H3K36me3) are enriched at internal exons and the downstream flanking intronic regions of highly expressed genes. However, the position of nucleosomes in the interior of genes has been thought to be largely random. Here we show, by analysis of data sets from human sperm and T cells and medaka (Japanese killifish, Oryzias latipes) blastulae, that internal exons of genes are characterized by sharply elevated average nucleosome occupancy in comparison to flanking intronic sequences. We also show that the preferential positioning of nucleosomes at internal exons is independent of their modification status, and of the GC content, conservation or the expression level of the exon. These findings show that the location of exons is recorded in the chromatin structure and may be inherited across generations. Such embedded information may underpin transcriptionally coupled exon recognition and splice site selection

Molecular evolution of the HBII-52 snoRNA Cluster

HBII-52 is a human brain-specific C/D box snoRNA that potentially regulates the editing and/or alternative splicing of the serotonin receptor. Forty-two nearly identical copies of the HBII-52 gene are located immediately downstream of the SNRPN protein-coding gene in an imprinted locus associated with Prader-Willi syndrome

Topological data analysis of task-based fMRI data from experiments on schizophrenia

We use methods from computational algebraic topology to study functional brain networks in which nodes represent brain regions and weighted edges encode the similarity of functional magnetic resonance imaging (fMRI) time series from each region. With these tools, which allow one to characterize topological invariants such as loops in high-dimensional data, we are able to gain understanding of low-dimensional structures in networks in a way that complements traditional approaches that are based on pairwise interactions. In the present paper, we use persistent homology to analyze networks that we construct from task-based fMRI data from schizophrenia patients, healthy controls, and healthy siblings of schizophrenia patients. We thereby explore the persistence of topological structures such as loops at different scales in these networks. We use persistence landscapes and persistence images to represent the output of our persistent-homology calculations, and we study the persistence landscapes and persistence images using k-means clustering and community detection. Based on our analysis of persistence landscapes, we find that the members of the sibling cohort have topological features (specifically, their one-dimensional loops) that are distinct from the other two cohorts. From the persistence images, we are able to distinguish all three subject groups and to determine the brain regions in the loops (with four or more edges) that allow us to make these distinctions