Inherent Signals in Sequencing-Based Chromatin-ImmunoPrecipitation Control Libraries

The growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.First, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.We found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights

Mapping Dynamic Histone Acetylation Patterns to Gene Expression in Nanog-depleted Murine Embryonic Stem Cells

Embryonic stem cells (ESC) have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac) on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in an concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.Comment: accepted at PLoS Computational Biolog

The Insulator Binding Protein CTCF Positions 20 Nucleosomes around Its Binding Sites across the Human Genome

Chromatin structure plays an important role in modulating the accessibility of genomic DNA to regulatory proteins in eukaryotic cells. We performed an integrative analysis on dozens of recent datasets generated by deep-sequencing and high-density tiling arrays, and we discovered an array of well-positioned nucleosomes flanking sites occupied by the insulator binding protein CTCF across the human genome. These nucleosomes are highly enriched for the histone variant H2A.Z and 11 histone modifications. The distances between the center positions of the neighboring nucleosomes are largely invariant, and we estimate them to be 185 bp on average. Surprisingly, subsets of nucleosomes that are enriched in different histone modifications vary greatly in the lengths of DNA protected from micrococcal nuclease cleavage (106–164 bp). The nucleosomes enriched in those histone modifications previously implicated to be correlated with active transcription tend to contain less protected DNA, indicating that these modifications are correlated with greater DNA accessibility. Another striking result obtained from our analysis is that nucleosomes flanking CTCF sites are much better positioned than those downstream of transcription start sites, the only genomic feature previously known to position nucleosomes genome-wide. This nucleosome-positioning phenomenon is not observed for other transcriptional factors for which we had genome-wide binding data. We suggest that binding of CTCF provides an anchor point for positioning nucleosomes, and chromatin remodeling is an important component of CTCF function

Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis

SICTIN: Rapid footprinting of massively parallel sequencing data

BACKGROUND: Massively parallel sequencing allows for genome-wide hypothesis-free investigation of for instance transcription factor binding sites or histone modifications. Although nucleotide resolution detailed information can easily be generated, biological insight often requires a more general view of patterns (footprints) over distinct genomic features such as transcription start sites, exons or repetitive regions. The construction of these footprints is however a time consuming task. METHODS: The presented software generates a binary representation of the signals enabling fast and scalable lookup. This representation allows for footprint generation in mere minutes on a desktop computer. Several different input formats are accepted, e.g. the SAM format, bed-files and the UCSC wiggle track. CONCLUSIONS: Hypothesis-free investigation of genome wide interactions allows for biological data mining at a scale never before seen. Until recently, the main focus of analysis of sequencing data has been targeted on signal patterns around transcriptional start sites which are in manageable numbers. Today, focus is shifting to a wider perspective and numerous genomic features are being studied. To this end, we provide a system allowing for fast querying in the order of hundreds of thousands of features

Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA–protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods

Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al

Bivalent-Like Chromatin Markers Are Predictive for Transcription Start Site Distribution in Human

Deep sequencing of 5′ capped transcripts has revealed a variety of transcription initiation patterns, from narrow, focused promoters to wide, broad promoters. Attempts have already been made to model empirically classified patterns, but virtually no quantitative models for transcription initiation have been reported. Even though both genetic and epigenetic elements have been associated with such patterns, the organization of regulatory elements is largely unknown. Here, linear regression models were derived from a pool of regulatory elements, including genomic DNA features, nucleosome organization, and histone modifications, to predict the distribution of transcription start sites (TSS). Importantly, models including both active and repressive histone modification markers, e.g. H3K4me3 and H4K20me1, were consistently found to be much more predictive than models with only single-type histone modification markers, indicating the possibility of “bivalent-like” epigenetic control of transcription initiation. The nucleosome positions are proposed to be coded in the active component of such bivalent-like histone modification markers. Finally, we demonstrated that models trained on one cell type could successfully predict TSS distribution in other cell types, suggesting that these models may have a broader application range

Epigenetic Transcriptional Regulation of the Growth Arrest-Specific gene 1 (Gas1) in Hepatic Cell Proliferation at Mononucleosomal Resolution

BACKGROUND: Gas1 (growth arrest-specific 1) gene is known to inhibit cell proliferation in a variety of models, but its possible implication in regulating quiescence in adult tissues has not been examined to date. The knowledge of how Gas1 is regulated in quiescence may contribute to understand the deregulation occurring in neoplastic diseases. METHODOLOGY/PRINCIPAL FINDINGS: Gas1 expression has been studied in quiescent murine liver and during the naturally synchronized cell proliferation after partial hepatectomy. Chromatin immunoprecipitation at nucleosomal resolution (Nuc-ChIP) has been used to carry out the study preserving the in vivo conditions. Transcription has been assessed at real time by quantifying the presence of RNA polymerase II in coding regions (RNApol-ChIP). It has been found that Gas1 is expressed not only in quiescent liver but also at the cell cycle G(1)/S transition. The latter expression peak had not been previously reported. Two nucleosomes, flanking a nucleosome-free region, are positioned close to the transcription start site. Both nucleosomes slide in going from the active to the inactive state and vice versa. Nuc-ChIP analysis of the acquisition of histone epigenetic marks show distinctive features in both active states: H3K9ac and H3K4me2 are characteristic of transcription in G(0) and H4R3me2 in G(1)/S transition. Sequential-ChIP analysis revealed that the "repressing" mark H3K9me2 colocalize with several "activating" marks at nucleosome N-1 when Gas1 is actively transcribed suggesting a greater plasticity of epigenetic marks than proposed until now. The recruitment of chromatin-remodeling or modifying complexes also displayed distinct characteristics in quiescence and the G(1)/S transition. CONCLUSIONS/SIGNIFICANCE: The finding that Gas1 is transcribed at the G(1)/S transition suggests that the gene may exert a novel function during cell proliferation. Transcription of this gene is modulated by specific "activating" and "repressing" epigenetic marks, and by chromatin remodeling and histone modifying complexes recruitment, at specific nucleosomes in Gas1 promoter