EpiChIP: gene-by-gene quantification of epigenetic modification levels

The combination of chromatin immunoprecipitation with next-generation sequencing technology (ChIP-seq) is a powerful and increasingly popular method for mapping protein–DNA interactions in a genome-wide fashion. The conventional way of analyzing this data is to identify sequencing peaks along the chromosomes that are significantly higher than the read background. For histone modifications and other epigenetic marks, it is often preferable to find a characteristic region of enrichment in sequencing reads relative to gene annotations. For instance, many histone modifications are typically enriched around transcription start sites. Calculating the optimal window that describes this enrichment allows one to quantify modification levels for each individual gene. Using data sets for the H3K9/14ac histone modification in Th cells and an accompanying IgG control, we present an analysis strategy that alternates between single gene and global data distribution levels and allows a clear distinction between experimental background and signal. Curve fitting permits false discovery rate-based classification of genes as modified versus unmodified. We have developed a software package called EpiChIP that carries out this type of analysis, including integration with and visualization of gene expression data

Strengthening global-change science by integrating aeDNA with paleoecoinformatics

Ancient environmental DNA (aeDNA) data are close to enabling insights into past global-scale biodiversity dynamics at unprecedented taxonomic extent and resolution. However, achieving this potential requires solutions that bridge bioinformatics and paleoecoinformatics. Essential needs include support for dynamic taxonomic inferences, dynamic age inferences, and precise stratigraphic depth. Moreover, aeDNA data are complex and heterogeneous, generated by dispersed researcher networks, with methods advancing rapidly. Hence, expert community governance and curation are essential to building high-value data resources. Immediate recommendations include uploading metabarcoding-based taxonomic inventories into paleoecoinformatic resources, building linkages among open bioinformatic and paleoecoinformatic data resources, harmonizing aeDNA processing workflows, and expanding community data governance. These advances will enable transformative insights into global-scale biodiversity dynamics during large environmental and anthropogenic changes

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

Assessing Computational Methods of Cis-Regulatory Module Prediction

Computational methods attempting to identify instances of cis-regulatory modules (CRMs) in the genome face a challenging problem of searching for potentially interacting transcription factor binding sites while knowledge of the specific interactions involved remains limited. Without a comprehensive comparison of their performance, the reliability and accuracy of these tools remains unclear. Faced with a large number of different tools that address this problem, we summarized and categorized them based on search strategy and input data requirements. Twelve representative methods were chosen and applied to predict CRMs from the Drosophila CRM database REDfly, and across the human ENCODE regions. Our results show that the optimal choice of method varies depending on species and composition of the sequences in question. When discriminating CRMs from non-coding regions, those methods considering evolutionary conservation have a stronger predictive power than methods designed to be run on a single genome. Different CRM representations and search strategies rely on different CRM properties, and different methods can complement one another. For example, some favour homotypical clusters of binding sites, while others perform best on short CRMs. Furthermore, most methods appear to be sensitive to the composition and structure of the genome to which they are applied. We analyze the principal features that distinguish the methods that performed well, identify weaknesses leading to poor performance, and provide a guide for users. We also propose key considerations for the development and evaluation of future CRM-prediction methods

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

Long-range DNA looping and gene expression analyses identify DEXI as an autoimmune disease candidate gene

The chromosome 16p13 region has been associated with several autoimmune diseases, including type 1 diabetes (T1D) and multiple sclerosis (MS). CLEC16A has been reported as the most likely candidate gene in the region, since it contains the most disease-associated single-nucleotide polymorphisms (SNPs), as well as an imunoreceptor tyrosine-based activation motif. However, here we report that intron 19 of CLEC16A, containing the most autoimmune disease-associated SNPs, appears to behave as a regulatory sequence, affecting the expression of a neighbouring gene, DEXI. The CLEC16A alleles that are protective from T1D and MS are associated with increased expression of DEXI, and no other genes in the region, in two independent monocyte gene expression data sets. Critically, using chromosome conformation capture (3C), we identified physical proximity between the DEXI promoter region and intron 19 of CLEC16A, separated by a loop of >150 kb. In reciprocal experiments, a 20 kb fragment of intron 19 of CLEC16A, containing SNPs associated with T1D and MS, as well as with DEXI expression, interacted with the promotor region of DEXI but not with candidate DNA fragments containing other potential causal genes in the region, including CLEC16A. Intron 19 of CLEC16A is highly enriched for transcription-factor-binding events and markers associated with enhancer activity. Taken together, these data indicate that although the causal variants in the 16p13 region lie within CLEC16A, DEXI is an unappreciated autoimmune disease candidate gene, and illustrate the power of the 3C approach in progressing from genome-wide association studies results to candidate causal genes

EphA4 targeting agents protect motor neurons from cell death induced by amyotrophic lateral sclerosis -astrocytes.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease that progressively destroys motor neurons (MNs). Earlier studies identified EphA4, a receptor tyrosine kinase, as a possible disease-modifying gene. The complex interplay between the EphA4 receptor and its ephrin ligands in motor neurons and astrocytes has not yet been fully elucidated and includes a putative pro-apoptotic activity of the unbound receptor compared to ephrin-bound receptor. We recently reported that astrocytes from patients with ALS induce cell death in co-cultured MNs. Here we found that first-generation synthetic EphA4 agonistic agent 123C4, effectively protected MNs when co-cultured with reactive astrocytes from patients with ALS from multiple subgroups (sALS and mutant SOD1). Newer generation and more potent EphA4 agonistic agents 150D4, 150E8, and 150E7 provided effective protection at a lower therapeutic dose. Combined, the data suggest that the development of EphA4 agonistic agents provides potentially a promising therapeutic strategy for patients with ALS

Cyclical Chromatin Looping and Transcription Factor Association on the Regulatory Regions of the p21 (CDKN1A) Gene in Response to 1α,25-Dihydroxyvitamin D3*S⃞

The nuclear receptor vitamin D receptor (VDR) is known to associate with three vitamin D response element (VDREs)-containing regions within the CDKN1A (p21) gene region. Here we show in MDA-MB453 breast cancer cells that the natural VDR ligand 1α,25-dihydroxyvitamin D3 causes cyclical transcription factor binding and chromatin looping of distal VDREs to the transcription start site (TSS) of the p21 gene, leading to cyclical accumulation of the p21 mRNA. At the chromatin level, association of the mediator protein MED1 precedes both the peaks of VDR binding to VDREs and phosphorylated RNA polymerase (p-Pol II) to the TSS. The loss of co-repressor NCoR1-histone deacetylase (HDAC) 3 complex and inhibitory chromatin looping from VDREs to the TSS are also initial events followed by increased acetylation of histone 3 at lysine 9 at the TSS prior to initiation of transcription. Simultaneous to VDR and p-Pol II peaks, chromatin loops between VDREs and the TSS are formed, and the lysine demethylase LSD1 and the histone acetyltransferase CBP are enriched in both regions. This is followed by a moderate peak in p21 transcript accumulation, repeated in cycles of 45-60 min. The transcript accumulation pattern is disturbed by siRNA inhibition of the mediator protein MED1, LSD1, NCoR1, or various HDACs, whereas CBP appears unnecessary for the response. Inhibition of MED1, HDAC4, or LSD1 by siRNA also attenuates ligand-induced chromatin looping. In conclusion, 1α,25-dihydroxyvitamin D3 regulates p21 transcription by inducing cyclical chromatin looping that depends on both histone deacetylation and demethylation