Evolutionary Conserved Motif Finder (ECMFinder) for genome-wide identification of clustered YY1- and CTCF-binding sites

We have developed a new bioinformatics approach called ECMFinder (Evolutionary Conserved Motif Finder). This program searches for a given DNA motif within the entire genome of one species and uses the gene association information of a potential transcription factor-binding site (TFBS) to screen the homologous regions of a second and third species. If multiple species have this potential TFBS in homologous positions, this program recognizes the identified TFBS as an evolutionary conserved motif (ECM). This program outputs a list of ECMs, which can be uploaded as a Custom Track in the UCSC genome browser and can be visualized along with other available data. The feasibility of this approach was tested by searching the genomes of three mammals (human, mouse and cow) with the DNA-binding motifs of YY1 and CTCF. This program successfully identified many clustered YY1- and CTCF-binding sites that are conserved among these species but were previously undetected. In particular, this program identified CTCF-binding sites that are located close to the Dlk1, Magel2 and Cdkn1c imprinted genes. Individual ChIP experiments confirmed the in vivo binding of the YY1 and CTCF proteins to most of these newly discovered binding sites, demonstrating the feasibility and usefulness of ECMFinder

Retrotransposons as a major source of epigenetic variations in the mammalian genome

Transcription of retrotransposons is usually repressed by DNA methylation, but a few elements, such as intracisternal A-particles (IAPs) associated with the Agouti and Axin-fused loci, partially escape this repression mechanism. The levels of this repression are also variable among individuals with an identical genome sequence, generating epigenetically different states of loci or epialleles. In the current study, we tested the existence of additional retrotransposon-derived epialleles in the mouse genome. Using a series of bioinformatics approaches, 143 candidate epialleles were first identified from the mouse genome based on their promoter activity and association with active histone modification marks. Detailed analyses suggest that a subset of these elements showed variable levels of DNA methylation among the individual mice of an isogenic background, revealing their stochastic nature (metastability) of DNA methylation. The analyses also identified two opposite patterns of DNA methylation during development, progressive gaining vs. losing, confirming the dynamic nature of their DNA methylation patterns. qRT-PCR analyses demonstrated that the expression levels of these elements are indeed variable among the individual mice, suggesting functional consequences on their associated endogenous genes. Overall, these data confirm the presence of a number of new retrotransposon-derived epialleles with suggestions ofthe presence of more, and further identify retrotransposons as a major source of epigenetic variations in the mammalian genome. © 2012 Landes Bioscience

Aebp2 as an epigenetic regulator for neural crest cells

Aebp2 is a potential targeting protein for the mammalian Polycomb Repression Complex 2 (PRC2). We generated a mutant mouse line disrupting the transcription of Aebp2 to investigate its in vivo roles. Aebp2-mutant homozygotes were embryonic lethal while heterozygotes survived to adulthood with fertility. In developing mouse embryos, Aebp2 is expressed mainly within cells of neural crest origin. In addition, many heterozygotes display a set of phenotypes, enlarged colon and hypopigmentation, similar to those observed in human patients with Hirschsprung\u27s disease and Waardenburg syndrome. These phenotypes are usually caused by the absence of the neural crest-derived ganglia in hindguts and melanocytes. ChIP analyses demonstrated that the majority of the genes involved in the migration and development process of neural crest cells are downstream target genes of AEBP2 and PRC2. Furthermore, expression analyses confirmed that some of these genes are indeed affected in the Aebp2 heterozygotes. Taken together, these results suggest that Aebp2 may regulate the migration and development of the neural crest cells through the PRC2-mediated epigenetic mechanism

Type I IFNs and TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation

Cross-regulation of Toll-like receptor responses by cytokines is essential for effective host defense, avoidance of toxicity, and homeostasis, but the underlying mechanisms are not well understood. A comprehensive epigenomic approach in human macrophages showed that the proinflammatory cytokines TNF and type I IFNs induce transcriptional cascades that alter chromatin states to broadly reprogram TLR4-induced responses. TNF tolerized inflammatory genes to prevent toxicity, while preserving antiviral and metabolic gene induction. Type I IFNs potentiated TNF inflammatory function by priming chromatin to prevent silencing of inflammatory NF-κB target genes. Priming of chromatin enabled robust transcriptional responses to weak upstream signals. Similar chromatin regulation occurred in human diseases. Our findings reveal that signaling crosstalk between IFNs and TNF is integrated at the level of chromatin to reprogram inflammatory responses, and identify new functions and mechanisms of action of these cytokines

AEBP2 as a potential targeting protein for Polycomb Repression Complex PRC2

AEBP2 is a zinc finger protein that has been shown to interact with the mammalian Polycomb Repression Complex 2 (PRC2). In the current study, we characterized this unknown protein and tested its potential targeting roles for the PRC2. AEBP2 is an evolutionarily well-conserved gene that is found in the animals ranging from flying insects to mammals. The transcription of mammalian AEBP2 is driven by two alternative promoters and produces at least two isoforms of the protein. These isoforms show developmental stage-specific expression patterns: the adult-specific larger form (51 kDa) and the embryo-specific smaller form (32 kDa). The AEBP2 protein binds to a DNA-binding motif with an unusual bipartite structure, CTT(N)15-23cagGCC with lower-case being less critical. A large fraction of AEBP2's target loci also map closely to the known target loci of the PRC2. In fact, many of these loci are co-occupied by the two proteins, AEBP2 and SUZ12. This suggests that AEBP2 is most likely a targeting protein for the mammalian PRC2 complex

P-TEFb Regulates Transcriptional Activation in Non-coding RNA Genes

Many non-coding RNAs (ncRNAs) serve as regulatory molecules in various physiological pathways, including gene expression in mammalian cells. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. It is thus important to understand transcriptional regulation in ncRNA genes but is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA polymerase II (Pol II) promoter-proximal pausing and in a tissue-specific manner. In this study, human ncRNA genes that are expressed in the early G1 phase, termed immediate early ncRNA genes, were monitored to assess the function of positive transcription elongation factor b (P-TEFb), a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is induced in the G0–G1 transition and regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb-dependent transcription of serum responsive ncRNA genes was observed: Pol II carboxyl-terminal domain phosphorylated at serine 2 (S2) was largely increased in the transcription start site (TSS, -300 to +300) whereas overall, it was decreased in the gene body (GB, > +350) upon chemical inhibition of P-TEFb. In addition, the three representative, immediate early ncRNAs, whose expression is dependent on P-TEFb, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), nuclear enriched abundant transcript 1 (NEAT1), and X-inactive specific transcript (XIST), were further analyzed for determining P-TEFb association. Taken together, our data suggest that transcriptional activation of many human ncRNAs utilizes the pausing and releasing of Pol II, and that the regulatory mechanism of transcriptional elongation in these genes requires the function of P-TEFb. Furthermore, we propose that ncRNA and mRNA transcription are regulated by similar mechanisms while P-TEFb inhibition unexpectedly increases S2 Pol II phosphorylation in the TSSs in many ncRNA genes.One Sentence Summary: P-TEFb regulates Pol II phosphorylation for transcriptional activation in many stimulus-inducible ncRNA genes

Functional Coordination of BET Family Proteins Underlies Altered Transcription Associated With Memory Impairment in Fragile X Syndrome

Bromodomain and extraterminal proteins (BET) are epigenetic readers that play critical roles in gene regulation. Pharmacologic inhibition of the bromodomain present in all BET family members is a promising therapeutic strategy for various diseases, but its impact on individual family members has not been well understood. Using a transcriptional induction paradigm in neurons, we have systematically demonstrated that three major BET family proteins (BRD2/3/4) participated in transcription with different recruitment kinetics, interdependency, and sensitivity to a bromodomain inhibitor, JQ1. In a mouse model of fragile X syndrome (FXS), BRD2/3 and BRD4 showed oppositely altered expression and chromatin binding, correlating with transcriptional dysregulation. Acute inhibition of CBP/p300 histone acetyltransferase (HAT) activity restored the altered binding patterns of BRD2 and BRD4 and rescued memory impairment in FXS. Our study emphasizes the importance of understanding the BET coordination controlled by a balanced action between HATs with different substrate specificity