Wenxiu Ma

An integrated software system for analyzing ChIP-chip and ChIP-seq data

We present CisGenome, a software system for analyzing genome-wide chromatin immunoprecipitation (ChIP) data. CisGenome is designed to meet all basic needs of ChIP data analyses, including visualization, data normalization, peak detection, false discovery rate computation, gene-peak association, and sequence and motif analysis. In addition to implementing previously published ChIP–microarray (ChIP-chip) analysis methods, the software contains statistical methods designed specifically for ChlP sequencing (ChIP-seq) data obtained by coupling ChIP with massively parallel sequencing. The modular design of CisGenome enables it to support interactive analyses through a graphic user interface as well as customized batch-mode computation for advanced data mining. A built-in browser allows visualization of array images, signals, gene structure, conservation, and DNA sequence and motif information. We demonstrate the use of these tools by a comparative analysis of ChIP-chip and ChIP-seq data for the transcription factor NRSF/REST, a study of ChIP-seq analysis with or without a negative control sample, and an analysis of a new motif in Nanog- and Sox2-binding regions.

TERT Promotes Epithelial Proliferation through Transcriptional Control of a Myc- and Wnt-Related Developmental Program

Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERTci) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERTci retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Neural-specific Sox2 input and differential Gli-binding affinity provide context and positional information in Shh-directed neural patterning

In the vertebrate neural tube, regional Sonic hedgehog (Shh) signaling invokes a time- and concentration-dependent induction of six different cell populations mediated through Gli transcriptional regulators. Elsewhere in the embryo, Shh/Gli responses invoke different tissue-appropriate regulatory programs. A genome-scale analysis of DNA binding by Gli1 and Sox2, a pan-neural determinant, identified a set of shared regulatory regions associated with key factors central to cell fate determination and neural tube patterning. Functional analysis in transgenic mice validates core enhancers for each of these factors and demonstrates the dual requirement for Gli1 and Sox2 inputs for neural enhancer activity. Furthermore, through an unbiased determination of Gli-binding site preferences and analysis of binding site variants in the developing mammalian CNS, we demonstrate that differential Gli-binding affinity underlies threshold-level activator responses to Shh input. In summary, our results highlight Sox2 input as a context-specific determinant of the neural-specific Shh response and differential Gli-binding site affinity as an important cis-regulatory property critical for interpreting Shh morphogen action in the mammalian neural tube.

Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes

High-throughput methods based on chromosome conformation capture have greatly advanced our understanding of the three-dimensional (3D) organization of genomes but are limited in resolution by their reliance on restriction enzymes. Here we describe a method called DNase Hi-C for comprehensively mapping global chromatin contacts. DNase Hi-C uses DNase I for chromatin fragmentation, leading to greatly improved efficiency and resolution over that of Hi-C. Coupling this method with DNA-capture technology provides a high-throughput approach for targeted mapping of fine-scale chromatin architecture. We applied targeted DNase Hi-C to characterize the 3D organization of 998 large intergenic noncoding RNA (lincRNA) promoters in two human cell lines. Our results revealed that expression of lincRNAs is tightly controlled by complex mechanisms involving both super-enhancers and the Polycomb repressive complex. Our results provide the first glimpse of the cell type–specific 3D organization of lincRNA genes.

The lncRNA Firre anchors the inactive X chromosome to the nucleolus by binding CTCF and maintains H3K27me3 methylation

BackgroundIn mammals, X chromosome genes are present in one copy in males and two in females. To balance the dosage of X-linked gene expression between the sexes, one of the X chromosomes in females is silenced. X inactivation is initiated by upregulation of the lncRNA (long non-coding RNA) Xist and recruitment of specific chromatin modifiers. The inactivated X chromosome becomes heterochromatic and visits a specific nuclear compartment adjacent to the nucleolus.ResultsHere, we show a novel role for the lncRNA Firre in anchoring the inactive mouse X chromosome and preserving one of its main epigenetic features, H3K27me3. Similar to Dxz4, Firre is X-linked and expressed from a macrosatellite repeat locus associated with a cluster of CTCF and cohesin binding sites, and is preferentially located adjacent to the nucleolus. CTCF binding present initially in both male and female mouse embryonic stem cells is lost from the active X during development. Knockdown of Firre disrupts perinucleolar targeting and H3K27me3 levels in mouse fibroblasts, demonstrating a role in maintenance of an important epigenetic feature of the inactive X chromosome. No X-linked gene reactivation is seen after Firre knockdown; however, a compensatory increase in the expression of chromatin modifier genes implicated in X silencing is observed. Further experiments in female embryonic stem cells suggest that Firre does not play a role in X inactivation onset.ConclusionsThe X-linked lncRNA Firre helps to position the inactive X chromosome near the nucleolus and to preserve one of its main epigenetic features.

Hedgehog pathway-regulated gene networks in cerebellum development and tumorigenesis

Many genes initially identified for their roles in cell fate determination or signaling during development can have a significant impact on tumorigenesis. In the developing cerebellum, Sonic hedgehog (Shh) stimulates the proliferation of granule neuron precursor cells (GNPs) by activating the Gli transcription factors. Inappropriate activation of Shh target genes results in unrestrained cell division and eventually medulloblastoma, the most common pediatric brain malignancy. We find dramatic differences in the gene networks that are directly driven by the Gli1 transcription factor in GNPs and medulloblastoma. Gli1 binding location analysis revealed hundreds of genomic loci bound by Gli1 in normal and cancer cells. Only one third of the genes bound by Gli1 in GNPs were also bound in tumor cells. Correlation with gene expression levels indicated that 116 genes were preferentially transcribed in tumors, whereas 132 genes were target genes in both GNPs and medulloblastoma. Quantitative PCR and in situ hybridization for some putative target genes support their direct regulation by Gli. The results indicate that transformation of normal GNPs into deadly tumor cells is accompanied by a distinct set of Gli-regulated genes and may provide candidates for targeted therapies.

Bipartite structure of the inactive mouse X chromosome

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

Motif-based analysis of large nucleotide data sets using MEME-ChIP

MEME-ChIP is a web-based tool for analyzing motifs in large DNA or RNA data sets. It can analyze peak regions identified by ChIP-seq, cross-linking sites identified by CLIP-seq and related assays, as well as sets of genomic regions selected using other criteria. MEME-ChIP performs de novo motif discovery, motif enrichment analysis, motif location analysis and motif clustering, providing a comprehensive picture of the DNA or RNA motifs that are enriched in the input sequences. MEME-ChIP performs two complementary types of de novo motif discovery: weight matrix–based discovery for high accuracy; and word-based discovery for high sensitivity. Motif enrichment analysis using DNA or RNA motifs from human, mouse, worm, fly and other model organisms provides even greater sensitivity. MEME-ChIPs interactive HTML output groups and aligns significant motifs to ease interpretation. This protocol takes less than 3 h, and it provides motif discovery approaches that are distinct and complementary to other online methods.

Escape from X Inactivation Varies in Mouse Tissues

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.

Mammalian X Upregulation Is Associated with Enhanced Transcription Initiation, RNA Half-Life, and MOF-Mediated H4K16 Acetylation

X upregulation in mammals increases levels of expressed X-linked transcripts to compensate for autosomal biallelic expression. Here, we present molecular mechanisms that enhance X expression at transcriptional and posttranscriptional levels. Active mouse X-linked promoters are enriched in the initiation form of RNA polymerase II (PolII-S5p) and in specific histone marks, including histone H4 acetylated at lysine 16 (H4K16ac) and histone variant H2AZ. The H4K16 acetyltransferase males absent on the first (MOF), known to mediate the Drosophila X upregulation, is also enriched on the mammalian X. Depletion of MOF or male-specific lethal 1 (MSL1) in mouse ES cells causes a specific decrease in PolII-S5p and in expression of a subset of X-linked genes. Analyses of RNA half-life data sets show increased stability of mammalian X-linked transcripts. Both ancestral X-linked genes, defined as those conserved on chicken autosomes, and newly acquired X-linked genes are upregulated by similar mechanisms but to a different extent, suggesting that subsets of genes are distinctly regulated depending on their evolutionary history.