High serum albumin ascites gradient ascites--an atypical presentation of metastatic pancreatic cancer.

Pancreatic adenocarcinoma has less than a 5% 5-year survival rate, and metastatic disease is associated with a median survival of 4.5 months. A typical presentation often includes evidence of biliary obstruction, abdominal pain, jaundice, and weight loss.

Dynamic chromatin states in human ES cells reveal potential regulatory sequences and genes involved in pluripotency.

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genome-wide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage.

ChIP-chip designs to interrogate the genome of Xenopus embryos for transcription factor binding and epigenetic regulation.

Background: Chromatin immunoprecipitation combined with genome tile path microarrays or deep sequencing can be used to study genome-wide epigenetic profiles and the transcription factor binding repertoire. Although well studied in a variety of cell lines, these genome-wide profiles have so far been little explored in vertebrate embryos.

Principal Findings: Here we report on two genome tile path ChIP-chip designs for interrogating the Xenopus tropicalis genome.

Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum.

Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. To explore the epigenome of Plasmodium falciparum asexual stages, we performed MS analysis of histone modifications and found a general preponderance of H3/H4 acetylation and H3K4me3. ChIP-on-chip profiling of H3, H3K4me3, H3K9me3, and H3K9ac from asynchronous parasites revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers.

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter.

Histone modifications at human enhancers reflect global cell-type-specific gene expression.

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome.

Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2.

Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.

Chromosome 12p deletions in TEL-AML1 childhood acute lymphoblastic leukemia are associated with retrotransposon elements and occur postnatally.

TEL-AML1 (ETV6-RUNX1) is the most common translocation in the childhood leukemias, and is a prenatal mutation in most children. This translocation has been detected at a high rate among newborns ( approximately 1%); therefore, the rate-limiting event for leukemia seems to be secondary mutations. One such frequent mutation in this subtype is partial deletion of chromosome 12p, trans from the translocation.

Overlapping euchromatin/heterochromatin- associated marks are enriched in imprinted gene regions and predict allele-specific modification.

Most genome-level analysis treats the two parental alleles equivalently, yet diploid genomes contain two parental genomes that are often epigenetically distinct. While single nucleotide polymorphisms (SNPs) can be used to distinguish these genomes, it would be useful to develop a generalized strategy for identifying candidate genes or regions showing allele-specific differences, independent of SNPs. We have explored this problem by looking for overlapping marks in the genome related to both euchromatin (histone H3 dimethyl lysine-4 [H3K4Me2]) and heterochromatin (DNA methylation [DNAm]).

Transcriptional enhancement by GATA1-occupied DNA segments is strongly associated with evolutionary constraint on the binding site motif.

Tissue development and function are exquisitely dependent on proper regulation of gene expression, but it remains controversial whether the genomic signals controlling this process are subject to strong selective constraint. While some studies show that highly constrained noncoding regions act to enhance transcription, other studies show that DNA segments with biochemical signatures of regulatory regions, such as occupancy by a transcription factor, are seemingly unconstrained across mammalian evolution. To test the possible correlation of selective constraint with enhancer activity, we used chromatin immunoprecipitation as an approach unbiased by either evolutionary constraint or prior knowledge of regulatory activity to identify DNA segments within a 66-Mb region of mouse chromosome 7 that are occupied by the erythroid transcription factor GATA1.

The genomic distribution and function of histone variant HTZ-1 during C. elegans embryogenesis.

In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date.

Analysis of the mechanisms mediating tumor-specific changes in gene expression in human liver tumors.

There is widespread interest in efficient characterization of differences between tumor and normal samples. Here, we show an effective methodology for genome-scale characterization of tumors. Using matched normal and tumor samples from liver cancer patients, as well as non-cancer-related normal liver tissue, we first determined changes in gene expression as monitored on RNA expression arrays.

SNP-specific array-based allele-specific expression analysis.

We have developed an optimized array-based approach for customizable allele-specific gene expression (ASE) analysis. The central features of the approach are the ability to select SNPs at will for detection, and the absence of need to PCR amplify the target. A surprisingly long probe length (39-49 nt) was needed for allelic discrimination.

An integrative genomic and epigenomic approach for the study of transcriptional regulation.

The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes.

Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells.

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A.

Systematic evaluation of variability in ChIP-chip experiments using predefined DNA targets.

The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups.

Genome-scale ChIP-chip analysis using 10,000 human cells.

The technique of chromatin immunoprecipitation (ChIP) is a powerful method for identifying in vivo DNA binding sites of transcription factors and for studying chromatin modifications. Unfortunately, the large number of cells needed for the standard ChIP protocol has hindered the analysis of many biologically interesting cell populations that are difficult to obtain in large numbers. New ChIP methods involving the use of carrier chromatin have been developed that allow the one-gene-at-a-time analysis of very small numbers of cells.

Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs.

By integrating genome-wide maps of RNA polymerase II (Polr2a) binding with gene expression data and H3ac and H3K4me3 profiles, we characterized promoters with enriched activity in mouse embryonic stem cells (mES) as well as adult brain, heart, kidney, and liver. We identified approximately 24,000 promoters across these samples, including 16,976 annotated mRNA 5' ends and 5153 additional sites validating cap-analysis of gene expression (CAGE) 5' end data. We showed that promoters with CpG islands are typically non-tissue specific, with the majority associated with Polr2a and the active chromatin modifications in nearly all the tissues examined.

A comprehensive ChIP-chip analysis of E2F1, E2F4, and E2F6 in normal and tumor cells reveals interchangeable roles of E2F family members.

Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F.

Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation.

Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at lysine 4 on histone H3 (H3K4) is found at the 5' end of active genes and contributes to transcriptional activation by recruiting chromatin-remodelling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells, but its location within genes and its function in transcription are unknown.

Identification of an OCT4 and SRY regulatory module using integrated computational and experimental genomics approaches.

ChIP-chip studies have revealed that many in vivo binding sites have a weak match to the consensus sequence for the transcription factor being analyzed. Possible explanations for these observations include (1) the in vitro-derived consensus site does not represent the in vivo binding site and/or (2) the factor is recruited to a weak binding site via interaction with another protein. To address these possibilities, we developed an approach (ChIPMotifs) that incorporates a bootstrap resampling method to statistically infer the optimal cutoff threshold for a position weight matrix (PWM) of a motif identified from ChIP-chip data by ab initio motif discovery programs.

Bcl-6 mediates the germinal center B cell phenotype and lymphomagenesis through transcriptional repression of the DNA-damage sensor ATR.

Antibody specificity and diversity is generated in B cells during germinal center maturation through clonal expansion while they undergo class-switch recombination and somatic hypermutation. Here we demonstrate that the transcriptional repressor Bcl-6 mediates this phenotype by directly repressing ATR in centroblasts and lymphoma cells. ATR is critical in replication and DNA damage-sensing checkpoints.

Genome-wide analysis of KAP1 binding suggests autoregulation of KRAB-ZNFs.

We performed a genome-scale chromatin immunoprecipitation (ChIP)-chip comparison of two modifications (trimethylation of lysine 9 [H3me3K9] and trimethylation of lysine 27 [H3me3K27]) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger (ZNF) genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27.

Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulator's function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported.