ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data

ChIPOTle (Chromatin ImmunoPrecipitation On Tiled arrays) takes advantage of two unique properties of ChIP-chip data: the single-tailed nature of the data, caused by specific enrichment but not specific depletion of genomic fragments; and the predictable enrichment of DNA fragments adjacent to sites of direct protein-DNA interaction. Implemented as a Microsoft Excel macro written in Visual Basic, ChIPOTle uses a sliding window approach that yields improvements in the identification of bona fide sites of protein-DNA interaction

Cisplatin DNA damage and repair maps of the human genome at single-nucleotide resolution

The chemotherapy drug cisplatin kills cancer cells by damaging their DNA. It has been used for treating a variety of cancer types for almost four decades. Although the drug is generally effective, it has strong adverse side effects, and some cancers exhibit or, after initial favorable response, develop drug resistance. The mechanism of drug resistance is multifactorial and involves the ability of cancer cells to repair the cisplatin-induced DNA damages. We have developed methods to map the sites of cisplatin damage and its repair for the entire human genome at single-nucleotide resolution. These methods can be used to study cancer sensitivity and resistance to the drugs, and to identify new strategies for efficient combination therapies

Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis

Nucleotide excision repair is the sole mechanism for removing bulky adducts from the human genome, including those formed by UV radiation and chemotherapeutic drugs. We used eXcision Repair-sequencing, a genomic assay for measuring DNA repair, to map the kinetics of repair after UV treatment. These genome-wide repair maps, in turn, allowed us to infer how excision repair is influenced by DNA packaging. Active and open chromatin regions were repaired more rapidly than other genomic regions. Repair in repressed and heterochromatic regions is slower and persists for up to 2 d. Furthermore, late-repaired regions are associated with a higher level of cancer-linked somatic mutations, highlighting the importance of efficient DNA repair and linking chromatin organization to cancer mutagenesis

Integral Nuclear Pore Proteins Bind to Pol III-Transcribed Genes and Are Required for Pol III Transcript Processing in C. elegans

Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of a subset of small nucleolar RNAs (snoRNAs) and tRNAs transcribed by RNA Polymerase (Pol) III. Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral nuclear pore components, and importin-β IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and snoRNA genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for Pol III recruitment. Instead, NPP-13 is required for cleavage of tRNA and snoRNA precursors into mature RNAs, whereas Pol II transcript processing occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and processing of Pol III transcripts in C. elegans

Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

Abstract Background Although Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known. Results We determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point. Conclusions Local DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus

A Common Set of DNA Regulatory Elements Shapes Drosophila Appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different "master regulator" transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. Here, we use genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. We propose that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules

A Common Set of DNA Regulatory Elements Shapes Drosophila Appendages

Animals have body parts made of similar cell types located at different axial positions, such as limbs. The identity and distinct morphology of each structure is often specified by the activity of different "master regulator" transcription factors. Although similarities in gene expression have been observed between body parts made of similar cell types, how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development is not known. Here, we use genome-wide open chromatin profiling to show that among the Drosophila appendages, the same DNA regulatory modules are accessible throughout the genome at a given stage of development, except at the loci encoding the master regulators themselves. In addition, open chromatin profiles change over developmental time, and these changes are coordinated between different appendages. We propose that master regulators create morphologically distinct structures by differentially influencing the function of the same set of DNA regulatory modules

Cell Cycle–Specified Fluctuation of Nucleosome Occupancy at Gene Promoters

The packaging of DNA into nucleosomes influences the accessibility of underlying regulatory information. Nucleosome occupancy and positioning are best characterized in the budding yeast Saccharomyces cerevisiae, albeit in asynchronous cell populations or on individual promoters such as PHO5 and GAL1–10. Using FAIRE (formaldehyde-assisted isolation of regulatory elements) and whole-genome microarrays, we examined changes in nucleosome occupancy throughout the mitotic cell cycle in synchronized populations of S. cerevisiae. Perhaps surprisingly, nucleosome occupancy did not exhibit large, global variation between cell cycle phases. However, nucleosome occupancy at the promoters of cell cycle–regulated genes was reduced specifically at the cell cycle phase in which that gene exhibited peak expression, with the notable exception of S-phase genes. We present data that establish FAIRE as a high-throughput method for assaying nucleosome occupancy. For the first time in any system, nucleosome occupancy was mapped genome-wide throughout the cell cycle. Fluctuation of nucleosome occupancy at promoters of most cell cycle–regulated genes provides independent evidence that periodic expression of these genes is controlled mainly at the level of transcription. The promoters of G(2)/M genes are distinguished from other cell cycle promoters by an unusually low baseline nucleosome occupancy throughout the cell cycle. This observation, coupled with the maintenance throughout the cell cycle of the stereotypic nucleosome occupancy states between coding and non-coding loci, suggests that the largest component of variation in nucleosome occupancy is “hard wired,” perhaps at the level of DNA sequence

A chromatin-mediated mechanism for specification of conditional transcription factor targets

Organisms respond to changes in their environment, and many such responses are initiated at the level of gene transcription. Here, we provide evidence for a previously undiscovered mechanism for directing transcriptional regulators to new binding targets in response to an environmental change. We show that repressor-activator protein 1 (Rap1), a master regulator of yeast metabolism, binds to an expanded target set after glucose depletion despite decreasing protein levels and no evidence of posttranslational modification. Computational analysis predicts that proteins capable of recruiting the chromatin regulator Tup1 act to restrict the binding distribution of Rap1 in the presence of glucose. Deletion of the gene(s) encoding Tup1, recruiters of Tup1 or chromatin regulators recruited by Tup1 cause Rap1 to bind specifically and inappropriately to low-glucose targets. These data, combined with whole-genome measurements of nucleosome occupancy and Tup1 distribution, provide evidence for a mechanism of dynamic target specification that coordinates the genome-wide distribution of intermediate-affinity DNA sequence motifs with chromatin-mediated regulation of accessibility to those sites

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization