Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing

<p>Abstract</p> <p>Background</p> <p>Short-read high-throughput DNA sequencing technologies provide new tools to answer biological questions. However, high cost and low throughput limit their widespread use, particularly in organisms with smaller genomes such as <it>S. cerevisiae</it>. Although ChIP-Seq in mammalian cell lines is replacing array-based ChIP-chip as the standard for transcription factor binding studies, ChIP-Seq in yeast is still underutilized compared to ChIP-chip. We developed a multiplex barcoding system that allows simultaneous sequencing and analysis of multiple samples using Illumina's platform. We applied this method to analyze the chromosomal distributions of three yeast DNA binding proteins (Ste12, Cse4 and RNA PolII) and a reference sample (input DNA) in a single experiment and demonstrate its utility for rapid and accurate results at reduced costs.</p> <p>Results</p> <p>We developed a barcoding ChIP-Seq method for the concurrent analysis of transcription factor binding sites in yeast. Our multiplex strategy generated high quality data that was indistinguishable from data obtained with non-barcoded libraries. None of the barcoded adapters induced differences relative to a non-barcoded adapter when applied to the same DNA sample. We used this method to map the binding sites for Cse4, Ste12 and Pol II throughout the yeast genome and we found 148 binding targets for Cse4, 823 targets for Ste12 and 2508 targets for PolII. Cse4 was strongly bound to all yeast centromeres as expected and the remaining non-centromeric targets correspond to highly expressed genes in rich media. The presence of Cse4 non-centromeric binding sites was not reported previously.</p> <p>Conclusion</p> <p>We designed a multiplex short-read DNA sequencing method to perform efficient ChIP-Seq in yeast and other small genome model organisms. This method produces accurate results with higher throughput and reduced cost. Given constant improvements in high-throughput sequencing technologies, increasing multiplexing will be possible to further decrease costs per sample and to accelerate the completion of large consortium projects such as modENCODE.</p

X chromosome-wide analyses of genomic DNA methylation states and gene expression in male and female neutrophils

The DNA methylation status of human X chromosomes from male and female neutrophils was identified by high-throughput sequencing of HpaII and MspI digested fragments. In the intergenic and intragenic regions on the X chromosome, the sites outside CpG islands were heavily hypermethylated to the same degree in both genders. Nearly half of X chromosome promoters were either hypomethylated or hypermethylated in both females and males. Nearly one third of X chromosome promoters were a mixture of hypomethylated and heterogeneously methylated sites in females and were hypomethylated in males. Thus, a large fraction of genes that are silenced on the inactive X chromosome are hypomethylated in their promoter regions. These genes frequently belong to the evolutionarily younger strata of the X chromosome. The promoters that were hypomethylated at more than two sites contained most of the genes that escaped silencing on the inactive X chromosome. The overall levels of expression of X-linked genes were indistinguishable in females and males, regardless of the methylation state of the inactive X chromosome. Thus, in addition to DNA methylation, other factors are involved in the fine tuning of gene dosage compensation in neutrophils

Diverse Roles and Interactions of the SWI/SNF Chromatin Remodeling Complex Revealed Using Global Approaches

A systems understanding of nuclear organization and events is critical for determining how cells divide, differentiate, and respond to stimuli and for identifying the causes of diseases. Chromatin remodeling complexes such as SWI/SNF have been implicated in a wide variety of cellular processes including gene expression, nuclear organization, centromere function, and chromosomal stability, and mutations in SWI/SNF components have been linked to several types of cancer. To better understand the biological processes in which chromatin remodeling proteins participate, we globally mapped binding regions for several components of the SWI/SNF complex throughout the human genome using ChIP-Seq. SWI/SNF components were found to lie near regulatory elements integral to transcription (e.g. 5′ ends, RNA Polymerases II and III, and enhancers) as well as regions critical for chromosome organization (e.g. CTCF, lamins, and DNA replication origins). Interestingly we also find that certain configurations of SWI/SNF subunits are associated with transcripts that have higher levels of expression, whereas other configurations of SWI/SNF factors are associated with transcripts that have lower levels of expression. To further elucidate the association of SWI/SNF subunits with each other as well as with other nuclear proteins, we also analyzed SWI/SNF immunoprecipitated complexes by mass spectrometry. Individual SWI/SNF factors are associated with their own family members, as well as with cellular constituents such as nuclear matrix proteins, key transcription factors, and centromere components, implying a ubiquitous role in gene regulation and nuclear function. We find an overrepresentation of both SWI/SNF-associated regions and proteins in cell cycle and chromosome organization. Taken together the results from our ChIP and immunoprecipitation experiments suggest that SWI/SNF facilitates gene regulation and genome function more broadly and through a greater diversity of interactions than previously appreciated

Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a New Group of Proteins Important for Chromosome Segregation in Saccharomyces cerevisiae

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene NNF1 was cloned by reverse genetics. Here it is shown that the conditional nnf1-17 mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes DSN1, DSN3, and NSL1 through genetic interactions with nnf1-17. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in nnf1 mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation

Mapping the chromosomal targets of STAT1 by Sequence Tag Analysis of Genomic Enrichment (STAGE)

Identifying the genome-wide binding sites of transcription factors is important in deciphering transcriptional regulatory networks. ChIP-chip (Chromatin immunoprecipitation combined with microarrays) has been widely used to map transcription factor binding sites in the human genome. However, whole genome ChIP-chip analysis is still technically challenging in vertebrates. We recently developed STAGE as an unbiased method for identifying transcription factor binding sites in the genome. STAGE is conceptually based on SAGE, except that the input is ChIP-enriched DNA. In this study, we implemented an improved sequencing strategy and analysis methods and applied STAGE to map the genomic binding profile of the transcription factor STAT1 after interferon treatment. STAT1 is mainly responsible for mediating the cellular responses to interferons, such as cell proliferation, apoptosis, immune surveillance, and immune responses. We present novel algorithms for STAGE tag analysis to identify enriched loci with high specificity, as verified by quantitative ChIP. STAGE identified several previously unknown STAT1 target genes, many of which are involved in mediating the response to interferon-γ signaling. STAGE is thus a viable method for identifying the chromosomal targets of transcription factors and generating meaningful biological hypotheses that further our understanding of transcriptional regulatory networks

Comprehensive annotation of the transcriptome of the human fungal pathogen Candida albicans using RNA-seq

Candida albicans is the major invasive fungal pathogen of humans, causing diseases ranging from superficial mucosal infections to disseminated, systemic infections that are often lifethreatening. We have used massively parallel high-throughput sequencing of cDNA (RNA-seq) to generate a high-resolution map of the C. albicans transcriptome under several different environmental conditions. We have quantitatively determined all of the regions that are transcribed under these different conditions, and have identified 602 novel transcriptionally active regions (TARs) and numerous novel introns that are not represented in the current genome annotation. Interestingly, the expression of many of these TARs is regulated in a condition-specific manner. This comprehensive transcriptome analysis significantly enhances the current genome annotation of C. albicans, a necessary framework for a complete understanding of the molecular mechanisms of pathogenesis for this important eukaryotic pathogen

The transcriptional activity of human Chromosome 22

A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A)(+) RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22

Sequencing Y chromosomes resolves discrepancy in time to common ancestor of males versus females.

The Y chromosome and the mitochondrial genome have been used to estimate when the common patrilineal and matrilineal ancestors of humans lived. We sequenced the genomes of 69 males from nine populations, including two in which we find basal branches of the Y-chromosome tree. We identify ancient phylogenetic structure within African haplogroups and resolve a long-standing ambiguity deep within the tree. Applying equivalent methodologies to the Y chromosome and the mitochondrial genome, we estimate the time to the most recent common ancestor (T(MRCA)) of the Y chromosome to be 120 to 156 thousand years and the mitochondrial genome T(MRCA) to be 99 to 148 thousand years. Our findings suggest that, contrary to previous claims, male lineages do not coalesce significantly more recently than female lineages

CREB Binds to Multiple Loci on Human Chromosome 22

The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5′ ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans