Animals have body parts made of similar cell types located at different axial positions (e.g. 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, it is not known how regulatory information in the genome is differentially utilized to create morphologically diverse structures in development. 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, while open chromatin profiles change over developmental time, 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.
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.
Eviction or destabilization of nucleosomes from chromatin is a hallmark of functional regulatory elements of the eukaryotic genome. Historically identified by nuclease hypersensitivity, these regulatory elements are typically bound by transcription factors or other regulatory proteins. FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) is an alternative approach to identify these genomic regions and has proven successful in a multitude of eukaryotic cell and tissue types. Cells or dissociated tissues are crosslinked briefly with formaldehyde, lysed, and sonicated. Sheared chromatin is subjected to phenol-chloroform extraction and the isolated DNA, typically encompassing 1–3% of the human genome, is purified. We provide guidelines for quantitative analysis by PCR, microarrays, or next-generation sequencing. Regulatory elements enriched by FAIRE display high concordance with those identified by nuclease hypersensitivity or ChIP, and the entire procedure can be completed in three days. FAIRE exhibits low technical variability, which allows its use in large-scale studies of chromatin from normal or diseased tissues.
FAIRE; open chromatin; nucleosome; next-generation sequencing
We performed a systematic evaluation of how variations in sequencing depth and other parameters influence interpretation of Chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) experiments. Using Drosophila S2 cells, we generated ChIP-seq datasets for a site-specific transcription factor (Suppressor of Hairy-wing) and a histone modification (H3K36me3). We detected a chromatin state bias, open chromatin regions yielded higher coverage, which led to false positives if not corrected and had a greater effect on detection specificity than any base-composition bias. Paired-end sequencing revealed that single-end data underestimated ChIP library complexity at high coverage. The removal of reads originating at the same base reduced false-positives while having little effect on detection sensitivity. Even at a depth of ~1 read/bp coverage of mappable genome, ~1% of the narrow peaks detected on a tiling array were missed by ChIP-seq. Evaluation of widely-used ChIP-seq analysis tools suggests that adjustments or algorithm improvements are required to handle datasets with deep coverage.
The C. elegans Dosage Compensation Complex (DCC) associates with both X chromosomes of XX animals to reduce X-linked transcript levels. Five DCC members are homologous to subunits of the evolutionarily conserved condensin complex, while two non-condensin subunits are required for DCC recruitment to X. Here, we investigated the molecular mechanism of DCC recruitment and spreading along X by examining gene expression and the binding patterns of DCC subunits in different stages of development, and in strains harboring X;autosome fusions. We show that DCC binding is dynamically specified according to gene activity during development, and that the mechanism of DCC spreading is independent of X-chromosome DNA sequence. Accordingly, in X;A fusion strains DCC spreading propagates from X-linked recruitment sites onto autosomal promoters as a function of distance. Quantitative analysis of spreading suggests that the condensin-like subunits spread from recruitment sites to promoters more readily than subunits involved in initial X-targeting. Via these mechanisms, a highly conserved chromatin complex is appropriated to accomplish domain-scale transcriptional regulation during development. Similarities to the X-recognition and spreading strategies used by the Drosophila DCC suggest mechanisms fundamental to chromosome-scale gene regulation.
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.
Regulatory elements; chromatin accessibility; nucleosome occupancy; histone; FAIRE; DNase hypersensitivity; transcriptional regulation
Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome.
We have systematically measured the effect of normal genetic variation present in a human population on the binding of a specific chromatin protein (CTCF) to DNA by measuring its binding in 51 human cell lines. We observed a large number of changes in protein binding that we can confidently attribute to genetic effects. The corresponding genetic changes are often clustered around the binding motif for CTCF, but only a minority are actually within the motif. Unexpectedly, we also find that at most binding sites on the X chromosome, CTCF binding occurs equally on both the X chromosomes in females at the same level as on the single X chromosome in males. This finding suggests that in general, CTCF binding is not subject to global dosage compensation, the process which equalizes gene expression levels from the two female X chromosomes and the single male X.
Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus which establishes latent infection in endothelial and B cells, as well as in primary effusion lymphoma (PEL). During latency, the viral genome exists as a circular DNA minichromosome (episome) and is packaged into chromatin analogous to human chromosomes. Only a small subset of promoters, those which drive latent RNAs, are active in latent episomes. In general, nucleosome depletion (“open chromatin”) is a hallmark of eukaryotic regulatory elements such as promoters and transcriptional enhancers or insulators. We applied formaldehyde-assisted isolation of regulatory elements (FAIRE) followed by next-generation sequencing to identify regulatory elements in the KSHV genome and integrated these data with previously identified locations of histone modifications, RNA polymerase II occupancy, and CTCF binding sites. We found that (i) regions of open chromatin were not restricted to the transcriptionally defined latent loci; (ii) open chromatin was adjacent to regions harboring activating histone modifications, even at transcriptionally inactive loci; and (iii) CTCF binding sites fell within regions of open chromatin with few exceptions, including the constitutive LANA promoter and the vIL6 promoter. FAIRE-identified nucleosome depletion was similar among B and endothelial cell lineages, suggesting a common viral genome architecture in all forms of latency.
Laminopathies are diseases characterized by defects in nuclear envelope structure. A well-known example is Emery-Dreifuss muscular dystrophy, which is caused by mutations in the human lamin A/C and emerin genes. While most nuclear envelope proteins are ubiquitously expressed, laminopathies often affect only a subset of tissues. The molecular mechanisms underlying these tissue-specific manifestations remain elusive. We hypothesize that different functional subclasses of genes might be differentially affected by defects in specific nuclear envelope components.
Here we determine genome-wide DNA association profiles of two nuclear envelope components, lamin/LMN-1 and emerin/EMR-1 in adult Caenorhabditis elegans. Although both proteins bind to transcriptionally inactive regions of the genome, EMR-1 is enriched at genes involved in muscle and neuronal function. Deletion of either EMR-1 or LEM-2, another integral envelope protein, causes local changes in nuclear architecture as evidenced by altered association between DNA and LMN-1. Transcriptome analyses reveal that EMR-1 and LEM-2 are associated with gene repression, particularly of genes implicated in muscle and nervous system function. We demonstrate that emr-1, but not lem-2, mutants are sensitive to the cholinesterase inhibitor aldicarb, indicating altered activity at neuromuscular junctions.
We identify a class of elements that bind EMR-1 but do not associate with LMN-1, and these are enriched for muscle and neuronal genes. Our data support a redundant function of EMR-1 and LEM-2 in chromatin anchoring to the nuclear envelope and gene repression. We demonstrate a specific role of EMR-1 in neuromuscular junction activity that may contribute to Emery-Dreifuss muscular dystrophy in humans.
The hsp-16.2 promoter is sufficient for recruitment of hsp-16.2 to nuclear pore complexes in a manner dependent on RNA pol II and ENY-2, but not on full-length mRNA production.
Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts.
DNaseI hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers, and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ~2.9 million DHSs that encompass virtually all known experimentally-validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation, and regulatory factor occupancy patterns. We connect ~580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is choreographed with dozens to hundreds of co-activated elements, and the trans-cellular DNaseI sensitivity pattern at a given region can predict cell type-specific functional behaviors. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.
Centromeres are chromosomal loci that direct segregation of the genome during cell division. The histone H3 variant CENP-A (also known as CenH3) defines centromeres in monocentric organisms, which confine centromere activity to a discrete chromosomal region, and holocentric organisms, which distribute centromere activity along the chromosome length1–3. Because the highly repetitive DNA found at most centromeres is neither necessary nor sufficient for centromere function, stable inheritance of CENP-A nucleosomal chromatin is postulated to epigenetically propagate centromere identity4. Here, we show that in the holocentric nematode Caenorhabditis elegans pre-existing CENP-A nucleosomes are not necessary to guide recruitment of new CENP-A nucleosomes. This is indicated by lack of CENP-A transmission by sperm during fertilization and by removal and subsequent reloading of CENP-A during oogenic meiotic prophase. Genome-wide mapping of CENP-A location in embryos and quantification of CENP-A molecules in nuclei revealed that CENP-A is incorporated at low density in domains that cumulatively encompass half the genome. Embryonic CENP-A domains are established in a pattern inverse to regions that are transcribed in the germline and early embryo, and ectopic transcription of genes in a mutant germline altered the pattern of CENP-A incorporation in embryos. Furthermore, regions transcribed in the germline but not embryos fail to incorporate CENP-A throughout embryogenesis. We propose that germline transcription defines genomic regions that exclude CENP-A incorporation in progeny, and that zygotic transcription during early embryogenesis remodels and reinforces this basal pattern. These findings link centromere identity to transcription and shed light on the evolutionary plasticity of centromeres.
Many animal species use a chromosome-based mechanism of sex determination, which has led to the coordinate evolution of dosage-compensation systems. Dosage compensation not only corrects the imbalance in the number of X chromosomes between the sexes but also is hypothesized to correct dosage imbalance within cells that is due to monoallelic X-linked expression and biallelic autosomal expression, by upregulating X-linked genes twofold (termed ‘Ohno’s hypothesis’). Although this hypothesis is well supported by expression analyses of individual X-linked genes and by microarray-based transcriptome analyses, it was challenged by a recent study using RNA sequencing and proteomics. We obtained new, independent RNA-seq data, measured RNA polymerase distribution and reanalyzed published expression data in mammals, C. elegans and Drosophila. Our analyses, which take into account the skewed gene content of the X chromosome, support the hypothesis of upregulation of expressed X-linked genes to balance expression of the genome.
Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly1-4. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor (TF) occupancy5, but are blind to kinetics and are poor predictors of TF function at a given locus. To measure TF binding dynamics genome-wide, we performed competition ChIP6,7 with a sequence-specific S. cerevisiae transcription factor, Rap18. Rap1 binding dynamics and Rap1 occupancy were only weakly correlated (R2 = 0.14), but binding dynamics were more strongly linked to function than occupancy. Long Rap1 residence was coupled to transcriptional activation, while fast binding turnover, which we term “treadmilling”, was linked to low transcriptional output. Thus, DNA-binding events that appear identical by conventional ChIP may have starkly different underlying modes of interaction that lead to opposing functional outcomes. We propose that TF binding turnover is a major point of regulation in determining the functional consequences of transcription factor binding, and is mediated in large part by control of competition between TFs and nucleosomes. Our model (Supplementary Fig. 1) predicts a clutch-like mechanism that rapidly engages a treadmilling transcription factor into a stable binding state, or vice-versa, to modulate TF function.
The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.
In many animals, males have one X chromosome and females have two. However, the same amount of gene expression from X chromosomes is needed in the two sexes. The process of dosage compensation (DC) globally regulates X-chromosome gene expression to make it equal between the sexes, and it occurs in different ways in different animals. In mammals, one X chromosome in females is randomly inactivated, leaving one active X chromosome. In contrast, in the nematode worm C. elegans, the two X chromosomes in hermaphrodites are repressed two-fold to match gene expression to the single X chromosome in males. Previous work in C. elegans identified proteins required for DC that bind to the X chromosome, but their mode of action is not known. Here we show that DC proteins lead to higher levels of histone H4 lysine 20 monomethylation (H4K20me1) on hermaphrodite X chromosomes and that H4K20me1 functions in repressing X-chromosome gene expression. This shows that histone modification is an important aspect of the mechanism of dosage compensation. Together with previous work linking H4K20me1 to chromatin structure regulation, our results suggest that dosage compensation might lower gene expression on hermaphrodite X chromosomes by compacting them.
Previous studies in Saccharomyces cerevisiae established that depletion of histone H4 results in the genome-wide transcriptional de-repression of hundreds of genes. To probe the mechanism of this transcriptional de-repression, we depleted nucleosomes in vivo by conditional repression of histone H3 transcription. We then measured the resulting changes in transcription by RNA–seq and in chromatin organization by MNase–seq. This experiment also bears on the degree to which trans-acting factors and DNA–encoded elements affect nucleosome position and occupancy in vivo. We identified ∼60,000 nucleosomes genome wide, and we classified ∼2,000 as having preferentially reduced occupancy following H3 depletion and ∼350 as being preferentially retained. We found that the in vivo influence of DNA sequences that favor or disfavor nucleosome occupancy increases following histone H3 depletion, demonstrating that nucleosome density contributes to moderating the influence of DNA sequence on nucleosome formation in vivo. To identify factors important for influencing nucleosome occupancy and position, we compared our data to 40 existing whole-genome data sets. Factors associated with promoters, such as histone acetylation and H2A.z incorporation, were enriched at sites of nucleosome loss. Nucleosome retention was linked to stabilizing marks such as H3K36me2. Notably, the chromatin remodeler Isw2 was uniquely associated with retained occupancy and altered positioning, consistent with Isw2 stabilizing histone–DNA contacts and centering nucleosomes on available DNA in vivo. RNA–seq revealed a greater number of de-repressed genes (∼2,500) than previous studies, and these genes exhibited reduced nucleosome occupancy in their promoters. In summary, we identify factors likely to influence nucleosome stability under normal growth conditions and the specific genomic locations at which they act. We find that DNA–encoded nucleosome stability and chromatin composition dictate which nucleosomes will be lost under conditions of limiting histone protein and that this, in turn, governs which genes are susceptible to a loss of regulatory fidelity.
Chromatin is formed by wrapping 146 bp of DNA around a disc-shaped complex of proteins called histones. These protein–DNA structures are known as nucleosomes. Nucleosomes help to regulate gene transcription, because nucleosomes compete with transcription factors for access to DNA. The precise positioning and level of nucleosome occupancy are known to be vital for transcriptional regulation, but the mechanisms that regulate the position and occupancy of nucleosomes are not fully understood. Recently, many studies have focused on the role of DNA sequence and chromatin remodeling proteins. Here, we manipulate the concentration of histone proteins in the cell to determine which nucleosomes are most susceptible to changes in occupancy and position. We find that the chromatin-associated proteins Sir2 and Tup1, and the chromatin remodelers Isw2 and Rsc8, are associated with stabilized nucleosomes. Histone acetylation and incorporation of the histone variant H2A.z are the factors most highly associated with destabilized nucleosomes. Certain DNA sequence properties also contribute to stability. The data identify factors likely to influence nucleosome stability and show a direct link between changes in chromatin and changes in transcription upon histone depletion.
We propose definitions and procedures for comparing nucleosome maps and discuss current agreement and disagreement on the effect of histone sequence preferences on nucleosome organization in vivo.
Next-generation sequencing-based assays to detect gene regulatory elements are enabling the analysis of individual-to-individual and allele-specific variation of chromatin status and transcription factor binding in humans. Recently, a number of studies have explored this area, using lymphoblastoid cell lines. Around 10% of chromatin sites show either individual-level differences or allele-specific behavior. Future studies are likely to be limited by cell line accessibility, meaning that white-bloodcell-based studies are likely to continue to be the main source of samples. A detailed understanding of the relationship between normal genetic variation and chromatin variation can shed light on how polymorphisms in non-coding regions in the human genome might underlie phenotypic variation and disease.
Insulin signaling has a profound effect on longevity and the oxidative stress resistance of animals. Inhibition of insulin signaling results in the activation of DAF-16/FOXO and SKN-1/Nrf transcription factors and increased animal fitness. By studying the biological functions of the endogenous RNA interference factor RDE-4 and conserved PHD zinc finger protein ZFP-1 (AF10), which regulate overlapping sets of genes in Caenorhabditis elegans, we identified an important role for these factors in the negative modulation of transcription of the insulin/PI3 signaling-dependent kinase PDK-1. Consistently, increased expression of pdk-1 in zfp-1 and rde-4 mutants contributed to their reduced lifespan and sensitivity to oxidative stress and pathogens due to the reduction in the expression of DAF-16 and SKN-1 targets. We found that the function of ZFP-1 in modulating pdk-1 transcription was important for the extended lifespan of the age-1(hx546) reduction-of-function PI3 kinase mutant, since the lifespan of the age-1; zfp-1 double mutant strain was significantly shorter compared to age-1(hx546). We further demonstrate that overexpression of ZFP-1 caused an increased resistance to oxidative stress in a DAF-16–dependent manner. Our findings suggest that epigenetic regulation of key upstream signaling components in signal transduction pathways through chromatin and RNAi may have a large impact on the outcome of signaling and expression of numerous downstream genes.
Reduced activity of the insulin-signaling pathway genes has been associated with a longer lifespan and increased resistance to oxidative stress in animals due to the activation of important transcription factors, which act as master regulators and affect large networks of genes. The ability to manipulate insulin signaling and reduce its activity may allow activation of oxidative-stress response programs in pathological conditions, such as neuronal degeneration, where oxidative stress plays a significant role. Here, we describe a new way of inhibiting insulin signaling that exists in the nematode Caenorhabditis elegans. We find that transcription of one of the insulin-signaling genes is inhibited by mechanisms involving chromatin and RNA interference, a silencing process that depends on short RNAs. We demonstrate that mutants deficient in RNA interference are more susceptible to stress due to increased insulin signaling and that increased dosage of a chromatin-binding protein repressing insulin signaling and promoting RNA interference leads to better survival of nematodes grown under oxidative stress conditions. Since there is a clear homolog of this chromatin-binding protein in mammals, it may also act to promote resistance to oxidative stress in human cells such as neurons.
ZINBA (Zero-Inflated Negative Binomial Algorithm) identifies genomic regions enriched in a variety of ChIP-seq and related next-generation sequencing experiments (DNA-seq), calling both broad and narrow modes of enrichment across a range of signal-to-noise ratios. ZINBA models and accounts for factors that co-vary with background or experimental signal, such as G/C content, and identifies enrichment in genomes with complex local copy number variations. ZINBA provides a single unified framework for analyzing DNA-seq experiments in challenging genomic contexts.
Software website: http://code.google.com/p/zinba/
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
We report testing of the specificity and utility of over 200 antibodies raised against 57 different histone modifications, in Drosophila melanogaster, Caenorhabditis elegans and human cells. While most antibodies performed well, over 25% failed specificity tests by dot blot or western blot. Among specific antibodies, over 20% failed in chromatin immunoprecipitation experiments. We advise rigorous testing of histone-modification antibodies before use and provide a website for posting new test results.