Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE

Summary Members of transcription factor families typically have similar DNA binding specificities yet execute unique functions in vivo. Transcription factors often bind DNA as multiprotein complexes, raising the possibility that complex formation might modify their DNA binding specificities. To test this hypothesis, we developed an experimental and computational platform, SELEX-seq, that can be used to determine the relative affinities to any DNA sequence for any transcription factor complex. Applying this method to all eight Drosophila Hox proteins, we show that they obtain novel recognition properties when they bind DNA with the dimeric cofactor Extradenticle-Homothorax (Exd). Exd-Hox specificities group into three main classes that obey Hox gene collinearity rules and DNA structure predictions suggest that anterior and posterior Hox proteins prefer DNA sequences with distinct minor groove topographies. Together, these data suggest that emergent DNA recognition properties revealed by interactions with cofactors contribute to transcription factor specificities in vivo.

Transcription factors (TFs) influence cell fate by interpreting the regulatory DNA within a genome. TFs recognize DNA in a specific manner; the mechanisms underlying this specificity have been identified for many TFs, based on three-dimensional structures of protein-DNA complexes. More recently, structural views have been complemented with data from high-throughput in vitro and in vivo explorations of the DNA binding preferences of many TFs. Together, these approaches have greatly expanded our understanding of TF-DNA interactions. However, the mechanisms by which TFs select in vivo binding sites and alter gene expression remain unclear. Recent work has highlighted the many variables that influence TF-DNA binding, while demonstrating that a biophysical understanding of these many factors will be central to understanding TF function.

The accurate control of cell proliferation and survival is critical for animal development. The Hippo tumor suppressor pathway regulates both of these parameters by controlling the nuclear availability of the transcriptional coactivator Yorkie (Yki), which regulates downstream target genes together with Scalloped (Sd), a DNA-binding protein. Here we provide evidence that Yki can also regulate target genes in conjunction with Homothorax (Hth) and Teashirt (Tsh), two DNA-binding transcription factors expressed in the uncommitted progenitor cells of the Drosophila eye imaginal disc. Clonal analyses demonstrate that Hth and Tsh promote cell proliferation and protect eye progenitor cells from apoptosis. Genetic epistasis experiments suggest that Hth and Tsh execute these functions with Yki, in part by up-regulating the microRNA bantam. A physical interaction between Hth and Yki can be detected in cell culture, and we show that Hth and Yki are bound to a DNA sequence ;14 kb upstream of the bantam hairpin in eye imaginal disc cells, arguing that this regulation is direct. These data suggest that the Hippo pathway uses different DNA-binding transcription factors depending on the cellular context. In the eye disc, Hth and Tsh provide spatial information to this pathway, promoting cell proliferation and survival in the progenitor domain.[Keywords: Drosophila eye development; Hippo signaling pathway; Homothorax; Yorkie; apoptosis; cell proliferation] Supplemental material is available at http://www.genesdev.org.

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.

SummaryDespite the large evolutionary distances, metazoan species show remarkable commonalities, which has helped establish fly and worm as model organisms for human biology1,2. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. We mapped the genome-wide binding locations of 165 human, 93 worm, and 52 fly transcription-regulatory factors (RFs) generating a total of 1,019 data sets from diverse cell-types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous RF families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding in the regulatory underpinnings of model organism biology and how these relate to human biology, development, and disease.

Summary Protein-DNA binding is mediated by the recognition of the chemical signatures of the DNA bases and the three-dimensional shape of the DNA molecule. Because DNA shape is a consequence of sequence, it is difficult to dissociate these modes of recognition. Here, we tease them apart in the context of Hox-DNA binding by mutating residues that, in a co-crystal structure, only recognize DNA shape. Complexes made with these mutants lose the preference to bind sequences with specific DNA shape features. Introducing shape-recognizing residues from one Hox protein to another swapped binding specificities in vitro and gene regulation in vivo. Statistical machine learning revealed that the accuracy of binding specificity predictions improves by adding shape features to a model that only depends on sequence, and feature selection identified shape features important for recognition. Thus, shape readout is a direct and independent component of binding site selection by Hox proteins.

SUMMARY The Hippo pathway regulates growth through the transcriptional co-activator Yorkie, but how Yorkie promotes transcription remains poorly understood. We address this by characterizing Yorkie’s association with chromatin, and by identifying nuclear partners that effect transcriptional activation. Co-immunoprecipitation and mass spectrometry identify GAGA Factor (GAF), Brahma complex, and Mediator complex as Yorkie-associated nuclear protein complexes. All three are required for Yorkie’s transcriptional activation of downstream genes, and GAF and the Brahma complex subunit Moira interact directly with Yorkie. Genome-wide chromatin binding experiments identify thousands of Yorkie sites, most of which are associated with elevated transcription, based on genome-wide analysis of mRNA and histone H3K4Me3 modification. Chromatin binding also supports extensive functional overlap between Yorkie and GAF. Our studies suggest a widespread role for Yorkie as a regulator of transcription, and identify recruitment of the chromatin modifying GAF protein and BRM complex as a molecular mechanism for transcriptional activation by Yorkie.