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.