To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

The transcription factor GATA1 regulates an extensive program of gene activation and repression during erythroid development. However, the associated mechanisms, including the contributions of distal versus proximal cis-regulatory modules, co-occupancy with other transcription factors, and the effects of histone modifications, are poorly understood. We studied these problems genome-wide in a Gata1 knockout erythroblast cell line that undergoes GATA1-dependent terminal maturation, identifying 2616 GATA1-responsive genes and 15,360 GATA1-occupied DNA segments after restoration of GATA1. Virtually all occupied DNA segments have high levels of H3K4 monomethylation and low levels of H3K27me3 around the canonical GATA binding motif, regardless of whether the nearby gene is induced or repressed. Induced genes tend to be bound by GATA1 close to the transcription start site (most frequently in the first intron), have multiple GATA1-occupied segments that are also bound by TAL1, and show evolutionary constraint on the GATA1-binding site motif. In contrast, repressed genes are further away from GATA1-occupied segments, and a subset shows reduced TAL1 occupancy and increased H3K27me3 at the transcription start site. Our data expand the repertoire of GATA1 action in erythropoiesis by defining a new cohort of target genes and determining the spatial distribution of cisregulatory modules throughout the genome. In addition, we begin to establish functional criteria and mechanisms that distinguish GATA1 activation from repression at specific target genes. More broadly, these studies illustrate how a ''master regulator'' transcription factor coordinates tissue differentiation through a panoply of DNA and protein interactions.

Isaac has an open source license and can be obtained at https://github.com/sequencing.

Interplays among lineage-specific nuclear proteins, chromatin modifying enzymes, and the basal transcription machinery govern cellular differentiation, but their dynamics of action and coordination with transcriptional control are not fully understood. Alterations in chromatin structure appear to establish a permissive state for gene activation at some loci, but they play an integral role in activation at other loci. To determine the predominant roles of chromatin states and factor occupancy in directing gene regulation during differentiation, we mapped chromatin accessibility, histone modifications, and nuclear factor occupancy genome-wide during mouse erythroid differentiation dependent on the master regulatory transcription factor GATA1. Notably, despite extensive changes in gene expression, the chromatin state profiles (proportions of a gene in a chromatin state dominated by activating or repressive histone modifications) and accessibility remain largely unchanged during GATA1-induced erythroid differentiation. In contrast, gene induction and repression are strongly associated with changes in patterns of transcription factor occupancy. Our results indicate that during erythroid differentiation, the broad features of chromatin states are established at the stage of lineage commitment, largely independently of GATA1. These determine permissiveness for expression, with subsequent induction or repression mediated by distinctive combinations of transcription factors.

Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map transfactor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1. (Blood. 2011;118(17):e139-e148) IntroductionMore than 2 million red blood cells are released into the circulation every second by a multistep process known as erythropoiesis, 1 which is accompanied by significant changes in the RNA expression profile. 2 The erythroid-specific DNA binding protein erythroid Kruppel-like factor (Klf1; EKLF), the founding member of the mammalian Kruppel/Sp1-like family of C2H2-type zinc finger DNA binding proteins, plays a significant role in this process. 3,4 Klf1 mRNA and EKLF are expressed in both erythroid progenitor cells and terminally differentiating erythrocytes. 5,6 In cell lines and in vitro, EKLF has been demonstrated to physically and functionally interact with DNA at a conserved CCNCNCCCN motif and with additional protein cofactors. 7,8 In EKLF-deficient (Klf1 Ϫ/Ϫ ) mice, definitive fetal liver erythroid progenitor cells fail to progress to erythroblasts, 9 leading to anemia and lethality by embryonic day 15. 10,11 Genome-wide mRNA profiling of WT and Klf1 Ϫ/Ϫ erythroid cells has revealed significant dysregulation of Ͼ 3000 mRNAs. 9,12-15 Two-thirds of the dysregulated transcripts were present at reduced levels in Klf1 Ϫ/Ϫ cells, consistent with the role of EKLF as a transcriptional activator, whereas one-third were present at increased levels, consistent with the role of EKLF as a transcriptional repressor. 16 Chromatin immunoprecipitation (ChIP) demonstrated EKLF occupancy at selected sites in genes encoding erythrocyte proteins AHSP, ankyrin, ␤-spectrin, Band 3, and dematin, all of which are down-regulated in EKLF-deficient cells. 9,12-15 EKLF also participates in chromatin modification and DNase hypersensitive site formation through interactions with CBP/p300 and an SWI/SNFrelated chromatin remodeling complex. 17,18 For example,...