Unknown,Transcriptomics,Genomics,Proteomics

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Epigenetic and genetic features that lead to discovery of enhancer function

ABSTRACT: The ability to measure epigenetic features, such as histone modifications and occupancy by transcription factors and co-activators, on a genome-wide scale is advancing the accuracy of CRM predictions. While integration of signals from multiple features is expected to improve predictions, the contribution of each feature to prediction accuracy is not known. We began with predictions of 4,915 erythroid enhancers based on genomic occupancy by TAL1, a key hematopoietic transcription factor that is strongly associated with gene induction in erythroid cells. Seventy of these DNA segments occupied by TAL1 (TAL1 OSs) were tested by transient transfections of cultured hematopoietic cells, and 56% of these were active as enhancers. Sixty-six TAL1 OSs were evaluated in transgenic mouse embryos, and 65% of these were active enhancers in various tissues. Inclusion of additional epigenetic features improved the prediction accuracy, with combinations of TAL1, GATA1, EP300, H3K4me1, and H3K27ac giving high accuracy of enhancer prediction (70%-75% success depending on method of clustering) while maintaining good sensitivity and specificity. Motifs that distinguish active from inactive TAL1 OSs implicate IRFs, STATs, and FOX protein families as candidate positive co-factors with TAL1, while REST (NRSF) and HOX family proteins are implicated in inactivity. While signals for evolutionary constraint were weak over the entire TAL1-bound DNA segments regardless of activity in either assay, phylogenetic preservation of a TF-binding site motif was associated with enhancer activity. The contribution of 8 epigenetic features including H3K27ac to identification of enhancers in 24h-induced G1E-ER4 cells.

ORGANISM(S): Mus musculus

SUBMITTER: Ross Hardison

PROVIDER: E-GEOD-61349 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO. The cells were further cultured in the presence of EPO, and formaldehyde crosslinked chromatin was isolated after cells differentiated into R3/R4 nucleated erythroid cells. Chromatin Immunoprecipitation followed by sequencing (chIP-seq) was performed using antibodies against GATA1, KLF1, NFE2, TAL1, p300, H3K4me2 and H3K4me3, along with a total input control. Raw data (fastq, SRA) is missing for the TAL1 chIP-seq dataset

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Epigenetic and genetic features that lead to discovery of enhancer function

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