title: Biased, non-equivalent gene-proximal and -distal binding motifs of orphan nuclear receptor TR4 in primary human erythroid cells.
abstract: We previously reported that TR2 and TR4 orphan nuclear receptors bind to direct repeat (DR) elements in the ε- and γ-globin promoters, and act as molecular anchors for the recruitment of epigenetic corepressors of the multifaceted DRED complex, thereby leading to ε- and γ-globin transcriptional repression during definitive erythropoiesis. Other than the ε- and γ-globin and the GATA1 genes, TR4-regulated target genes in human erythroid cells remain unknown. Here, we identified TR4 binding sites genome-wide using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) as human primary CD34(+) hematopoietic progenitors differentiated progressively to late erythroid precursors. We also performed whole transcriptome analyses by RNA-seq to identify TR4 downstream targets after lentiviral-mediated TR4 shRNA knockdown in erythroid cells. Analyses from combined ChIP-seq and RNA-seq datasets indicate that DR1 motifs are more prevalent in the proximal promoters of TR4 direct target genes, which are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing and primary metabolic processes). In contrast, other non-DR1 repeat motifs (DR4, ER6 and IR1) are more prevalent at gene-distal TR4 binding sites. Of these, approximately 50% are also marked with epigenetic chromatin signatures (such as P300, H3K27ac, H3K4me1 and H3K27me3) associated with enhancer function. Thus, we hypothesize that TR4 regulates gene transcription via gene-proximal DR1 sites as TR4/TR2 heterodimers, while it can associate with novel nuclear receptor partners (such as RXR) to bind to distant non-DR1 consensus sites. In summary, this study reveals that the TR4 regulatory network is far more complex than previously appreciated and that TR4 regulates basic, essential biological processes during the terminal differentiation of human erythroid cells.
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Lihong
authors.initials: L
authors.lastname: Shi
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: M C
authors.initials: MC
authors.lastname: Sierant
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America; Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Katherine
authors.initials: K
authors.lastname: Gurdziel
authors.affiliation: Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhu
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Shuaiying
authors.initials: S
authors.lastname: Cui
authors.affiliation: PharmaMatch, Amsterdam, Netherlands.
authors.forename: Katarzyna E
authors.initials: KE
authors.lastname: Kolodziej
authors.affiliation: Institute of Molecular Oncology, BSRC Alexander Fleming, Varkiza, Greece.
authors.forename: John
authors.initials: J
authors.lastname: Strouboulis
authors.affiliation: Department of Computational Medicine and Bioinformatics, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Yuanfang
authors.initials: Y
authors.lastname: Guan
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America; Department of Integrative Genomics, Tohoku Medical Megabank, Tohoku University, Sendai, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Tanabe
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: Kim-Chew
authors.initials: KC
authors.lastname: Lim
authors.affiliation: Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
authors.forename: James Douglas
authors.initials: JD
authors.lastname: Engel
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D004742:Enhancer Elements, Genetic; D041905:Erythroid Cells / Q000378:metabolism*; D015894:Genome, Human; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: C472782:NR2C2AP protein, human; D009687:Nuclear Proteins; D012097:Repressor Proteins