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.

The orphan nuclear receptor TR4 regulates erythroid cell proliferation and maturation

“…To investigate the mechanisms that result in these observed deficiencies in erythroid cell physiology, we compared these results characterizing the murine mutants with previously published human RNA-seq data generated by our laboratory in which CD34 1 cells were induced to undergo erythroid differentiation with or without infection with a lentivirus containing short hairpin RNA (shRNA) against TR4 (shRNA-TR4) or a scrambled control (shRNA-Ctrl, GSE5476). 36 Differentially expressed genes (when compared with shRNA-Ctrl) whose function has been described in erythroid proliferation and differentiation were analyzed as candidate TR4 target genes. 36 shRNA-TR4 in the human erythroid progenitor cells resulted in a 50% to 60% reduction in the abundance of TR4 transcripts (notably without altering TR2 expression; Figure 6A).…”

“…36 Differentially expressed genes (when compared with shRNA-Ctrl) whose function has been described in erythroid proliferation and differentiation were analyzed as candidate TR4 target genes. 36 shRNA-TR4 in the human erythroid progenitor cells resulted in a 50% to 60% reduction in the abundance of TR4 transcripts (notably without altering TR2 expression; Figure 6A). As anticipated, one known TR4 direct target gene, 4 human «-globin, exhibited increased expression after TR4 knockdown, although there was no change observed in adult b-globin (b) transcription (Figure 6B).…”

Genome Wide Mapping of NR4A Binding Reveals Cooperativity with ETS Factors to Promote Epigenetic Activation of Distal Enhancers in Acute Myeloid Leukemia Cells

“…The association of nuclear receptors with ETS factor motifs in various hematopoietic cells has previously been reported on a genome wide scale for vitamin D receptor and the orphan nuclear receptor TR4 [ 64 , 65 ], but the importance or functionality of this potential cooperation has not been addressed in detail. However, treatment of t(16;21) AML with all-trans retinoic acid (ATRA) has recently been shown to recruit ERG to new genome-wide binding sites, suggesting that the RARa/RXR heterodimer is capable of reprogramming ERG binding.…”

Fetal Globin Gene Repressors as Drug Targets for Molecular Therapies To Treat the β-Globinopathies

“…In erythroid cells, hundreds of genes are repressed (either directly or indirectly) by TR4, while even more genes are activated (58). Analysis of erythroid differentiation in comparative data sets from chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA-seq experiments indicated that the DR motif is more prevalent in the proximal promoters of TR4 direct target genes that are involved in basic biological functions (e.g., mRNA processing, ribosomal assembly, RNA splicing, and primary metabolic processes [58,59]). We also previously identified TRIM28 as an interacting protein of TR2/TR4 (39).…”