Biased, non-equivalent gene-proximal and -distal binding motifs of orphan nuclear receptor TR4 in primary human erythroid cells.

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.

Developmental transcriptome analysis of human erythropoiesis.

To globally survey the changes in transcriptional landscape during terminal erythroid differentiation, we performed RNA sequencing (RNA-seq) on primary human CD34(+) cells after ex vivo differentiation from the earliest into the most mature erythroid cell stages. This analysis identified thousands of novel intergenic and intronic transcripts as well as novel alternative transcript isoforms. After rigorous data filtering, 51 (presumptive) novel protein-coding transcripts, 5326 long and 679 small non-coding RNA candidates remained. The analysis also revealed two clear transcriptional trends during terminal erythroid differentiation: first, the complexity of transcript diversity was predominantly achieved by alternative splicing, and second, splicing junctional diversity diminished during erythroid differentiation. Finally, 404 genes that were not known previously to be differentially expressed in erythroid cells were annotated. Analysis of the most extremely differentially expressed transcripts revealed that these gene products were all closely associated with hematopoietic lineage differentiation. Taken together, this study will serve as a comprehensive platform for future in-depth investigation of human erythroid development that, in turn, may reveal new insights into multiple layers of the transcriptional regulatory hierarchy that controls erythropoiesis.