Biased, Non-equivalent Gene-Proximal and -Distal Binding Motifs of Orphan Nuclear Receptor TR4 in Primary Human Erythroid Cells

<div><p>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 <i>GATA1</i> 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 <i>via</i> 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.</p></div

Number and percentage of epigenetic enhancer marks that co-localize with TR4 distal peaks.^*

<p>*Within ±2 Kbp of TR4 distal peaks.</p

Sequence conservation among TR4 peaks.

<p>(<b>A</b>) The percentage of peaks located >10 Kbp from the nearest RefSeq genes decreased from D8 to D14 of differentiation. (<b>B</b>) For peaks located at the proximal promoter, the average PhastCons score of each nucleotide within a peak (500 bp from peak center) across vertebrate, mammalian or primate species are graphed. The center of each peak is defined as “0”. (<b>C</b>) Comparison of the average PhastCons scores of peak sequences and random control sequences in the TR4 peaks located at proximal promoter, >10 Kbp from genes or in all identified peaks at D14 differentiation.</p

The expression of genes with TR4 bound at proximal promoter is reduced after TR4 depletion.

<p>(<b>A</b>) The distribution of the expression fold change between genes with TR4 bound in the proximal promoter region (dashed red lines) <i>vs</i>. that of all expressed genes (solid black line) after TR4 depletion by either lentivirus sh#174 (right panel) or lentivirus sh#658 (left panel), where positive values indicate an increase in expression after TR4 depletion. (<b>B</b>) Genes with TR4 bound at the proximal promoter are enriched in basic biological functions.</p

Genome-wide distribution of TR4 binding sites in differentiating human erythroid cells.

<p>(<b>A</b>) TR4 peak assignment is based on the distance from the peak center to the nearest transcription start site (TSS) of RefSeq genes. Once a peak has been assigned to the nearest gene, its location is classified into: within the gene (from TSS to transcription end site (TES)), 5′ upstream or 3′ downstream. Peaks in the 5′ upstream regions are further grouped into: promoter (from −0.001 to −2 Kbp), 5′ distal I (from −2 to −10 Kbp 5′) and 5′ distal II (from −10 to −100 Kbp 5′), and peaks located 3′ of the TES are grouped as 3′ proximal (from TES to 2 Kbp after TES), 3′ distal I (from 2 to 10 Kbp after TES) and 3′ distal II (from 10 to 100 Kbp 3′ to TES). Peaks >100 Kbp from TSS or TES are reported here to fall within gene deserts. (<b>B</b>) Distribution of TR4 binding peaks across the genome in day 8 (D8), 11 (D11) and 14 (D14) erythroid cells. (<b>C</b>) TR4 binding peaks that fall within genes are mapped within those gene exons and introns. Here, only the first 5 exons and the first 10 introns are shown. (<b>D</b>) Histogram illustrating the distribution of peaks in a window ±1 Kbp from the TSS (proximal promoter) at D8, D11 and D14 of erythroid differentiation. Peaks were combined into 15 bp bins. (<b>E</b>) Percentage of peaks that mapped to proximal promoter in D8, D11 and D14 differentiated erythroid cells.</p

Characterization of TR4 downstream targets with lentiviral-mediated shRNAs.

<p>(<b>A</b>) The upper panel shows the immunoblots of TR4 and β-actin (internal control) in shRNA lentivirus (#174 and #658) or control virus infected cells at D11. The bar graph shows the relative abundance of TR4 normalized to β-actin and to control cells (*<i>p</i><0.05 and error bars represent s.e.m.). (<b>B</b>) Venn diagram summarizing the common and unique differentially expressed genes after TR4 depletion with lentivirus sh#174 and sh#658. (<b>C</b>) The enriched GO terms for TR4 repressed and activated genes, respectively.</p

Total raw reads and peaks from TR4 ChIP-seq analyses of differentiating human erythroid cells.

<p>Total raw reads and peaks from TR4 ChIP-seq analyses of differentiating human erythroid cells.</p

Identification of proteins precipitated with streptavidin beads from MEL cells expressing biotin-tagged TR4.

<p>Nuclear extracts were prepared from MEL cells expressing the biotin ligase gene (birA) without (control) or with biotin-tagged TR4 and then incubated with streptavidin beads. Proteins precipitated with the beads (Bound), 3∼6% of the input, and equal amount of supernatants (Sup) were subjected to SDS-PAGE, followed by immunoblotting with antibodies that recognize TR2, RXR and RAR.</p

Distribution of potential NR binding sites in TR4-bound peaks.

<p>Peak sequences (250 bp from peak center) were interrogated using NHR-scan for the presence of direct repeat (DR), everted repeat (ER) and inverted repeat (IR) motifs with 0–8 spacer nucleotides in D8, D11 and D14 (<b>A</b>, <b>B</b>) or in D8 (<b>C</b>) cells. The percentages of each motif type in peaks located at the proximal promoter (<b>A</b>), in all peaks (<b>B</b>), and in peaks at proximal promoter <i>vs</i>. >10 Kbp from genes (<b>C</b>) are represented graphically.</p

<i>De novo</i> motif analysis of TR4 peaks located at the proximal promoter.

<p>Motif analysis of peak sequences (250 bp from peak center) identifies DR1 (<b>A</b>) and ETS (<b>B</b>) motifs as overrepresented among the peaks located at the proximal promoter in D14 erythroid cells.</p