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.我们此前已有研究报道，TR2与TR4孤儿核受体可结合ε-珠蛋白和γ-珠蛋白启动子中的同向重复（direct repeat, DR）元件，并作为分子锚点招募多组分DRED复合物的表观遗传共阻遏蛋白，进而在定型红细胞生成过程中介导ε-珠蛋白与γ-珠蛋白的转录抑制。除ε-珠蛋白、γ-珠蛋白及GATA1基因外，人红系细胞中TR4调控的靶基因仍未明确。本研究通过染色质免疫共沉淀联合高通量测序（chromatin immunoprecipitation followed by massively parallel sequencing, ChIP-seq），在逐步分化为晚期红系前体细胞的人原代CD34阳性造血祖细胞中，全基因组范围鉴定了TR4的结合位点。同时，我们在红系细胞中通过慢病毒介导的TR4短发夹RNA（short hairpin RNA, shRNA）敲低后，利用RNA测序（RNA-seq）开展全转录组分析，以鉴定TR4的下游靶基因。联合分析ChIP-seq与RNA-seq数据集后发现，DR1基序在TR4直接靶基因的近端启动子中更为富集，这些靶基因参与基础生物学功能（如mRNA加工、核糖体组装、RNA剪接及初级代谢过程）。与之相反，其他非DR1重复基序（DR4、ER6及IR1）则更多出现在基因远端的TR4结合位点中。其中约50%的远端结合位点还带有与增强子功能相关的表观遗传染色质标记（如P300、H3K27ac、H3K4me1及H3K27me3）。据此我们提出假说：TR4以TR4/TR2异二聚体的形式通过基因近端的DR1位点调控基因转录，同时也可与新型核受体伴侣蛋白（维A酸X受体，retinoid X receptor, RXR）结合，从而识别远端的非DR1共识结合位点。综上，本研究揭示TR4的调控网络远比此前认知更为复杂，且在人红系细胞终末分化过程中，TR4可调控基础且必需的生物学过程。