A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation

Abstract

Retinoic acid (RA) induces rapid differentiation of embryonic stem cells (ESCs), partly by activating expression of the transcription factor Hoxa1, which regulates downstream target genes that promote ESCs differentiation. However, mechanisms of RA-induced Hoxa1 expression and ESCs early differentiation remain largely unknown. Here, we identify a distal enhancer interacting with the Hoxa1 locus through a long-range chromatin loop. Enhancer deletion significantly inhibited expression of RA-induced Hoxa1 and endoderm master control genes such as Gata4 and Gata6. Transcriptome analysis revealed that RA-induced early ESCs differentiation was blocked in Hoxa1 enhancer knockout cells, suggesting a requirement for the enhancer. Restoration of Hoxa1 expression partly rescued expression levels of ∼40% of genes whose expression changed following enhancer deletion, and ∼18% of promoters of those rescued genes were directly bound by Hoxa1. Our data show that a distal enhancer maintains Hoxa1 expression through long-range chromatin loop and that Hoxa1 directly regulates downstream target genes expression and then orchestrates RA-induced early differentiation of ESCs. This discovery reveals mechanisms of a novel enhancer regulating RA-induced Hoxa genes expression and early ESCs differentiation.

Contribution of CTCF binding to transcriptional activity at theHOXAlocus inNPM1-mutant AML cells

“…Specifically, long-range loops were diminished, and were replaced by compensatory interactions with regions of the SKAP2 gene. Some of these candidate enhancers have been reported in other studies (32), but have not previously been shown to be active in hematopoietic cells. Further investigation of these sequences, and their associated regulatory proteins, may shed light into factors that promote HOXA gene activation in normal and malignant myeloid cells.…”

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

“…To investigate whether CBE +47 has a regulatory effect, CRISPR-Cas9 mediated deletion method was used for further research ( Su et al, 2019 ). Two sgRNAs were designed in upstream and downstream of CBE +47 ( Figure 2A ), after Cas9 cleavage and DNA recombination, we obtained two homozygous CBE +47 knockout cell lines (CBE +47 -KO).…”

“…Studies by us and others have found that there are three enhancers at the 3’-end of HoxA ( Figure 5A ). The three enhancers (e.g., E1, E2 and E3) are required for HoxA expression during RA-induced ESC differentiation ( Cao et al, 2017; Liu et al, 2016; Su et al, 2019; Yin et al, 2015 ). Among them, it has been reported that E1 and E3 have significant long-range interactions with HoxA chromatin.…”

“…Many experimental techniques, such as Capture-C, have been widely used to study the precise chromatin interactions between enhancers and target genes ( Davies et al, 2016; Hughes et al, 2014; Liang et al, 2020; Qian et al, 2019; Su et al, 2019; Zhang et al, 2020 ). We used the Capture-C method to study different interactions between enhancers and HoxA chromatin in WT and CBE +47 -KO cells.…”

“…Recent studies have shown that CBE plays the core regulatory roles in tumorigenesis ( Li et al, 2020; Huacheng Luo et al, 2018 ), immunogenesis ( Ba et al, 2020; Jain, Ba, Zhang, Dai, & Alt, 2018 ) and stem cell development ( Arzate-Mejia, Recillas-Targa, & Corces, 2018; H. Zheng & Xie, 2019 ) by controlling target genes expression through organizing high-order chromatin structures. We and other research groups have found that multiple enhancers are required for RA-induced HoxA expression and early ESC differentiation by long-range chromatin interactions with HoxA ( Cao et al, 2017; Liu et al, 2016; Su et al, 2019; Yin et al, 2015 ). Besides, previous studies also have reported many regulatory patterns for HoxA activation during ESC differentiation, among them, several CBE (also known as CBS, CTCF-binding site) within HoxA locus can organize HoxA chromatin structures, and play significant regulatory roles in ESC differentiation and leukemogenesis ( Ghasemi, Struthers, Wilson, & Spencer, 2020; Huacheng Luo et al, 2018; Narendra et al, 2015 ).…”