Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. We tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/beta-globin proximity. CRISPR/Cas9 editing of the beta-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated the LDB1 complex and mediator occupancy and resulted in loss of LCR/beta-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/beta-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the beta-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our ChIP-seq data indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Lineage-specific transcription factors are critical for long-range enhancer interactions but direct or indirect contributions of architectural proteins such as CTCF to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the beta-globin locus through LDB1 self-interaction. We find that an LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1 deficient cells. Genome wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression. Long non-coding RNAs (lncRNA) are increasingly being appreciated as participants in regulation of important cellular processes, including transcription. Because lncRNAs are highly cell-type specific, they have the potential to contribute to the unique transcriptional repertoire of diverse cells, but underlying mechanisms are unclear. We studied BGLT3, an erythroid lncRNA encoded downstream of Agamma-globin (HBG1). BGLT3 and gamma-globin genes are dynamically co-transcribed in erythroid cells in vivo. Deletion of BGLT3 using CRISPR/Cas9 editing shows that it specifically contributes to regulation of gamma-globin genes. We used reduction or over-expression of the RNA and inhibition of transcription through the locus by CRISPRi to distinguish functions of the transcript versus the underlying sequence. Transcription of the BGLT3 locus is critical for looping between the gamma-globin genes and BGLT3 sequences. In contrast, the BGLT3 transcript is dispensable for gamma-globin/BGLT3 looping but interacts with the Mediator complex on chromatin. Manipulation of the BGLT3 locus does not compromise gamma-globin gene long-range looping interactions with the beta-globin locus control region (LCR). These data reveal that BGLT3 regulates gamma-globin transcription in a developmental stage-specific fashion together with the LCR by serving as a separate means to increase RNA Pol II density at the gamma-globin promoters.
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