LDB1 Establishes Multi-Enhancer Networks to Regulate Erythroid Gene Expression

How specific enhancer-promoter pairing is established is still mostly unclear. While cohesin has been widely implicated in assisting CRE connectivity, it only influences a limited fraction of such contacts. Besides components of the CTCF/cohesin machinery, only a few nuclear factors have been studied via acute degradation to interrogate a direct/proximal role in physically connecting cis-regulatory elements (CRE). Here, we investigated the transcription co-factor LDB1 as a mediator of CRE connectivity. LDB1 is a widely expressed nuclear factor that is recruited to specific genomic sites by lineage specific transcription factors such as GATA1 and TAL1. Using an acute degradation system combined with Micro-C, we found that LDB1 directly and broadly promotes enhancer-promoter and enhancer-enhancer contacts to drive erythroid gene expression. To explore LDB1’s interplay, if any, with cohesin, CTCF, and the transcription factor YY1 (which has been implicated in CRE interactions), we engineered acute degrons for all these factors. Notably, most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin or YY1. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1 occupied sites but aids in the formation of a subset of LDB1 dependent loops. Thus, structural loops and the process of cohesin-driven chromatin loop extrusion have a limited influence on regulatory connectivity established by LDB1. Leveraging the extensive and highly dynamic reorganization of nuclear architecture during the transition from mitosis to G1-phase, we defined the relationship between LDB1-dependent CRE interactions and contacts formed via cohesin-driven loop extrusion. During the mitosis-to-G1-phase transition, many LDB1 dependent CRE interactions formed prior to cohesin-driven contacts, demonstrating LDB1’s ability to newly forge long range contacts independently of loop extrusion. Tri-C and ultra-high-resolution Region Capture Micro-C revealed that LDB1 organizes complex multi-enhancer networks to activate erythroid transcription. Lastly, super-resolution microscopy showed that LDB1 forms multi-molecular clusters which we speculate underly LDB1’s ability to coordinate multi-enhancer interactions. Together, this work establishes LDB1 as a direct and potent driver of regulatory network inter-connectivity in erythroid cells and likely other lineages as well.