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. Here we tested 
the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and 
cohesin to establishment of locus control region (LCR)/β-globin proximity. 
CRISPR/Cas9 editing of the β-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 LDB1 complex and mediator occupancy and resulted in loss of 
LCR/β-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/β-globin proximity was restored 
without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 
to the β-globin promoter forced LCR loop formation in the absence of mediator or 
cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation 
results 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.