NLI/Ldb1, a widely expressed nuclear factor, is a potential mediator of long range interaction between enhancers and target genes. We have shown that Ldb1 and erythroid partners SCL, GATA-1 and LMO2 form a complex that is required for beta-globin transcription and for chromatin looping between the gene and the beta-globin LCR enhancer. In recent work, we have discovered that elongation competent Ser-2P pol II and its kinase P-TEFb co-occupy the LCR and beta-globin promoter with the same kinetics as Ldb1 and that Ldb1 is required for their recruitment. Ldb1 complex formation on the beta-globin promoter and Ser-2P pol II recruitment are independent of the LCR since they are unaffected in fetal liver erythroid cells of mice with a deletion of the LCR despite strongly reduced transcription. These results indicate that the LCR and Ldb1 each provide a critical function required for robust transcription of beta-globin that is manifest at the time of, or following, the establishment of a chromatin loop between the LCR and gene. In addition, using mouse Ldb1 null ES cells we observe that both embryonic and adult globins normally require Ldb1. Thus, it is likely that Ldb1 provides its critical function during both primitive and definitive erythropoiesis. In other experiments, we explored the mechanisms underlying enhancer blocking by insulators. We found that human beta-globin HS5, the orthologue of the CTCF dependent chicken HS4 insulator, has intrinsic, portable enhancer blocking activity that is manifest though chromatin loop formation. In fact, looping between two CTCF sites engineered to surround the beta-globin LCR topologically isolates the LCR from its target globin genes and nullifies its enhancer function. To investigate whether the looping activity of CTCF sites is a general property of these sites in the genome, we carried out chromatin conformation capture (3C) on CTCF/cohesin sites over 2 Mb on chromosome 11 encompassing beta-globin locus and flanking olfactory receptor genes. We found that the interaction frequencies among the sites are highly cell type specific revealing a more densely clustered organization of CTCF sites in the absence of globin gene activity. These results document a general chromosome organizational role for CTCF/cohesin sites through loop formation that relies on both for maintenance and the disruption of which can have functional consequences dependent on surrounding chromatin.
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