Chromatin Structure In Regulation Of Mammalian Gene Expression

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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. Using mice homozygous for a deletion of the LCR, we demonstrated that these components are recruited to the beta-globin promoter independent of the LCR. However, occupancy by the Ldb1 complex is insufficient for high level transcription, highlighting the importance of the LCR/gene long range interaction. In addition, reduction of Ldb1 levels using RNAi results in the inability of the locus to migrate away from the nuclear periphery which is necessary to achieve robust transcription of beta-globin in nuclear transcription factories. These data suggest chromatin loop formation promotes high level transcription because it is required for mediating intra-nuclear migration. 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 the 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. To assess the function of the CTCF dependent loops, we reduced CTCF using RNAi in cells actively transcribing the fetal -globin genes. Expression of gamma-globin was strongly reduced in concert with the acquisition of repressive histone marks in the locus. These data provide an example of the regulatory importance of CTCF sites in the human genome to define functional domains and document a general chromosome organizational role for CTCF/cohesin sites through loop formation.