Understanding the complex regulation of beta-globin genes is critically important to design therapeutic approaches to beta-thalassemia and sickle cell disease. We have shown that a complex of erythroid proteins GATA-1, LMO2 and TAL1 as well as the more widely expressed nuclear factor Ldb1 mediates beta-globin gene looping to the distant locus control region (LCR) enhancer, which is required for transcription activation. Dimerization of Ldb1 was required for looping and beta-globin transcription rescue in the background of Ldb1 knock down erythroid cells. Deletion of a small conserved region within the dimerization domain yields an Ldb1 mutant protein that can dimerize and rescue long-range LCR/beta-globin looping but is unable to rescue RNA pol II occupancy or beta-globin gene expression. This result indicates that looping does not require transcription. We are studying the functions of this small domain. ChIP experiments now suggest the necessity of this small region for SWI/SNF, NuRD and FOG1 recruitment. Using RNA-seq, Ldb1-dependent genes sensitive and insensitive for loss of this region have been identified. The results so far suggest that in addition to establishing long-range interactions, Ldb1 serves as transcription activator. The insulator binding protein CTCF is also known to be involved in long range interaction at several different loci, primarily as an enhancer blocker. We found that CTCF and LDB1 are cooperatively involved in chromatin loop formation and gene transcription activation at the Ldb1-dependent carbonic anhydrase 2 (CA2) gene. By performing 3C, we observed that intergenic LDB1 binding sites interact with the CA2 promoter which is a CTCF binding site. Enhancer activity of the intergenic sites was confirmed by dual-luciferase assay and enhancer deletion assay using the CRISPR-Cas system. To verify this interaction we performed dual cross-linking ChIP and showed LDB1 occupancy at CTCF sites and vice versa. CA2 is strongly down-regulated by knockdown of either LDB1 or CTCF in mouse erythroid leukemia (MEL) cells. We are studying the domains of LDB1 and CTCF required for their interaction. These experiments suggest that LDB1 and CTCF can cooperate directly in chromatin loop formation and gene transcription. The principles underlying the architectural landscape of chromosomes in living cells remain largely unknown despite its potential to play a role in mammalian gene regulation. We investigated the 3-dimensional conformation of a 1 Mbp region of human chromosome 11 containing the beta-globin genes by integrating looping interactions of the insulator protein CTCF determined comprehensively by 3C into a polymer model of chromatin folding . Knock down of CTCF showed that regional CTCF contacts in mammalian nuclei functionally affect spatial distances between globin genes and their control elements and, hence, contribute to chromosomal reorganization required for transcription. We are using Talens to delete CTCF sites with strong 3C interaction frequencies in erythroid cells and testing the effect on globin gene expression and interaction frequencies of the remaining CTCF sites. The predictive nature of the polymer model will be tested with the new data.
Showing the most recent 10 out of 21 publications
