Polymorphisms and DNA methylation: two ways for functional differences in the 3' regulatory region of the IgH locus

The IgH locus in mouse and human has a 3' regulatory region (3'RR) with multiple DNaseI hypersensitive sites. In the human, but not in the mouse, the sites (HS3, HS1.2 and HS4) are duplicated. One unit is downstream of the Cα-1 gene and a second unit is downstream of the Cα-2 gene. Human HS1,2 enhancers show polymorphic features. In the mouse, HS3A, HS1.2, HS3B and HS4 are enhancers involved in the expression and class switching of immunoglobulin heavy chain genes. A recently identified downstream region, which contains hypersensitive sites HS5, HS6 and HS7, has been hypothesized to serve as an insulator of the Igh locus. This downstream region is associated with marks of active chromatin throughout B cell development and contains binding sites for CTCF, a protein associated with mammalian insulators. CTCF binding to many of its cognate DNA sites is prevented by DNA methylation. Previous studies using genomic Southern analysis have shown changes in DNA methylation in the upstream region of the murine 3' RR during B cell development. In the first part of this work I identified the polymorphic structure of human HS1,2, and its distribution in some populations and in some immunological diseases. The data suggest that the HS1,2 enhancer that lies downstream of the Cα-1 gene has four alleles, one of which, allele *2, is more frequent in some immunological disorders and less frequent in the sub-Saharan region. I have also observed using EMSA that protein binding is different in the four alleles. Furthermore I have studied changes in DNA methylation in the murine 3'RR during B cell development by digesting genomic DNA with methylation-sensitive restriction enzymes, such as HpaII and MaeII, followed by PCR. The data revealed that the 3’RR is methylated in embryonic stem cells. ES cells derived from histone H1 depleted mice showed a reduction in methylation as compared to their respective wildtype counterparts. I have detected a progressive loss of DNA methylation during B cell development. DNase I HS sites HS4, HS5 and HS7 are the earliest regulated and unmethylated sites in cell lines reflecting early stages of B development, while the HS1.2 and HS3B enhancers are unmethylated only in plasma cell lines. DNA methylation is also reduced in splenic B cells stimulated with LPS, and LPS plus IL4 to undergo class switch recombination. These experiments suggest that the DNA methylation pattern of the 3'RR is regulated by the H1 linker histone and related to B cell development and activation. I have also used EMSA to detect the influence of DNA methylation on protein binding to HS5. Results indicate that USF and CTCF proteins bind HS5 in vitro when their specific binding sites are unmethylated, but not when these sites are methylated. These observations suggest that DNA methylation and polymorphic regions could change the binding of insulator and/or transcription factors to the 3’RR, thereby impacting on the functions of the 3’RR during B cell development.