In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1.

Abstract

The promoter region of the X-linked human phosphoglycerate kinase-I [PGK-I) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomie sequencing study in which 450 bp of the human PGK-1 promoter region was analyzed for the presence of in vivo protein footprints and cytosine methylation at all CpG sites. A technique was devised to selectively visualize the DNA of the inactive X chromosome (Xi), even in the presence of the active X chromosome (Xa). We found that the human Xa in both normal male lymphoeytes and hamster-human hybrids is completely unmethylated at all 120 CpG sites. In contrast, 118 of the CpG sites are methylated on the human Xi in hamster-human hybrids. The Xi in normal female lymphoeytes is also highly methylated, but some GCG or CGC trinueleotides partially escape methylation; all other CpGs are fully methylated. In vivo footprinting studies with dimethylsulfate (DMS) revealed eight regions of apparent protein-DNA contacts on the Xa. Four of the footprints contained the consensus sequence of the binding site for transcription factor Spl. The other regions include potential binding sites for transcription factors ATF, NF1, and a CCAAT-binding protein. The Xi did not show any specifically protected sequences, and with the exception of four hyperreaetive sites, the in vivo DMS reactivity profile of Xi DNA was very similar to that of purified, linear Xi DNA. The implications of these findings with regard to the maintenance of methylation-free islands, X chromosome inactivation, and the ehromatin structure of faeultative heteroehromatin are discussed.

Role of CTCF Binding Sites in the Igf2/H19 Imprinting Control Region

“…4A and 5); therefore, CTCF binding maintains the hypomethylated state of the ICR in somatic cells, i.e., binding to the maternal ICR is required for excluding methyltransferases from the region. Similarly, the hypomethylated state of CpG islands, which are often coincident with promoters, might result from the exclusion of methyltransferases by bound transcription factors (5,22,27). The paternal ICR became methylated in the male germ line, and this methylation persisted into somatic development.…”

“…It was shown that establishment of differential methylation is a function autonomous to the ICR (15), but the germ line-specific processes that establish this differential ICR methylation are undefined. Based on the paradigm that transcription factor binding can inhibit DNA methylation, possibly by physically blocking access of DNA methyltransferases (5,22,27), it is conceivable that differential protein binding at these sites during germ cell development may play a role in differential acquisition of DNA methylation. This could involve CTCF or another protein with an affinity for the site.…”

A Novel Element and a TEF-2-like Element Activate the Major Histocompatibility Complex Class II Transactivator in B-lymphocytes

“…UV Irradiation and Dimethylsulfate in Vivo Genomic Footprinting-In vivo methylation of cells with dimethylsulfate and DNA preparation were as described previously (24). In vivo UV irradiation footprinting was done essentially as described by Pfeifer and Tornaletti (25).…”

DNA methylation and gene silencing in cancer: which is the guilty party?

“…It is thought that the CpG islands spanning the housekeeping type genes are protected from methylation through either (1) the process of active transcription, including the possible steric hindrance of transcription factor or DNA polymerase binding, (2) active demethylation via RNA transcripts from CpG islands possibly in combination with 5MeC-glycosylase (Jost et al, 1997), (3) replicating timing or (4) local chromatin structure that may inhibit access to the DNA methyltransferase (Kass et al, 1997). The importance of gene activity, however as the mode of protection is exemplified by the fact that the CpG islands on the inactive X chromosome are silenced prior to methylation (Pfeifer et al, 1990). Therefore, CpG islands are not intrinsically 'unmethylatable' in the early embryo but active transcription appears to be a major protective factor.…”