Figure 4

ChIP analysis of histone modification at the SYBL1 promoter region. Allele-specific ChIP analysis for H3 acetylation and H3 K4 methylation was performed using an XhoI polymorphism in the 5' UTR of the SYBL1 gene in the Xq28 pseudoautosomal region that is also present on the Y chromosome. In normal male cells, the Y-linked locus is inactivated, hypermethylated, and late-replicating as is the inactive X allele in female cells [23,31,49]. To determine if this region has abnormal histone modifications on the X-inactivated but DNA hypomethylated ICF female X, ChIP assays were performed for acetylated histone H3 (acH3) and K4-methylated histone H3 (mK4) in normal male lymphoblasts and in PT3 ICF female fibroblasts; the ethidium bromide-stained gels of each sample are shown before and after digestion with XhoI. The undigested alleles (XhoI+) are 268 bp; the digested alleles (XhoI-) result in fragments of 108 and 260 bp. The ChIP assay for acetylated histone H3 (acH3) shows that only the XhoI-digested allele (XhoI+) is hyperacetylated in a normal male lymphoblast (NMLB1), and this corresponds to the active X allele (Xa) by RT-PCR (data not shown). An hTERT-transformed clone of PT3 ICF fibroblasts was also analyzed by ChIP. This clone has normal monoallelic expression of SYBL1 even though the promoter region is extremely hypomethylated as determined by bisulfite methylation analysis of DNA. The inactive X allele in the PT3 clone is hypoacetylated at histone H3 and hypomethylated at H3K4 because only the active X allele (XhoI-) is immunoprecipitated with either the acetylated or K4-methylated histone H3 antibodies (although a small portion of the inactive X also appears to have been precipitated by the acetylated H3 antibody).