Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.

Abstract

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele.Of the footprints on the active allele, at least four occur over canonical GC boxes or Spl consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in coijunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.The random inactivation of a single X chromosome during normal mammalian female embryogenesis results in a unique system of differential gene expression in which a transcriptionally active X chromosome and transcriptionally inactive X chromosome occupy the same nucleus. The inactivation of genes on one X chromosome in female somatic cells compensates for the dosage imbalance of X-linked genes between the sexes (8, 9). The molecular mechanisms responsible for initiating, spreading, and maintaining X chromosome inactivation are unknown. However, DNA-protein interactions (8, 28), chromatin structure (20,34,36), DNA replication (7, 47), and DNA methylation (19,24,25,31,38,55,57) have all been postulated to be involved. Though X inactivation is a chromosome-wide phenomenon and process, some degree of regulation at the level of individual X-linked genes must also be involved, as indicated by the ability to independently reactivate individual genes on the inactive X chromosome by 5-azacytidine (5-azaC) (12,13,31,49,50).The differential expression of genes on the active and inactive X chromosomes is manifested by a difference in nuclease sensitivity of chromatin from the active and inactive alleles of the X-linked hypoxanthine-guanine phosphoribosyltransferase (HiPRT) and phosphoglycerate kinase (PGK-1) genes (12,23,39,40,54,56). Furthermore, the presence of DNase I hypersensitive sites in the 5' region of * ...

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“…Southern Blot Analysis-DNA from mice tails was prepared by standard techniques (31,32). The DNA was digested with HindIII, size-fractionated on an agarose gel, and alkali-transferred to a positively charged nylon membrane.…”

5-Azadeoxycytidine-induced Chromatin Remodeling of the Inactive X-linked HPRT Gene Promoter Occurs prior to Transcription Factor Binding and Gene Reactivation

“…Cell Lines-DNA samples were prepared from cultures of cell lines described previously (24,26). Briefly, GM00468 (National Institute of General Medical Sciences Human Genetic Mutant Cell Repository) is a normal diploid human male fibroblast cell line containing an active X chromosome.…”

“…To visualize the final DNA sequencing ladder, strand-specific hybridization probes were synthesized from M13 clones containing the human HPRT promoter region. Probe synthesis, hybridization, washing, and autoradiography were performed as described previously (24,26,29).…”

“…To visualize the final DNA sequencing ladder, strand-specific hybridization probes were synthesized from M13 clones containing the human HPRT promoter region. Probe synthesis, hybridization, washing, and autoradiography were performed as described previously (24,26,29).Quantification of Footprints by Autoradiogram Densitometry-Quantification of transcription factor binding at position Ϫ91 was carried out by densitometry of the DNA sequencing autoradiogram. The densitometric value for the band intensity at position Ϫ91 in each sample was first normalized for loading differences using the average band intensity of eight nonfootprinted bands flanking both sides of the band at position Ϫ91 in each lane.…”

“…3. RT-PCR analysis of human HPRT mRNA in X8 -6T2 cells after treatment with 5aCdr. Levels of human HPRT mRNA were assayed by 12,24,32, 48, and 60 h after initiating 5aCdr treatment of X8 -6T2 cells (lanes marked by numbered brackets). Numbers above each lane denote micrograms of RNA used in RT-PCR reaction; RNA from each 5aCdr-treated sample was assayed by RT-PCR in duplicate.…”