Anita A. Piper

Demethylation of the human MDR1 5' region accompanies activation of P-glycoprotein expression in a HL60 multidrug resistant subline.

Chemotherapy is frequently limited by the development of multidrug resistance, a major cause of which is activation of the P-glycoprotein-encodingMDR1 gene. We have previously developed a P-glycoprotein-expressing multidrug resistant subline (HL60/E8) from the non-P-glycoprotein-expressing human HL60 promyelocytic leukemia cell line. A possible cause ofMDR1 silencing in HL60 cells is methylation of the promoter proximal region, thus demethylation occurring as a result of drug treatment may be responsible forMDR1 activation in the multidrug resistant subline. Using the bisulphite genomic sequencing technique we demonstrated that HL60 DNA is methylated at multiple sites within two distinct areas, one upstream and one downstream of the transcription start point. Only a single site in each area was methylated in all strands examined, with the remaining adjacent sites showing partial methylation. In contrast, DNA from the multidrug resistant HL60/E8 subline was unmethylated at essentially all sites in both areas. Thus the development of the P-glycoprotein-expressing multidrug resistant subline was associated with demethylation of theMDR1 5′ region.

Isolation of a clone partially encoding hill kangaroo X-linked hypoxanthine phosphoribosyltransferase : sex differences in methylation in the body of the gene

An X-linked clone encoding exons 4–9 of the hypoxanthine phosphoribosyltransferase (HPRT) gene was isolated from a kangaroo (Macropus robustus: Marsupialia) λEMBL4 genomic library. Sequence similarity between the kangaroo and eutherian HPRT coding sequences was high; however, intron sizes varied significantly between the kangaroo and other eutherian species. HpaII and HhaI sites in the body of the gene were generally hypermethylated in vivo on the active, relative to the inactive X, with sites within intron 3 showing essentially complete correspondence of activity with methylation and inactivity with unmethylation. At approximately 5 kb downstream from the gene, a switch to unmethylation of active X-linked sites occurred. This switch occurred within a cluster of HpaII and HhaI sites that may represent a CG island associated with a subsequent gene.

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X‐linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T‐rich elements, thought to facilitate DNA unwinding.

Methylation sensitive protein binding to an intragenic active X-specific methylated region in the M-robustus Hprt gene

An intragenic region of the wallaroo (Macropus robustus) hypoxanthine phosphoribosyltransferase gene which contains three active X-specific methylated cytosines was examined for protein(s) binding.In vitro DNase I footprinting of unmethylated DNA identified three footprints, one of which (footprint I) contained two of the known differentially methylated sites (a HpaII and a HhaI site). Methylation of the footprint I HpaII site only, abolished the formation of several, specific DNA-protein complexes in mobility shift assays. UV cross-linking experiments indicated that polypeptides involved in the methylation sensitive interactions with footprint I had molecular weights ranging from 72 to 48 kDa. Analogous results were obtained with nuclear extracts from both eutherian and metatherian cells, indicating that these proteins are conserved. We suggest that the binding of these proteins to the inactive X may play some role in gene silencing, with the active gene being protected from this effect by methylation of the binding site.