5-Azadeoxycytidine-induced chromatin remodeling of the inactive X-linked HPRT gene promoter occurs prior to transcription factor binding and gene reactivation.

During the process of 5-aza-2'-deoxycytidine (5aCdr)-induced reactivation of the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, acquisition of a nuclease-sensitive chromatin conformation in the 5' region occurs before the appearance of HPRT mRNA. In vivo footprinting experiments reported here show that the 5aCdr-induced change in HPRT chromatin structure precedes the appearance of three footprints in the immediate 5' flanking region that are characteristic of the active HPRT allele. These and other data suggest the following sequence of events that lead to the reactivation of the HPRT gene after 5aCdr treatment: (a) hemi-demethylation of the promoter, (b) an "opening" of chromatin structure detectable as increased nuclease sensitivity, (c) transcription factor binding to the promoter, (d) assembly of the transcription complex, and (e) synthesis of HPRT RNA. This sequence of events supports the view that inactive X-linked genes are silenced by a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter.

Multiple eruptive dermatofibromas in three men with HIV infection.

BACKGROUND: Multiple eruptive dermatofibromas (MEDF) are rare and their etiology is unknown. An association with immunosuppression has led to the speculation that they are the result of an abortive immunoreactive process. In the literature, there have been 5 isolated case reports of multiple dermatofibromas and human immunodeficiency virus (HIV) infection. Three of these cases had other immune modulators present (i.e. prednisone, systemic lupus erythematosus, alpha-interferon, UVB phototherapy). The other 2 cases had disseminated mycobacteriosis. OBSERVATIONS: A series of 3 men with HIV infection and MEDF is described. In contrast to previous case reports, our patients did not have other immune modulators besides HIV infection nor did they have disseminated mycobacteriosis. CONCLUSIONS: This series lends support to the speculation that MEDF may be associated with immunosuppression. Further study is needed to delineate the exact mechanism for this relationship. These patients presented within a 4-month period and illustrate the frequency at which MEDF may be seen in the HIV-positive population. As clinicians who care for patients with HIV infection, it is important to be aware that MEDF may be seen in this immunosuppressed population.

In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.

To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5' region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.

High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.

DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus. To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5' CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142 CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5' CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5' CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.

High resolution methylation analysis of the FMR1 gene trinucleotide repeat region in fragile X syndrome.

Fragile X syndrome is the most common form of inherited mental retardation in man. The disease is associated with expansion in the number of tandem CGG trinucleotide repeats in the 5' untranslated region of the human FMR1 gene. Transmitting males, individuals who are unaffected carriers of the disease, show a moderate increase in the number of repeat units, while fully penetrant males show a major expansion in repeat number. Major expansion of the repeat in affected males is correlated with methylation of certain restriction enzyme recognition sites in the 5' CpG island containing the trinucleotide repeat in these patients. Phenotypic expression of the mutation appears to be due to transcriptional silencing of the FMR1 gene. We now report direct high resolution methylation analysis of the trinucleotide repeat and its flanking regions using ligation-mediated PCR genomic sequencing. We find the cytosine residue of all CpG dinucleotides examined within and surrounding the FMR1 trinucleotide repeat to be unmethylated in the DNA of normal male leukocytes and transmitting male lymphoblasts; these same cytosines are methylated in affected male lymphoblasts, in a somatic cell hybrid containing a fragile X chromosome from an affected male, and in a somatic cell hybrid containing a normal inactive X chromosome. The methylation pattern of the FMR1 5' CpG island in affected patients as determined by genomic sequencing is remarkably similar to that seen for the X-linked human phosphoglycerate kinase and hypoxanthine phosphoribosyltransferase gene 5' CpG islands on the inactive human X chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 Sp1 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 conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.