Undermethylation and DNase I hypersensitivity of myeloperoxidase gene in HL-60 cells before and after differentiation.

Methylation and DNase I-hypersensitive sites of the myeloperoxidase gene in human myeloid leukemia HL-60 cells were studied by Southern blot hybridization using the myeloperoxidase gene probes. Digestion of DNA with a methylation-sensitive restriction endonuclease indicated that a CpG in the CCGG sequence located 3.53 kbp upstream of the myeloperoxidase gene was unmethylated in HL-60 cells expressing the gene, whereas it was methylated in K562 cells and human placenta not expressing the gene. The site in HL-60 cells remained unmethylated after retinoic acid- or 12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation that arrests myeloperoxidase synthesis. Digestion of isolated nuclei with various amounts of DNase I indicated that four DNase I-hypersensitive sites were in an upstream region of the myeloperoxidase gene in HL-60 cells and three sites were within the gene. In retinoic acid-induced cells, the bands of the hypersensitive site near the 5' side of the gene and that in the first intron became weak, while that of the site in the fifth intron became strong. The bands of these hypersensitive sites were weak in K562 cells. The implications of these changes in tissue-specific expression and developmental down-regulation of the myeloperoxidase gene are discussed.     

DNA methylation and the regulation of aldolase B gene expression

DNA methylation was studied as a potential factor for the regulation of tissue-specific and developmentally specific expression of the rat aldolase B gene. We examined cytosine methylation in the HpaII and HhaI recognition sequences in the aldolase B gene in aldolase expressing and nonexpressing tissues and cells. Out of the 15 methyl-sensitive restriction sites examined, the sites in the 3'-half and 3'-flanking regions were found to be heavily methylated in all the tissues or cells, regardless of the level of aldolase B gene expression. However, the methylation pattern in the region immediately upstream and in the 5'-half of the gene exhibited tissue-specificity: the site located about 0.13 kb upstream of the cap site (just next to the CCAAT box), and the sites in the first intron (intron 1) were heavily methylated in nonexpressing cells and tissues (ascites hepatoma AH130 and brain), whereas those in an expressing tissue (liver) were considerably less methylated. These results suggest that cytosine methylation at the specific sites in the 5'-flanking and 5'-half regions of the gene is associated with repression of the gene activity. However, the gene is still substantially methylated in the fetal liver on day 16 of gestation, when it is in a committed state for rapid activation in the period immediately afterwards (Numazaki et al. (1984) Eur. J. Biochem. 152, 165-170). This suggests that demethylation of the methylated cytosine residues in the specific gene region is not necessarily required before activation of the gene during development, but it may occur along with or after the activation.     

Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.

The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.     

Albumin and alpha-fetoprotein gene transcription in rat hepatoma cell lines is correlated with specific DNA hypomethylation and altered chromatin structure in the 5'' region.

We examined DNA methylation and DNase I hypersensitivity of the alpha-fetoprotein (AFP) and albumin gene region in hepatoma cell lines which showed drastic differences in the level of expression of these genes. We assayed for methylation of the CCGG sequences by using the restriction enzyme isoschizomers HpaII and MspI. We found two methylation sites located in the 5' region of the AFP gene and one in exon 1 of the albumin gene for which hypomethylation is correlated with gene expression. Another such site, located about 4,000 base pairs upstream from the AFP gene, seems to be correlated with the tissue specificity of the cells. DNase I-hypersensitive sites were mapped by using the indirect end-labeling technique with cloned genomic DNA probes. Three tissue-specific DNase I-hypersensitive sites were mapped in the 5' flanking region of the AFP gene when this gene was transcribed. Similarly, three tissue-specific DNase I-hypersensitive sites were detected upstream from the albumin gene in producing cell lines. In both cases, the most distal sites were maintained after cessation of gene activity and appear to be correlated with the potential expression of the gene. Interestingly, specific methylation sites are localized in the same DNA region as DNase I hypersensitive sites. This suggests that specific alterations of chromatin structure and changes in methylation pattern occur in specific critical regulatory regions upstream from the albumin and AFP genes in rat hepatoma cell lines.     

Genomic analysis of the mouse protamine 1, protamine 2, and transition protein 2 gene cluster reveals hypermethylation in expressing cells

To understand the role of chromatin structure in the expression of the mouse protamine 1, protamine 2, and transition protein 2 genes during spermatogenesis, we have examined the genomic organization of this cluster of ``haploid-specific' genes. As seen in the human genome, protamine 2, transition protein 2, and approximately 2.8 kb of a CpG island, hereafter called CpG island-dTP2, were clustered in a small region. Methylation analyses of this region have demonstrated that i) unlike most other tissue-specific genes, the protamine 1, protamine 2, and transition protein 2 genes were located in a large methylated domain in round spermatids, the cell type where they are transcribed, ii) the protamine 1 gene was only partially methylated in somatic cells and in testes from 7-day-old mice, and iii) the approximately 2 kb upstream and downstream of the CpG island-dTP2 were only partially methylated in somatic tissues. DNase I analysis revealed the presence of at least five strong DNase I hypersensitive sites over the CpG island-dTP2 in somatic tissues, but not in germ cells, and sequence analysis indicated that the CpG island-dTP2 is homologous to a CpG island located approximately 10.6 kb downstream of the human transition protein 2 gene. Although the nature of a CpG island-dTP2 and the function of a CpG island-dTP2-containing somatic tissue-specific DNase I hypersensitive sites in close proximity to the germ cell-specific gene cluster are unclear, the ``open' chromatin structure of the CpG island-dTP2 may be responsible for the partial methylation pattern of the flanking sequences including the transition protein 2 gene in somatic tissues. Received: 6 September 1996 / Accepted: 14 January 1997     

Genetic variability at the human tumor necrosis factor loci.

Variability in the structure of the human tumor necrosis factor (TNF-alpha) or lymphotoxin (TNF-beta) genes may contribute to the functional polymorphism of the HLA gene complex. We have characterized an allelic restriction fragment length polymorphism (RFLP) of the TNF-beta gene by using the restriction endonuclease NcoI. Digestion of genomic DNA with NcoI and Southern blotting by using TNF-alpha gene probes show 5.4-kb and 10.5-kb hybridizing fragments. In Caucasian populations, the 10.5-kb fragment is present in 64 to 72% of haplotypes. The polymorphic NcoI site is located within the first intron of the TNF-beta gene. Additional restriction fragment variability was demonstrated by digestion with AccI; however, this restriction fragment variability was not allelic in nature. Rather, it was a consequence of variable DNA methylation at AccI sites within and upstream of the TNF-beta gene. In peripheral blood leukocytes, methylation of the TNF-beta AccI sites was greatest in neutrophils (TNF-beta nonproducers), and lowest in T lymphocytes (the major producers of TNF-beta). These results suggest strongly that variation in DNA methylation may play an important role in regulation of the expression of the TNF-beta gene.     

GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia

Dysfunction of prefrontal cortex in schizophrenia includes changes in GABAergic mRNAs, including decreased expression of GAD1, encoding the 67 kDa glutamate decarboxylase (GAD67) GABA synthesis enzyme. The underlying molecular mechanisms remain unclear. Alterations in DNA methylation as an epigenetic regulator of gene expression are thought to play a role but this hypothesis is difficult to test because no techniques are available to extract DNA from GAD1 expressing neurons efficiently from human postmortem brain. Here, we present an alternative approach that is based on immunoprecipitation of mononucleosomes with anti-methyl-histone antibodies differentiating between sites of potential gene expression as opposed to repressive or silenced chromatin. Methylation patterns of CpG dinucleotides at the GAD1 proximal promoter and intron 2 were determined for each of the two chromatin fractions separately, using a case-control design for 14 schizophrenia subjects affected by a decrease in prefrontal GAD1 mRNA levels. In controls, the methylation frequencies at CpG dinucleotides, while overall higher in repressive as compared to open chromatin, did not exceed 5% at the proximal GAD1 promoter and 30% within intron 2. Subjects with schizophrenia showed a significant, on average 8-fold deficit in repressive chromatin-associated DNA methylation at the promoter. These results suggest that chromatin remodeling mechanisms are involved in dysregulated GABAergic gene expression in schizophrenia.     

In vitro methylation inhibits the promotor activity of a cloned intracisternal A-particle LTR.

We studied the relation between LTR methylation and expression of the family of endogenous retrovirus-like elements related to mouse intracisternal A-particles (IAP). Comparative HpaII/MspI and HhaI restriction analysis of genomic DNA's showed that in cells and tissues with a low level of IAP gene expression, HpaII and HhaI sites within the 5' LTR were heavily methylated, while in cells abundantly expressing IAP's 20 to 30% of the 5' LTRs were demethylated at these sites. The effects of methylation on the promoter activity of a cloned IAP 5' LTR was studied directly, using the plasmid pMIA5' L-cat in which this LTR was linked to the chloramphenicol acetyl transferase (CAT) gene. In vitro methylation of three HhaI sites located between -137 and -205 bp from the RNA start site of this LTR completely inactivated the promoter activity of pMIA5' L-cat transfected into COS7 cells. Methylation of a HpaII site located 94 bp downstream from the RNA start site reduced the promoter activity by 75%. The results show that methylation at sites both upstream and downstream from the RNA start site profoundly effects the promoter activity of this LTR and suggest that methylation within the 5' LTR can serve to regulate IAP gene expression in vivo.     

Developmental switch in chromatin structure associated with alternate promoter usage in the Drosophila melanogaster alcohol dehydrogenase gene.

During the development of Drosophila melanogaster a switch in alcohol dehydrogenase gene promoter usage occurs, such that proximally initiated mRNA is replaced by mRNA initiated from a more distal location. Investigation of the nucleo-protein organization at this gene in cells inactive for Adh expression, or derived from tissues active at either the proximal or distal promoter, reveals distinct changes in patterns of nucleosome organization and regions of nuclease sensitivity that are strongly correlated with the activity of the gene and its promoter usage. A positioned array of nucleosomes covers the coding region of the inactive gene, but is partially disassembled on gene activation. A series of proximally located hypersensitive sites, detected in early third instar larval fat body cells, are replaced by new, distally located regions of hypersensitivity in late third instar larval fat body, the change apparently coinciding with the promoter switch. Further developmental stage differences are detected in regions over 1 kb upstream of the distal start site. In addition, for both proximally and distally expressing cells, separate and different regions of apparent resistance to DNase I cleavage in chromatin are detected in locations that, in some instances, were previously demonstrated to bind specific factors in vitro.