Spreading of Alu methylation to the promoter of the MLH1 gene in gastrointestinal cancer

The highly repetitive Alu retroelements are regarded as methylation centres in the genome. Methylation in the gene promoters could be spreading from them. Promoter methylation of MLH1 is frequently detected in cancers, but the underlying mechanism is unclear. The aim of this study is to understand whether the methylation in the Alu elements is associated with promoter methylation in the MLH1 gene. Bisulfite genomic sequencing was used to analyse the CpG sites of the 5' end (promoter, exon 1 and Alu-containing intron 1) of the MLH1 gene in colorectal cancer cells and tissues, and gastric cancer tissues. Hypomethylation in the Alu elements and hypermethylation in the promoters and the regions between the promoters and the Alu elements were detected in two cancer cell lines and seven cancer tissues. However, demethylation or hypomethylation of the MLH1 promoter and regions between promoter and the Alu elements, and hypermethylation in the Alu elements, were identified in the normal tissues. MLH1 promoter methylation may spread from Alu elements that are located in intron 1 of the MLH1 gene. The trans-acting elements binding to the mutation sites could play a role in the methylation spreading.     

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.     

Intragenic control of expression of a rice MADS box gene OsMADS1

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.     

Long-range downstream enhancers are essential for Pax6 expression

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus. In this 420 kb YAC a tauGFP-IRES-Neomycin reporter cassette has been inserted into the PAX6 translational start site in exon 4. The YAC has been further engineered to insert LoxP sites flanking a 35 kb long, distant downstream regulatory region (DRR) containing previously described DNaseI hypersensitive sites, to allow direct comparison between the presence or absence of this region in the same genomic context. Five independent transgenic lines were obtained that vary in the extent of downstream PAX6 locus that has integrated. Analysis of transgenic embryos carrying full-length and truncated versions of the YAC indicates the location and putative function of several novel tissue-specific enhancers. Absence of these distal regulatory elements abolishes expression in specific tissues despite the presence of more proximal enhancers with overlapping specificity, strongly suggesting interaction between these control elements. Using plasmid-based reporter transgenic analysis we provide detailed characterization of one of these enhancers in isolation. Furthermore, we show that overexpression of a short PAX6 isoform derived from an internal promoter in a multicopy YAC transgenic line results in a microphthalmia phenotype. Finally, direct comparison of a single-copy line with the floxed DRR before and after Cre-mediated deletion demonstrates unequivocally the essential role of these long-range control elements for PAX6 expression.     

In vivo analysis of DNase I hypersensitive sites in the human CFTR gene.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows a complex pattern of expression. The regulatory elements conferring tissue-specific and temporal regulation are thought to lie mainly outside the promoter region. Previously, we identified DNase I hypersensitive sites (DHS) that may contain regulatory elements associated with the CFTR gene at -79.5 and at -20.5 kb with respect to the ATG and at 10 kb into the first intron. MATERIALS AND METHODS: In order to evaluate these regulatory elements in vivo we examined these DHS in a human CFTR gene that was introduced on a yeast artificial chromosome (YAC) into transgenic mice. The 310 kb human CFTR YAC was shown to restore the pheno-type of CF-null mice and so is likely to contain most of the regulatory elements required for tissue-specific expression of CFTR. RESULTS: We found that the YAC does not include the -79.5 kb region. The DHS at -20.5 kb is present in the chromatin of most tissues of the transgenic mice, supporting its non-tissue-specific nature. The DHS in the first intron is present in a more restricted set of tissues in the mice, although its presence does not show complete concordance with CFTR expression. The intron I DHS may be important for the higher levels of expression found in human pancreatic ducts and in lung submucosal glands. CONCLUSION: These data support the in vivo importance of these regulatory elements.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3′ enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3′ enhancer was active only in CD34⁺ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34⁺ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3′ enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160 kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

Characterization of a Cerebellar Granule Cell-Specific Gene Encoding the γ-Aminobutyric Acid Type A Receptor α6 Subunit

Abstract: The α6 subunit of γ-aminobutyric type A receptors is a marker for cerebellar granule cells and is an attractive candidate to study cell-specific gene expression in the brain. The mouse α6 subunit gene has nine exons and spans ～14 kb. The largest intron (intron 8) is ～7 kb. For a minority of mRNAs, a missplice of the first exon was identified that disrupts the signal peptide and most likely results in the production of nonfunctional protein. The gene is transcribed from a TATA-less promoter that uses multiple start sites. Using transgenic mice, it was found that the proximal 0.5 kb of the rat α6 gene upstream region confers expression on a β-galactosidase reporter gene. One founder gave rise to a line with cerebellar granule cell-specific expression, although expression varied with lobule region. Other founders had ectopic but neuron-specific expression, with β-galactosidase found in cerebellar Purkinje cells, neocortex, thalamus, hippocampus, caudate-putamen, and inferior colliculi. Thus, we have defined a region containing the basal promoter of the α6 subunit gene and that confers neuron-specific expression.     

Negative and positive regulation by a short segment in the 5''-flanking region of the human cytomegalovirus major immediate-early gene.

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.     

Analysis of spatiotemporal regulation of aromatase in the brain using transgenic mice

Brain aromatase is widely distributed in the vertebrates, from fish to mammals, and plays important roles in functional reproductive behavior through production of estrogen as a neurosteroid. It is expressed only in the nerve cells of specific brain regions with a transient peak in the neonatal period when sexual behavior becomes organized, and therefore provides a good model system to study regulatory mechanism of cell-specific, brain region-specific, and developmental stage-specific expression.

To elucidate spatiotemporal regulation of brain aromatase, we prepared transgenic mice carrying a reporter gene under the promoter of brain-specific exon 1f of the mouse aromatase gene. The reporter transgene carrying a 6.5 kb upstream region of the brain-specific promoter accurately reproduced the spatiotemporal expression patterns of aromatase in mouse brain, whereas transgenes carrying smaller fragments of the promoter showed ambiguous or inconsistent expression patterns.

The binding sites of Aro-AI, Aro-AII, and Aro-B for nuclear factors were also identified in the proximal region of the exon 1f brain-specific promoter. Introduction of a mutation into the Aro-AII site in the reporter transgene carrying −6.5 kb promoter region of exon 1f caused complete alteration of the spatiotemporal expression pattern of the reporter gene in the transgenic mice.

These results indicate that the −6.5 kb promoter region of exon 1f is the minimal essential element for brain-specific regulation, with both proximal and distal promoter regions required for accurate spatiotemporal expression of aromatase in the mouse brain.