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DNA methylation is an essential epigenetic modification involved in regulating gene expression and maintaining epigenetic information across generations. However, how these marks are recognized and interpreted to activate or repress imprinted genes is not fully understood. Preliminary evidence describes the transcriptional repressor TRIM28 as a key regulator of imprinted gene expression during and after early genome-wide reprogramming. Aberrant expression of imprinted genes maybe one possible cause of incomplete epigenetic reprogramming and low efficiency in somatic cell nuclear transfer. Here, we perform a series of experiments to determine whether knockdown of Trim28 alters imprinted gene expression and DMR methylation in sheep embryonic fibroblast (SEF) cells. siRNA-mediated Trim28 silencing in SEF cells resulted in significantly decreased expression of Gtl2 to 30% and increased expression of Dlk1 (~1.7-fold). Moreover, knocking down Trim28 induced DNA methylation at the IG-DMR and the Gtl2 promoter was disrupted. Here, we uncover an important role for Trim28 in the maintenance of DNA methylation at IG-DMR during replication-dependent dilution of methylated cytosine during cellular proliferation. Unlike Dlk1-Gtl2 however, knocking down Trim28 does not affect DMR methylation in the Igf2-H19 gene cluster, yet results in increased expression of Igf2 and H19. Interestingly, Peg3 expression decreased by 60% in Trim28 knockdown cells. PEG3 as a transcriptional repressor to the H19-ICR that interacts with the co-repressor protein TRIM28 through KRAB-A. Trim28 therefore appears to control the Igf2-H19 imprinted cluster indirectly via PEG3, which is distinct from its classical role in preserving DNA methylation during DNA replication. Our results therefore indicate that Trim28 regulates imprinted gene expression through at least two distinct mechanisms during cells proliferation.

The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the -700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-aza2-deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methyltransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner.

Epigenetic modifications have been highlighted in chronic non-communicable diseases. The aim of this study was to investigate genome-wide DNA methylation for the identification of methylation markers in obesity. With obese Chinese preschool children, we performed comprehensive DNA methylation profiling of gene promoters and CpG islands to determine the differentially methylated genes using methylated DNA immunoprecipitation followed by hybridization to the NimbleGen Human DNA Methylation 385K Promoter Plus CpG Island Microarray. We found that compared to lean children, 251 promoters and 575 CGIs were demethylated, and 141 promoters and 277 CGIs were hypermethylated in obese children, and their distribution on chromosomes was imbalanced, showing more promoters and CGIs with demethylation on chromosomes 3, 16, 17 and 19 and more differentially methylated promoters and CGIs on chromosome X compared with chromosome Y. Further analysis indicated that aberrant methylations occurred mostly in HCP promoters and promoter CGIs. Among the top 80 promoters and CGIs that had differentiated methylation between obese and lean children, nearly half have been previously studied, and almost all of them are involved in the pathogenesis of cancers that are associated with many organs. Furthermore, four genes (FZD7, PRLHR, EXOSC4, and EIF6) with differential promoter methylation were validated, and their associations with obesity must be clarified. In conclusion, this study represents the first effort to determine methylation markers in obese Chinese children, which has potential relevance for identifying markers that are useful in elucidating the mechanisms of obesity pathogenesis and its complications.

Differentiation of umbilical cord blood derived mononuclear cells to endothelial cells is accompanied by massive changes in gene expression. Although methylation and demethylation of DNA likely play crucial roles in regulating gene expression, their interplay during differentiation remains elusive. To address this question, we performed deep sequencing of DNA methylation and mRNA expression to profile global changes in promoter methylation and gene expression during differentiation from mononuclear cells to outgrowing cells. We identified 61 downregulated genes with hypermethylation, including CD74, VAV1, TLR8, and NCF4, as well as 21 upregulated genes with hypomethylation, including ECSCR, MCAM, PGF, and ARHGEF15. Interestingly, gene ontology analysis showed that downregulated genes with hypermethylation were enriched in immune-related functions, and upregulated genes with hypomethylation were enriched in the developmental process and angiogenesis, indicating the important roles of DNA methylation in regulating differentiation. We performed polymerase chain reaction analyses and bisulfite sequencing of representative genes (CD74, VAV1, ECSCR, and MCAM) to verify the negative correlation between DNA methylation and gene expression. Further, inhibition of DNA methyltransferase and demethylase activities using 5'-aza-dc and shRNAs, specific for TET1 and TET2 mRNAs, respectively, revealed that DNA methylation was the main regulator of the reversible expression of functionally important genes. Collectively, our findings implicate DNA methylation as a critical regulator of gene expression during umbilical cord blood derived mononuclear cells to endothelial cell differentiation.

The human cytokine precursor ECRG4 has been associated with multiple physiological, developmental and pathophysiological processes involving cell proliferation, cell migration, innate immunity, inflammation, cancer progression and metastases. Although down-regulation of ECRG4 gene expression has been largely attributed to hypermethylation of CpG islands in the 5'untranslated region of the ECRG4 promoter, the mechanisms that underlie the dynamics of its regulation have never been systematically described. Here we show that the ECRG4 gene is widely expressed in human tissues and report that its core promoter lies between the -780 to +420 base pairs relative to the ATG start codon of the ECRG4 open reading frame. This sequence, which contains several CpG islands, also includes multiple overlapping Sp1 consensus binding sequences and a putative binding site for NF-kB activation. 5'RACE of mRNA derived from human leukocytes shows that ECRG4 transcription initiates from the guanidine at -11 from the initiation ATG of the ECRG4 open reading frame. While there is no canonical TATA- or CAAT-boxes proximal to this translational initiation site, there is a distal TATA-sequence in the 5'UTR. This region was identified as the sequence targeted by hypermethylation because in vitro methylation of plasmids encoding the ECRG4 promoter abolish promoter activity and the treatment of Jurkat cells (which naturally express ECRG4) with the methylation inhibitor 5-AzaC, increases endogenous ECRG4 expression. Because ChIP assays show that Sp1 binds the ECRG4 promoter, that forced Sp1 expression trans-activates the ECRG4 promoter and Sp1 inhibition with mithramycin inhibits ECRG4 expression, we conclude that the dynamic positive and negative regulatory elements controlling ECRG4 expression include a counter regulation between promoter methylation and Sp1 activation.

LHX3 is a LIM-homeodomain transcription factor with critical roles in pituitary and nervous system development. Mutations in the LHX3 gene are associated with pediatric diseases featuring severe hormone deficiencies, hearing loss, developmental delay, and other symptoms. The mechanisms that govern LHX3/Lhx3 transcription are poorly understood. In this study, we examined the role of DNA methylation in the expression status of the mouse Lhx3 gene. Pituitary cells that do not normally express Lhx3 (Pit-1/0 cells) were treated with 5-aza-2'-deoxycytidine, a demethylating reagent. This treatment leads to activation of Lhx3 gene expression suggesting that methylation contributes to Lhx3 regulation. Treatment of Pit-1/0 pituitary cells with a combination of a demethylating reagent and a histone deacetylase inhibitor led to rapid activation of Lhx3 expression, suggesting possible crosstalk between DNA methylation and histone modification processes. To assess DNA methylation levels, treated and untreated Pit-1/0 genomic DNAs were subjected to bisulfite conversion and sequencing. Treated Pit-1/0 cells had decreased methylation at specific sites in the Lhx3 locus compared to untreated cells. Chromatin immunoprecipitation assays demonstrated interactions between the MeCp2 methyl binding protein and Lhx3 promoter regions in the Pit-1/0 cell line. Overall, this study demonstrates that DNA methylation patterns of the Lhx3 gene are associated with its expression status.

Six1, an evolutionary conserved transcription factor, has been shown to play an important role in organogenesis and diseases. However, no reports were shown to investigate its transcriptional regulatory mechanisms. In the present study, we first identified porcine Six1 gene core promoter region (+170/-360) using luciferase reporter assay system and found that promoter activities were significantly higher in the mouse myoblast C2C12 cells than that in the mouse fibroblast C3H10T1/2 cells, implying that Six1 promoter could possess muscle-specific characteristics. Moreover, our results showed that promoter activities of Six1 were decreased as induction of differentiation of C2C12 cells, which was accompanied by the down-regulation of mRNA expression of Six1 gene. In addition, we found that the DNA methylation of Six1 promoters in vitro obviously influences the promoter activities and the DNA methylation level of Six1 promoter core region was negatively correlated to Six1 gene expression in vivo. Taken together, we preliminarily clarified transcriptional regulatory mechanisms of Six1 gene, which should be useful for investigating its subtle transcriptional regulatory mechanisms in the future. On the other hand, based on Six1 involved in tumorigenesis, our data also provide a genetic foundation to control the generation of diseases via pursuing Six1 as therapeutic target gene.

We have correlated cytosine methylation of two epialleles, P1-rr and P1-pr , with variation in gene expression and therefore phenotype. The p1 gene in maize encodes a transcription factor that controls phlobaphene pigment accumulation in floral tissues. While cytosine methylation was assayed in various regions spanning 17 kb, the only difference in DNA methylation pattern between the expressed P1-rr allele and the silenced P1-pr allele was detected in a region that consists of a complex arrangement of transposons and adjacent repeats. This region, which comprises the distal enhancer element of P1-rr , is hypermethylated in P1-pr compared to P1-rr . Based on other precedents, we hypothesize that DNA methylation spreads from the transposable elements into the flanking P1-rr enhancer, thereby transcriptionally silencing the gene. Interestingly, P1-pr is reactivated in mutants of the dominant epigenetic modifier Ufo1 . DNA methylation in the distal enhancer sequence is significantly reduced, which inversely correlates with increased transcript levels and pigmentation in P1-pr Ufo1 plants. If in general DNA methylation spreads from transposons into adjacent sequences containing regulatory elements for neighboring genes, the corresponding genes could be silenced by chance. Given the large amount of transposable elements in the maize genome, epialleles may be far more frequent than previously estimated.

The discovery of DNA methylation has provided a new perspective on how DNA may be dynamically regulated in the mammalian genome. DNA methylation is a dynamic process with a demethylation and de novo methylation from primordial germ cell to differentiation. DNA methylation and demethylation have been proposed to play important roles in somatic cell reprogramming. Some essential components were discussed, such as hydroxymethylation which has recently been confirmed as a modification of developmental importance.

Aberrant transcription read-through of a gene promoter as a result of genetic structural rearrangements can cause the epigenetic inactivation of a neighbouring gene. All reported cases have involved copy number alterations that remove the 3' poly(A) transcription terminator sequence of a gene leading to transcription read-through (TRT) and methylation of the gene promoter of a downstream gene. We aimed to determine whether deletion of poly (A) transcription terminator sequences was associated with the methylation of neighbouring genes in a CRC with extensive copy number alterations. We performed a high resolution CGH array and methylation analysis on a CRC specimen to identify such alterations. Analysis of the CRC using high-resolution CGH identified 6 genes with deletions in the 3' part of the gene that encompassed the poly(A) transcription terminator sequence. Bisulphite sequencing of the promoter region of neighbouring (affected) genes at these six regions showed all candidate genes were unmethylated. Considering the fact that six TRT affected genes in a CRC with multiple deletions show no signs of hypermethylated promoters, it would be fairly appropriate to suggest that epigenetic inactivation by TRT might be a rare phenomenon in sporadic CRCs.

Background Caveolin-1 (CAV1) is an important structural component of cellular caveolae involved in cell signaling. CAV1 gene on/off regulatory mechanism in multiple diseases, including cancer is not clearly understood. The tumor suppressor versus oncogene paradox of CAV1 during tumor development tempted us to investigate the role for the epigenetic drift of CAV1 gene regulation. Methods We have analyzed CAV1 gene expression and associated epigenetic marks (DNA methylation and histone 3 modifications) in the CAV1 promoter in two colon cancer cell lines, under treatment with well established epigenetic modulators, AZA, SAM, TSA and SFN at varying concentrations. CAV1 gene promoter DNA methylation and histone modifications were analyzed by DNA methylation specific PCR, bisulphite modification of DNA and ChIP analyses following PCR respectively. Results Ectopic expression of CAV1 by epigenetic modulators inhibits colon cancer cell growth. CAV1 promoter DNA remains unmethylated before and after treatment with epigenetic modulators, which confirmed that DNA methylation is not the regulator of CAV1 expression in colon cancer. There was enrichment of H3K4me3 and H3K9AcS10P and depletion of H3K9me3 modifications around the CAV1 promoter. Conclusions Our data provides novel insight into the regulation of CAV1 gene by histone H3 modifications and enhance the amplitude of the cancer epigenome.

SVEP1 gene encodes a cell adhesion molecule (CAM) that was previously shown to be expressed by cells related to skeletal tissues. Here we focus on SVEP1 expression regulation in pre-osteoblastic MBA-15 and mammary adenocarcinoma DA3 cells. We show that SVEP1 message and protein are highly expressed by MBA-15 when compared with DA3 cells. DNA methylation of CpGs sites is an epigenetic mechanism associated with gene silencing. Therefore, we analyzed the methylation status of a region potentially harbors SVEP1 promoter and further activity alterations induced by estrogen (17βE(2)) and TNFα. We also mapped in silico the transcription binding sites namely TFIIB, NF-κB, ERE, AP1 and Sp1 at the putative promoter. Treatments with demethylation reagents, 5'-aza-deoxy-Cytidine (5'-aza-dC), or histone deacetylase inhibitor, Trichostatin A (TSA) resulted with an elevation of SVEP1 mRNA expression in both cell types. Methylation levels of specific CpGs sites located at transcription binding sites were assessed using sodium bisulfite genomic DNA sequencing, methylated DNA immunoprecipitation (meDIP) and Methylation-Specific PCR (MSP). Our results show that the putative promoter of SVEP1 is hypermethylated in DA3- compared with MBA-15 cells, thus regulating SVEP1 expression levels. In addition, by affecting SVEP1 promoter methylation status, 17βE(2) and TNFα regulate ectopic SVEP1 promoter and mRNA expression. Our data sheds light on understanding the cell-type specific promoter status for regulation of the SVEP1. Since SVEP1 protein mediates cellular adhesion, this data might be beneficial for the future characterization of SVEP1 expression in the interactions existing in bone.

The maintenance and differentiation of embryonic stem cells (ES cells) depends on the regulation of gene expression through the coordinated binding of transcription factors to regulatory promoter elements. One of the genes involved in embryonic development is the chromatin factor CP27. Previously, we have shown that NF-Y interacted with the CP27 proximal promoter CCAAT-box. Here we report that CP27 gene expression in mouse ES cells is controlled by CCAAT and E-box cis-acting regulatory elements and their corresponding transcription factors NF-Y and USF1. Specifically, USF1 interacts with the E-box of the CP27 proximal promoter and NF-Y interacts with the CCAAT-box. NF-Y and USF1 also interacted with each other and activated the CP27 promoter in a synergistic fashion. Together, these studies demonstrate that gene expression of the chromatin factor CP27 is regulated through the interaction of the transcription factors NF-Y and USF1 with the CP27 proximal promoter.

Prolyl oligopeptidase (POP) is a widely distributed serine peptidase which hydrolyzes small peptides on the carboxyl side of an internal proline residue. While its physiological role has been intensely studied, the regulatory mechanism of the gene expression is poorly understood. This time we assessed the POP mRNA expression in mouse embryos and tissues related to reproduction and development and found that POP mRNA was highly expressed in the ovarian granulosa cell, placental spongiotrophoblast, and blastocyst embryo. To elucidate the mechanism by which POP expression is regulated, we investigated DNA methylation and histone modification patterns of the two CpG islands (CGIs) found at the, mouse POP locus. Whereas the CGI including the POP promoter (CGI-1) was completely hypomethylated in all the tissues examined, DNA methylation level of the CGI in the gene body (CGI-2) was lower in the granulosa cell, placenta, and blastocyst than in the liver. Some specific CpGs in CGI-2 were significantly demethylated in the three tissues. An in vitro reporter analysis indicated that CGI-2 enhanced POP promoter activity and its effect was significantly reduced by DNA methylation. Moreover, histone H3 acetylation and H3K4 methylation levels of CGI-2 were higher in the granulosa cell than liver. The results suggest that the CGI-2 region is a cis-element for the POP gene expression. (C) 2010 Elsevier B.V. All rights reserved.

In this study, we report the expression and genomic structure of the gene encoding human suppressor of cytokine signaling 6 (SOCS6), and the characterization of the functional promoter region. The human SOCS6 gene, spanning 40 kb on chromosome 18q22.2, is composed of two exons separated by an intron of 35 kb. Two transcripts are ubiquitously expressed, and both encode the full-length open reading frame of SOCS6. A primer extension assay revealed that the major transcription initiation site is located 469 bp upstream the ATG codon. Luciferase promoter analysis demonstrated that the 5'-flanking region is able to drive transcription, and the CpG-rich sequences near the transcription initiation site are important for the TATA-less SOCS6 promoter activity. Analogous to SOCS1 and SOCS3, which are down-regulated in several human cancers, SOCS6 is expressed at lower levels in carcinomas of stomach and colon. We demonstrated that hypermethylation of the SOCS6 promoter is one of the mechanisms for the epigenetic regulation of SOCS6 expression. Firstly, in vitro methylation of the reporter promoter plasmid significantly suppressed the promoter activity. Secondly, SOCS6 expression in vivo was enhanced by treating cells with a methyltransferase inhibitor. The SOCS6 gene from various species shares significant homology in amino acid sequences, transcription factor binding motifs in promoter regions and the two-exon genomic structure, suggesting that the SOCS6 gene is highly conserved.

The recently identified transcription factor YY2 shares important features with the well characterized YY1 zinc finger protein. Both proteins mediate activating as well as repressing transcriptional properties and bind specifically to an identical DNA consensus motif, suggesting synergistic or competitive function of both factors in controlling target genes. In fact, the human yy2 gene has evolved from a retroposed copy of yy1, unusually inserted within an intron of another gene, the membrane-bound transcription factor protease site 2 (mbtps2). To elucidate the biological function of human yy2, we studied its regulation within the extraordinary genomic context. The intronic upstream region of yy2 indeed mediates significant promoter activity. In vitro, transcriptional activity of the CpG-rich yy2 promoter is clearly repressed by DNA methylation. In vivo, DNA demethylation experiments performed by treating human embryonic kidney (HEK293) cells with 5-Aza-2-deoxycytidine (Aza) revealed that endogenous yy2 expression is more than 3-fold enhanced whereas the mbtps2 level appears unaffected. In summary, our findings indicate that yy2 expression is mediated by its own methylation-sensitive promoter.

TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out. (C) 2008 Elsevier B.V. All rights reserved.

Insulin-like growth factors (IGFs) belong to a family of growth factors with structural homology to proinsulin. Up till now, no specific details regarding the transcriptional regulation by autocrine, paracrine or endocrine effector molecules in vivo have been described for the IGF-II gene. This is in big contrast to IGF-I gene transcription which has been studied more extensively. To better understand how the IGF-II gene is controlled at the gene transcription level, we have isolated the common carp IGF-II gene together with the 5'-flanking region by genomic library screening. The mature IGF-II protein was encoded by exon 2 and exon 3. Transient transfection of the 5'-flanking region containing a TATA box-like sequence into cultured eukaryotic cells revealed that it is a strong promoter with definitive tissue specificity. Nucleotides between -301 and -62 in the promoter are essential to drive the basal IGF-II gene expression; whereas nucleotides between -891 and -416 in the promoter are responsible for the growth hormone activation. Using electrophoretic mobility shift assay and yeast one-hybrid screening, it was demonstrated that alpha1-antitrypsin could bind specifically to the nucleotide position -301 to -262 of the gene promoter. Co-transfection studies revealed that the over-expression of alpha1-antitrypsin increased the IGF-II promoter activity by 3.4-fold, further confirming that alpha1-antitrypsin acts as a trans-acting factor on the IGF-II promoter.

Epigenetic modifications are critical for regulating many different aspects of normal cell biology and tumourigenesis. Gene expression may be epigenetically silenced by DNA-methylation and histone modifications, resulting in remodelling of chromatin into a repressed state. We performed DNA-methylation analysis of the ICSBP/IRF8 gene, a member of the IRF family of transcriptional regulators expressed in monocytic and lymphocytic cells, in human monoblastic U-937 cells. We found complete methylation of all 39 CpG positions located in a 308 bp sequence encompassing the proximal promoter and transcriptional start site of the ICSBP/IRF8 gene. However, strikingly, the ICSBP/IRF8 gene is still expressed. Chromatin Immuno-precipitation (ChIP) showed that RNA-Polymerase II was present at the major transcriptional start site. Investigating the histone modifications across the ICSBP/IRF8 gene we found the positive histone marks H3K9ac and H3K4me3 to be enriched at the promoter, whereas the level of H3K9me3 was low. This suggests that an active chromatin structure, indicated by histone H3 modifications and enrichment for RNA Pol II, can over-ride the silencing effect of DNA-methylation at the promoter, thereby permitting transcription of the ICSBP/IRF8 gene.

Epigenetic reprogramming has a crucial role in establishing nuclear totipotency in normal development and in cloned animals. Insulin-like growth factor-2 receptor ( Igf-2r ) is a tissue-specifically and species-dependently imprinted gene, regulated by epigenetic modifications. The diversity of Igf-2r imprinting suggests that the success of animal cloning may be species-dependent. To determine the relation between epigenetic modifications and Igf-2r expression in cattle, and explore whether this gene was correctly imprinted and reprogrammed after nuclear transfer, we quantified Igf-2r mRNA in a cattle cell line after treated with an inhibitor of DNA methylation transferase or an inhibitor of histone deacetylase, and confirmed that DNA methylation and histone acetylation could regulate this gene expression. CpG island searching showed that there is a conservative imprinting control region (ICR) within the second intron of Igf-2r in cattle, analogous to mice and sheep, regulating this gene imprinting. DNA methylation analysis in sperm and blood cells showed that DNA methylation at Igf-2r ICR2 was reprogrammed in normal cattle. The methylation at Igf-2r ICR2 showed significant variation in tissues, such as blood, liver, brain, heart and heart. It suggested that Igf-2r imprinting was tissue-specifically regulated. In cloned cattle, DNA methylation at Igf-2r ICR2 was markedly altered in comparison with normal fetus, while patterns of DNA methylation at Igf-2r 3′-UTR (3-terminal untranslated region) were similar to normal fetus, it indicated that 3′-UTR was not significantly altered by cloning procedures, but DNA methylation at the locus of gene imprinting was disrupted and not completely reprogrammed after nuclear transfer.

The human NHEDC1 (hNHEDC1) protein is thought to be essential for sperm motility and fertility however the mechanisms regulating its gene expression are largely unknown. In this study we have identified multiple DNA regulatory elements in the 5' end of the gene encoding hNHEDC1 (SLC9B1) and have explored the role that DNA methylation at these elements plays in the regulation of its expression. We first show that the full-length hNHEDC1 protein is testis-specific for the tissues that we tested and that it localizes to the cells of the seminiferous tubules. In silico analysis of the SLC9B1 gene locus identified two putative promoters (P1 and P2) and two CpG islands - CpGI (overlapping with P1) and CpGII (intragenic) - at the 5' end of the gene. By deletion analysis of P1, we show that the region from -23 bp to +200 bp relative to the transcription start site (TSS) is sufficient for optimal promoter activity in a germ cell line. Additionally, in vitro methylation of the P1 (the -500 bp to +200 bp region relative to the TSS) abolishes its activity in germ cells and somatic cells strongly suggesting that DNA methylation at this promoter could regulate SLC9B1 expression. Furthermore, bisulfite-sequencing analysis of the P1/CpGI uncovered reduced methylation in the testis vs. lung whereas CpGII displayed no differences in methylation between these two tissues. Additionally, treatment of HEK 293 cells with 5-aza-2-Deoxycytidine led to upregulation of NHEDC1 transcript and reduced methylation in the promoter CpGI. Finally, we have uncovered both enhancer and silencer functions of the intragenic SLC9B1 CpGII. In all, our data suggests that SLC9B1 gene expression could be regulated via a concerted action of DNA methylation-dependent and independent mechanisms mediated by these multiple DNA regulatory elements.

CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) is a member of the p34cdc2-related kinases. It has been previously shown to be involved in a variety of different cellular processes including RNA processing, apoptosis, and cell cycle progression. It is encoded by two different but highly similar genes, Cdc2L1 (cell division control 2 like 1) and Cdc2L2 (cell division control 2 like 2). Previous studies from our group identified and characterized the transcriptional regulation of the human Cdc2L2 gene promoter. The current studies identify and characterize the Cdc2L1 gene promoter. We cloned the promoter and elucidated the different transcriptional regulatory elements that reside within the 5' region of the gene. Deletion analysis of the promoter showed a region of nucleotides -152 to +11 to be necessary for basal transcription of the Cdc2L1 gene. Sequencing analysis found this region of the promoter to be highly GC-rich but is lacking both TATA and CAAT boxes. There are several different transcription factor binding sites that are consensus or near consensus found within this region. The potential binding sites include two Ets-1 sites, one Skn-1 site, and one E2F-1 site. Transfection studies of various site-directed mutagenesis clones for these different sites revealed that both Ets-1 sites play critical roles in sustained transcriptional activity as well as Skn-1. Chromatin immunoprecipitation of the endogenous promoter with Ets-1 and Skn-1 verified an in vivo association of Ets-1 and Skn-1 transcription factors with the endogenous promoter. These results, in addition to our Cdc2L2 results, lead to the further comprehension of the fundamental mechanisms dictating CDK11 gene expression through the Cdc2L1 gene promoter.

The CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) protein kinases are part of the large family of p34(cdc2)-related kinases and have been shown to play a role in cell cycle progression, RNA processing and apoptosis. They are encoded by two genes-cell division control like 1 (Cdc2L1) and cell division control like 2 (Cdc2L2). To date, little is known about the transcription factors controlling their expression. To understand the mechanisms underlying the regulation of CDK11 gene expression, we cloned and identified the Cdc2L2 promoter and determined its transcriptional regulatory elements. By deletion analysis, a region between nucleotides -145 and +10 was identified to be critical for basal level transcription of the Cdc2L2 gene. Sequencing analysis revealed that the proximal promoter of the Cdc2L2 gene is GC rich and does not contain TATA and CAAT boxes. However, multiple consensus and near consensus transcription factor binding sites were found to be present in this region, such as two Ets-1, one cAMP-responsive element (CRE) and one TCF11/LCR-F1/Nrf1 binding sites. Site-directed mutagenesis and transfection studies revealed that all these binding sites were necessary to achieve sustained transcriptional activity. Electrophoretic mobility shift assay confirmed that transcription factors Ets-1 and CREB bind to the Cdc2L2 promoter elements, indicating their potential role in the transcriptional regulation of Cdc2L2 gene. More importantly, Ets-1, CREB and phosphorylated CREB were found binding to the endogenous Cdc2L2 promoter using chromatin immunoprecipitation (CHIP) assay. Our results provide the foundation for further studies into the regulation of Cdc2L2 gene expression in normal homeostasis and cancer.

Epigenetic regulation is the mechanism by which gene function is selectively activated or inactivated in the cells. It provides higher-ordered and more specified genetic information, compared with the whole genome itself. Recently, a variety of regulatory proteins including DNA methyltransferases, methyl-CpG binding proteins, histone-modifying enzymes, chromatin remodeling factors, and their multimolecular complexes have been identified. These facilitate our understanding of the molecular basis for transcription, DNA replication, mutation and repair, DNA recombination, and chromosome dynamics, which are crucial for normal cell regulation. Abnormalities in the epigenetic states represent human disease phenotypes, especially developmental defects and tumorigenesis. Therefore, epigenetics will become the focus and a major target for emerging biological and medical discoveries.

Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner.

DNA methylation is an important component of the epigenetic control of genome functions. Understanding the regulation of the DNA Methyltransferase (dnmt1) gene expression is critical for comprehending how DNA methylation is coordinated with other critical biological processes. In this paper, we investigate the transcriptional regulatory region of the human dnmt1 gene using a combination of RACE, RNase protection analysis and CAT assays. We identified one major and three minor transcription initiation sites in vivo (P1-P4), which are regulated by independent enhancers and promoter sequences. The minimal promoter elements of P1, P2 and P4 are mapped within 256 bp upstream of their respective transcription initiation sites. P1 is nested within a CG-rich area, similar to other housekeeping genes, whereas P2-P4 are found in CG-poor areas. Three c-Jun-dependent enhancers are located downstream to P1 and upstream to P2-P4, thus providing a molecular explanation for the responsiveness of dnmt1 to oncogenic signals that are mediated by the Ras-c-Jun oncogenic signaling pathway.

DNA is organized into a hierarchy of structures, resulting in the level of compaction required to pack 2m of DNA into a nucleus with a diameter of 10 micrometer. The orderly packaging of DNA in the nucleus plays an important role in the functional aspects of gene regulation. A small percentage of chromatin is made available to transcription factors and the transcription machinery, while the remainder of the genome is in a state that is essentially invisible to the RNA polymerases. Modification of histones has a key role in altering chromatin higher order structure and function. In this review, we will present the latest developments in the study of histone modifications (ubiquitination, acetylation, methylation, and phosphorylation) and the enzymes involved in these processes.

Mouse ES cells with a null mutation of the known DNA methyltransferase retain some residual DNA methylation and can methylate foreign sequences de novo. We have used bisulfite genomic sequencing to examine the sequence specificity and distributions of methylation of a hypermethylated CG island sequence, mouse A-repeats. There were 13 CG dinucleotides in the region examined, 12 of which were methylated to variable extents in all DNAs. We found that: (1) there is considerable residual DNA methylation in ES cells lacking the known DNA methyltransferase (29% of normal methylation in the complete knockout ES DNA); (2) this other activity methylates at exactly the same CG sites as the major methyltransferase; and (3) differences in the distribution of methylated sites between A-repeats in these DNAs are consistent with this other activity methylating in a random de novo fashion. Also, the lack of any methylation in non-CG sites argues that, in other studies where non-CG methylation sites have been found by bisulfite sequencing, detection of such sites of non-CG methylation is not an inherent artifact in this methodology.

The Ku70/80 complex, known as Ku, constitutes the DNA end binding component of the DNA-dependent protein kinase (DNA-PK). We have characterized the promoter region of the mouse and human Ku80 genes to delineate transcriptional elements necessary for basal gene expression and proliferation-dependent regulation. Consensus Sp1 recognition elements were identified in both promoters, and were determined to be essential for basal expression. We further identified a near-perfect palindrome of 21 base pairs located immediately 5' to one Sp1 element. This sequence was present once within the mouse Ku80 promoter and seven times, in a head-to-tail tandem array, within the human Ku80 promoter. This sequence possessed homology with a methylation-sensitive promoter element, Enh2, present in the LTR of mouse intractisternal A-particles. Promoter deletion studies and expression analysis of in-vitro methylated reporter gene constructs provided strong evidence that, in vivo, this repeat sequence regulates Ku80 gene expression in cis, through a mechanism involving CpG methylation. Evidence is also presented, suggesting that Ku is directly involved in this regulatory process.

Previously it has been found that binding of the Sp1 transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5'-GGGCGG (lower strand, 5'-CCGCCC). Since it has been established that mammalian cells also have the capacity to methylate cytosines (C) at CpNpG sites we examined the effect of methylation of the outer C of the CpCpG on Sp1 binding. We find that methylation of the outer C is inhibitory and in particular methylation of both cytosines (m)Cp(m)CpG inhibits binding by 95%. Furthermore, we have identified endogenous (m)Cp(m)CpG methylation of an Sp1 site in the CpG island promoter of the retinoblastoma (Rb) gene by genomic sequencing. This occurs in a proportion of retinoblastoma tumors which are extensively CpG methylated in the Rb promoter. The results raise the possibility that (m)Cp(m)CpG methylation could have a biological function in preventing Sp1 binding, thereby contributing to the subsequent abnormal methylation of CpG islands often observed in tumor cells.

The methylation status of the cytosines located on the 5′ region of the pS2 gene have been investigated in two human breast cancer cell lines. The genomic sequencing method used is based on an oxydative deamination by NaHSO 3 of the unmethylated cytosines, but not 5-methylcytosine. Data obtained indicate that in the DNA extracted from the pS2 -expressing cell line, MCF7, the CpG located at the 5′ flanking sequence of pS2 are unmethylated. In contrast in the non-expressing cell line, BT20, these CpG are largely methylated. However, this correlation is not observed at all CpG sites, since a significant portion of the cloned PCR fragments obtained from BT20 cells are unmethylated at specific CpG sites. These results suggest that the methylation status of only some of the CpG located at the 5′ flanking sequences of pS2 might differ between expressing and nonexpressing cell lines.

The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.

Upon HPLC fractionation of human placenta or calf thymus H1 histone preparations, only some fractions enriched in the H1e-c variants were able to exert a severe inhibition on in vitro enzymatic DNA methylation. These fractions, though similar to the other variants in interacting with genomic DNA, were also the only ones which could bind CpG-rich ds-oligodeoxyribonucleotides (oligos). Both the 6-CpG ds-oligo and the DNA purified from chromatin fractions enriched in 'CpG islands' were good competitors for the binding of H1e-c to the 6meCpG ds-oligo. This ability to bind any DNA sequence and to suppress the enzymatic methylation in any sequence containing CpG dinucleotides suggests, for these particular H1 variants, a possible role in maintaining CpG island DNA and linker DNA at low methylation levels.

Human arylamine N-acetyltransferase 1 (NAT1), a polymorphic xenobiotic metabolising enzyme, has been investigated in relation to susceptibility and prognosis in certain types of cancer. Both human NAT1 and its murine equivalent NAT2 have previously been shown to play roles in the catabolism of folate, which is required for the synthesis of S-adenosylmethionine, the methyl donor for cellular methylation reactions. We have tested whether the expression of mouse Nat2 is subject to epigenetic regulation, specifically CpG methylation in the promoter region, by determining levels of 5-methylcytosine by bisulphite sequencing and methylation-specific PCR. Under normal conditions, methylation levels of the Nat2 promoter were low, and varied in different tissues. However, CpG methylation was significantly increased by dietary folate supplementation, and increased methylation corresponded to decreased use of the core promoter. Functional deletion of the Nat2 gene gave rise to a significant increase in Nat2 methylation, extending our previous observations that folate catabolism is decreased in Nat2 null mice. Mouse NAT2 is likely to influence epigenetic gene control, particularly of its own locus, and this is consistent with recent evidence associating aberrant mouse Nat2/human NAT1 gene expression with certain developmental malformations and cancers.

DNA methylation plays important roles in various developmental processes in many organisms. In carrots, the treatment of embryogenic cells (ECs) with DNA methylation inhibitors induces hypomethylation and blocks somatic embryogenesis. CARROT-LEAFY COTYLEDON 1 (C-LEC1) is an important transcription factor for embryo development that shows embryo-specific expression in ECs and somatic and zygotic embryos. However, the regulation of embryo-specific transcription factor genes such as C-LEC1 in plants is not well understood. In this study, we used embryogenic carrot cells (Daucus carota L. cv. US-Harumakigosun) to investigate the DNA methylation status of the embryogenesis-related genes C-LEC1, Carrot ABA INSENSITIVE 3 (C-ABI3), and Daucus carota Embryogenic cell protein 31 (DcECP 31) during the transition from embryogenesis to vegetative growth. The C-LEC1 promoter region showed a reduced level of DNA methylation during somatic embryogenesis followed by an increase during the transition from embryonic to vegetative growth. To test whether the increased level of DNA methylation down-regulates C-LEC1 expression, RNA-directed DNA methylation (RdDM) was used to induce the hypermethylation of two segments of the C-LEC1 5′-upstream region: Regions 1 and 2, corresponding to nucleotides − 1,904 to − 1,272 and − 896 to − 251, respectively. When the hypermethylation of Region 1 was induced by RdDM, C-LEC1 expression was reduced in the transgenic ECs, indicating a negative correlation between DNA methylation and C-LEC1 expression. In contrast, the hypermethylation of Region 2 did not greatly affect C-LEC1 expression. Based on these results, we hypothesize that DNA methylation may be involved in the control of C-LEC1 expression during carrot embryogenesis.

The expression of genes in transgenic mice is known to be influenced by the site of integration even when they carry their own promoter elements and transcription factor binding sites. The cytomegalovirus (CMV) promoter, a strong promoter often used for transgene expression in mammalian cells in culture, is known to be silenced by DNA methylation and histone deacetylation but there is no report on the role of histone methylations in its regulation. We generated two transgenic lines carrying green florescence protein coding gene as reporter driven by cytomegalovirus major immediate-early promoter/enhancer. We observe that silencing of CMV promoter is dependent on the site of transgene integration, except in testis, and the nature of DNA and histone methylations strongly correlate with the expression status of the reporter. We find that silenced CMV promoter interacts in vivo, with Methyl CpG binding protein 2 (MeCP2), a recruiter of histone deacetylases (HDACs) and histone (H3K9) methyl transferase. Histone H3K4methylation, the active chromatin mark, is also associated with silenced promoter, suggesting bivalent marking of the promoter and its susceptibility to reactivation on induction.

The methyl-CpG binding domain protein MBD3 has been shown to be essential for embryonic development and differentiation, and to act by suppressing gene expression through the recruitment of co-repressor complexes. We have recently shown that MBD3 is also involved in maintaining the demethylated and active state of rRNA genes, and that depletion of MBD3 results in hypermethylation of rRNA promoters. The possibility that MBD3 could also trigger DNA demethylation of RNA polymerase II-transcribed genes has not been addressed. In this study we used a gain-of-function approach to examine whether MBD3 expression alters DNA methylation states in a living cell and whether it has specific targets for DNA methylation or demethylation in the genome. We used a combination of methylated DNA immunoprecipitation (mDIP) and hybridization to a human promoter tiling microarray to examine the landscape of DNA methylation patterns in response to MBD3 overexpression. We demonstrate that MBD3 induces genomic DNA demethylation and that it has specific targets in the genome with which it associates. Demethylation is localized to promoter regions with intermediate CpG density, and promoters with predicted transcription factor binding sites for NF-Y were significantly affected. These data demonstrate a causal relationship between MBD3 and DNA demethylation of genomic targets in cells.

A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

The SLC22A18/SLC22A18AS genes are a sense–antisense pair located at human chromosome segment 11p15.5. These genes are paternally imprinted: paternal alleles are silenced and maternal alleles are expressed. Although SLC22A18 is a well-characterized gene, very little is known regarding its antisense partner SLC22A18AS. We therefore sought to identify the potential cis-regulating elements including the promoter of this gene, differentially methylated regions (DMRs) and the translation of its putative ORF. Dual promoters (P1 and P2) were identified for this gene and both are devoid of consensus TATA and CCAAT boxes. However, the P1 promoter harbors a putative Sp1 binding site. Sp1 binds to the P1 promoter in vivo and positively regulates its activity. Promoter and CpG II island regions showed heavy methylation of CpG sites, but no DMRs were observed. Treatment of a non-SLC22A18AS expressing HuH7 cells with the methyltransferase inhibitor 5-aza-2′-deoxycytidine restored expression of this gene. The histone deacetylase (HDAC) inhibitor Trichostatin-A, on the other hand, failed to induce its expression. We suggest that the expression of this gene is methylation-dependent, but histone acetylation-independent. This gene was found to be translated with a cytoplasmic localization. The present data will help to understand the regulation of this gene and its role in tumorigenesis.

We have studied the dynamics of de novo DNA methylation of 16 contiguous CpGs in the non-CpG island-coding region of the proto-oncogene c-fos during mouse development by Na-bisulfite sequencing. Methylation commences from 16.5 dpc and occurs in stepwise-manner. In liver 7 sites are methylated between 16.5 dpc and day 5 after birth, but all the sites are completely methylated on 20 dpp and remain so in the adult liver. The present study provides evidence that (1) pattern of methylation of c-fos is distinct from those DNA sequences which methylate pre- and post-implantation, both in terms of the timing and spreading, and (2) spacing of CpGs is an important factor in determining the course of methylation. We suggest that there could be other isoforms of Dnmtases for the c-fos like embryonic genes, not only because they methylate later in development but also because of the difference in kinetics of the reaction, and that the nucleation of certain methylated sites facilitate methylation of neighbouring sites and their maintenance in subsequent cell generations.

Latent TGF-beta binding proteins (LTBPs) are extracellular matrix glycoproteins, which are essential for the targeting and activation of TGF-betas. LTBP-3 regulates the bioavailability of TGF-beta especially in the bone. To understand the regulation of LTBP-3 expression, we have isolated and characterized the promoter region of human LTBP-3 gene. The GC-rich TATA-less promoter contained several transcription initiation sites and putative binding sites for multiple sequence specific transcription factors including Sp1, AP-1, c-Ets, MZF-1, Runx1 and members of the GATA-family. Reporter gene analyses of the promoter indicated that it was more active in MG-63 than in Saos-2 osteosarcoma cells, suggesting that it is regulated as the endogenous gene. TGF-beta1 stimulated the transcriptional activity of LTBP-3 promoter in MG-63 cells, while certain other bone-derived growth factors and hormones were ineffective. TGF-beta1 increased LTBP-3 mRNA levels accordingly. Analyses of deletion constructs of the promoter and mutational deletion of specific transcription factor binding sites indicated that Smad3/4 and AP-1 binding sites mediated the TGF-beta1 response. The involvement of AP-1 activity was further indicated by decreased TGF-beta responsiveness of the LTBP-3 promoter in the presence of a MEK/Erk signaling pathway inhibitor. Our results suggest an important new role for TGF-beta1 in the regulation of its binding protein, LTBP-3.

We have studied the effects of methylation of the promoter region on the expression of human synaptotagmin XI (syt11), a gene implicated in the onset of schizophrenia. Sequence analysis showed that cytosine residues not in the CpG sequence, but still within the promoter region of the gene, are partially methylated. The methylated cytosine residues are located in the mRNA-coding (minus) strand of the promoter region (mCmCTTmCTTmCmC). Gel mobility shift assays showed that when the cytosine residues are methylated, the binding activity of an Sp family protein, a transcription factor, to the region is significantly reduced. Furthermore, transient transcription assays using artificially methylated promoter sequences showed that methylation did reduce the expression of the reporter gene. The biological significance of the finding is discussed in respect to the effect of methylation of non-CpG sequences in promoter regions on gene expression.

Rac2 is a Rho family GTPase expressed specifically in hematopoietic cells. The 4.5 kb proximal Rac2 gene promoter exhibits strong but promiscuous activity following either transient or stable transfection into tissue culture cells, indicating that additional cis-elements are required to silence Rac2 expression in non-hematopoietic cells. A bacterial artificial chromosome (BAC) containing the human Rac2 gene, including as little as 1.6 kb of upstream and 8 kb of downstream sequence, exhibits hematopoietic-restricted expression in transgenic mice. The Rac2 genomic locus exhibits distinct patterns of DNA methylation in expressing versus non-expressing cells. Cells that lack Rac2 expression exhibit increased cytosine methylation in the sequences flanking the gene, whereas cells that express Rac2 exhibit increased cytosine methylation within the body of the Rac2 gene. Treatment of non-expressing cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine reduces cytosine methylation of the Rac2 gene locus and is sufficient to induce Rac2 gene expression. Conversely, treatment with the histone deacetylase (HDAC) inhibitor trichostatin A fails to induce Rac2 gene expression. These findings define a genomic fragment sufficient to direct hematopoietic-specific expression of Rac2, and reveal the importance of cytosine methylation in the repression of Rac2 expression in non-hematopoietic cells.

The divergently transcribed Surf-1 and Surf-2 genes are separated by a bi-directional, TATA-less promoter which contains three important factor-binding sites, Sul , Su2 and Su3 . The transcription initiation factor YY1 binds to the Sul site and stimulates transcription in the direction of Surf-1 and, to a lesser extent, Surf-2 . Members of the ETS family of transcription factors bind to the Su2 and Su3 sites. Here we show that in transient transfection assays, transcription in both the Surf-1 and the Surf-2 direction is severely reduced by CpG methylation. Although the Sul site contains three CpG dinucleotides, the binding of YY1 is not affected by CpG methylation. In contrast, the binding of two ETS factors (ETS-2 and PEA-3) to the Su2 site (which also contains three CpG dinucleotides) is totally abolished by CpG methylation. Finally, we show that methylation of a single C within the Su2 site is sufficient to prevent ETS factor binding.

The GADD34 gene is transcriptionally induced by growth arrest and DNA damage. However, the mechanisms underlying the transcriptional regulation are still unclear. We analyzed the promoter of mouse GADD34 gene and the methylmethane sulfonate (MMS)-induced transcriptional regulation of this gene. By introducing genome mutants, which were linked to the luciferase reporter, into NIH3T3 cells, we defined a 100-bp fragment upstream of the transcriptional initiating site as the minimal promoter of the GADD34 gene. Subsequent study revealed that CRE-binding site located in this minimal promoter was critical for MMS-induced transcription of the GADD34 gene. In vitro binding experiments showed that phosphorylated c-Jun was contained in the CRE/DNA complex. Overexpression of the dominant negative form of c-Jun led to a decrease of MMS-responsive promoter activity. From these results, we conclude that the CRE site of the GADD34 promoter is indispensable to the MMS-responsive cis-element that c-Jun is the essential transcription factor for MMS-stimulated regulation of GADD34 gene expression and that the upstream signaling is dependent on JNK.

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na(+)-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (-1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between -133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2'-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.

L1 (LINE-1) is an interspersed non-LTR retrotransposon and several genetic defects caused by L1 transposition have been reported. L1 is thus considered as a potential mutagen. However, this potentially hazardous insertional event seems to be rare in spite of the presence of 3000 or more L1 elements of full or nearly full length in the human genome. Thus there must exist a mechanism(s) for repressing the expression of most, if not all, L1 elements. Some studies suggested that methylation plays a major role in the repression of L1 expression. However, no direct evidence has been presented and further study is required to draw a conclusion. We thus studied the effect of methylation on L1 transcription in vivo and in vitro. Transfection of plasmid which contained a L1 promoter linked to cat gene into HeLa cells showed that methylation did repress the L1 promoter activity. In vitro transcription studies using mutagenized templates indicated that methylation of the first seven CpGs in L1 promoter, particularly four CpGs at +52, +58, +61 and +70 was essential for the inhibition. These results suggest that there exists a mechanism to regulate the L1 transcription through the region-specific methylation.

The binding of the transcription factors Sp1 and CTF immediately upstream from the TATA box of the Herpes simplex virus (type 1) thymidine kinase-coding gene (tk) facilitates efficient transcription of this gene in microinjected Xenopus laevis oocytes. To establish whether the presence of methylated CpG dinucleotides within the binding sites of these two factors affects transcription of the tk gene in vivo, we replaced a 33-bp promoter segment, consisting solely of the Sp1 and CTF binding sites, with synthetic oligodeoxynucleotide duplexes containing 5-methylcytosine residues at selected positions. We show that symmetrical methylation (modification of both strands) of any of the four CpGs within this promoter segment resulted in an approximately 20-fold reduction in the specific transcription of the tk gene in Xenopus oocytes, as shown by primer extension analysis of the isolated mRNA. As no other methylated CpG dinucleotides were present within the entire 9.2-kb vector, our results demonstrate that the presence of a single mCpG dinucleotide within the promoter region is sufficient for transcriptional inactivation of the tk gene. The possible mechanisms of this downregulation are discussed.