DNA methylation in blood from neonatal screening cards and the association with BMI and insulin sensitivity in early childhood.

BACKGROUND/OBJECTIVES: There is increasing evidence that metabolic diseases originate in early life, and epigenetic changes have been implicated as key drivers of this early life programming. This led to the hypothesis that epigenetic marks present at birth may predict an individual's future risk of obesity and type 2 diabetes. In this study, we assessed whether epigenetic marks in blood of newborn children were associated with body mass index (BMI) and insulin sensitivity later in childhood. SUBJECTS/METHODS: DNA methylation was measured in neonatal blood spot samples of 438 children using the Illumina Infinium 450 k BeadChip. Associations were assessed between DNA methylation at birth and BMI z-scores, body fat mass, fasting plasma glucose, insulin and homeostatic model assessment of insulin resistance (HOMA-IR) at age 5 years, as well as birth weight, maternal BMI and smoking status. RESULTS: No individual methylation sites at birth were associated with obesity or insulin sensitivity measures at 5 years. DNA methylation in 69 genomic regions at birth was associated with BMI z-scores at age 5 years, and in 63 regions with HOMA-IR. The methylation changes were generally small (<5%), except for a region near the non-coding RNA nc886 (VTRNA2-1) where a clear link between methylation status at birth and BMI in childhood was observed (P=0.001). Associations were also found between DNA methylation, maternal smoking and birth weight. CONCLUSIONS: We identified a number of DNA methylation regions at birth that were associated with obesity or insulin sensitivity measurements in childhood. These findings support the mounting evidence on the role of epigenetics in programming of metabolic health. Whether many of these small changes in DNA methylation are causally related to the health outcomes, and of clinical relevance, remains to be determined, but the nc886 region represents a promising obesity risk marker that warrants further investigation.

Epigenetics and human obesity.

BACKGROUND: Recent technological advances in epigenome profiling have led to an increasing number of studies investigating the role of the epigenome in obesity. There is also evidence that environmental exposures during early life can induce persistent alterations in the epigenome, which may lead to an increased risk of obesity later in life. METHOD: This paper provides a systematic review of studies investigating the association between obesity and either global, site-specific or genome-wide methylation of DNA. Studies on the impact of pre- and postnatal interventions on methylation and obesity are also reviewed. We discuss outstanding questions, and introduce EpiSCOPE, a multidisciplinary research program aimed at increasing the understanding of epigenetic changes in emergence of obesity. RESULTS: An electronic search for relevant articles, published between September 2008 and September 2013 was performed. From the 319 articles identified, 46 studies were included and reviewed. The studies provided no consistent evidence for a relationship between global methylation and obesity. The studies did identify multiple obesity-associated differentially methylated sites, mainly in blood cells. Extensive, but small, alterations in methylation at specific sites were observed in weight loss intervention studies, and several associations between methylation marks at birth and later life obesity were found. CONCLUSIONS: Overall, significant progress has been made in the field of epigenetics and obesity and the first potential epigenetic markers for obesity that could be detected at birth have been identified. Eventually this may help in predicting an individual's obesity risk at a young age and opens possibilities for introducing targeted prevention strategies. It has also become clear that several epigenetic marks are modifiable, by changing the exposure in utero, but also by lifestyle changes in adult life, which implies that there is the potential for interventions to be introduced in postnatal life to modify unfavourable epigenomic profiles.

Methylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.

BACKGROUND: Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS). METHODS: Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay. RESULTS: The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1-8.2; P<0.0001). A decrease in mGSTP1 DNA levels after cycle 1 was associated with a PSA response (P=0.008). In the Phase II cohort, baseline mGSTP1 DNA was a stronger predictor of OS than PSA change after 3 months (P=0.02). Undetectable plasma mGSTP1 after one cycle of chemotherapy was associated with PSA response (P=0.007). CONCLUSIONS: We identified plasma mGSTP1 DNA as a potential prognostic marker in men with CRPC as well as a potential surrogate therapeutic efficacy marker for chemotherapy and corroborated these findings in an independent Phase II cohort. Prospective Phase III assessment of mGSTP1 levels in plasma DNA is now warranted.

Characterization of the human N-ras promoter region.

Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1.

Detailed methylation analysis of the glutathione S-transferase pi (GSTP1) gene in prostate cancer.

Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the pi-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not confined to specific CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no effect on gene expression.

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

Binding of proteins from embryonic and differentiated cells to a bidirectional promoter contained within a CpG island.

We have analysed binding sites of nuclear protein factors to a CpG island (HTF9), which contains the promoter for a pair of overlapping, divergently-transcribed "housekeeping" genes. Using DNaseI protection assays with extracts from a range of differentiated and undifferentiated cell lines, including mouse embryonic stem (ES) and embryonal carcinoma (EC) cells, we located multiple protein binding sites on HTF9. Most of the sites were outside the defined core promoter and could bind to previously identified transcription factors. These included constitutive, inducible and apparently tissue-specific factors in an extremely asymmetric array relative to the transcription start sites of the two genes. A number of sites showed different binding specificities or affinities in different cell types, including ES cells. However, we found no factors that were specific for both ES and EC cells, and no protein-binding site protected exclusively in undifferentiated embryonic cells.

Avian keratin genes. I. A molecular analysis of the structure and expression of a group of feather keratin genes.

The nucleotide sequence of the four complete chicken feather keratin genes A to D contained in the previously isolated recombinant lambda CFK1 has been determined. All four genes have a very similar structure; each gene encodes a polypeptide of 97 amino acid residues and contains an intron in the 5' non-coding region, 37 base-pairs from the cap site. Comparison of the previously determined feather keratin gene C sequence to genes A, B and D indicates that a high level of gene correction has occurred in the protein coding and 5' non-coding regions, which show more than 90% homology, whereas the intron and 3' non-coding regions are by contrast poorly conserved with one or two exceptions. The dramatic conservation of the 5' non-coding region between the feather keratin sequences and an unrelated but co-expressed gene encoding a histidine-rich protein suggests that this segment may play an important role in transcriptional regulation. In addition, both gene types contain an identically positioned intron in the 5' non-coding region. Northern blots performed using gene-specific probes show that the four characterized genes A to D plus gene E, which is partially contained in the recombinant lambda CFK1, are all expressed in feather tissue from 14-day old chick embryos. In addition, we report that a scale keratin gene (originally isolated from a scale complementary DNA library) is expressed at a low level in the embryonic feather.

Relative efficiency of tumor cell killing in vitro by two enzyme-prodrug systems delivered by identical adenovirus vectors.

Enzyme-prodrug therapy for the treatment of cancer is an experimental procedure that is under intensive investigation. However, the relative merits of the various systems for use under specific conditions are still being determined. We have compared the efficacy of cell killing by the herpesvirus thymidine kinase (HSVTK)/ganciclovir and the purine nucleoside phosphorylase (PNP)/9-(beta-M-2-deoxy-erythropentofuranosyl)6-methylpurine enzyme/prodrug systems. These were chosen because of their differential dependence on DNA replication for their mechanism of action. The HSVTK and PNP genes, expressed from the identical prostate-specific antigen promoter, were transduced into human prostate and breast cancers cells using the same human adenovirus vector. The kinetics of cell killing in the presence of the respective prodrugs was monitored using a nondestructive assay that measured total cell bioactivity. The PNP/9-(beta-D-2-deoxy-erythropentofuranosyl)6-methylpurine system was clearly superior in its ability to cause cell death in vitro. Cells were killed in about half the time and at a 5-10-fold lower input of virus relative to the HSVTK/ganciclovir system. The PNP system may offer advantages for the treatment of slow-growing tumors in which the daily proliferative rate is low or in situations in which gene delivery or expression is inefficient.

In vivo gene therapy for prostate cancer: preclinical evaluation of two different enzyme-directed prodrug therapy systems delivered by identical adenovirus vectors.

Advanced prostate cancer is invariably lethal once it becomes androgen independent (AI). With the aim of developing a new treatment we have used the human androgen-independent prostate cancer cell line, PC-3, to evaluate the effectiveness of two enzyme-directed prodrug therapy (EPT) systems as a novel means for promoting tumor cell destruction in vivo. We have confined our study to the use of a PSA promoter, in a preliminary attempt to achieve prostate specificity. The two EPT systems used were the HSVTK/GCV and PNP/6MPDR systems. These were chosen for their differential dependence on DNA replication for their mechanism of action. In the present work, either the HSVTK or PNP gene, each controlled by a PSA promoter fragment, was delivered by an E1-, replication-deficient human adenovirus (Ad5) into PC-3 tumors growing subcutaneously in BALB/c nude mice. Tumors were injected with a single dose of recombinant Ad5 and mice were treated intraperitoneally with the appropriate prodrug, twice daily, for 6 days thereafter. The growth of established PC-3 tumors was significantly suppressed and host survival increased with a single course of HSVTK/GCV or PNP/6MPDR treatment. HSVTK/GCV-treated PC-3 tumor growth was 80% less than that of control treatments on day 33, while PNP/6MPDR-treated tumor growth was approximately 75% less than that of control treatments on day 52. Survival data showed that 20% of HSVTK/GCV- or PNP/6MPDR-treated animals lived >45 and >448 days, respectively, longer than control animals. These results demonstrate that both HSVTK/GCV and PNP/6MPDR therapies interrupt the growth of an aggressive human prostate cancer cell line in vivo.

Sp1 binding is inhibited by (m)Cp(m)CpG methylation.

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.

An engineered PGK promoter and lac operator-repressor system for the regulation of gene expression in mammalian cells.

Previous reports have demonstrated that the Escherichia coli lac repressor can operate effectively in mammalian cells to repress expression of genes driven by modified viral or metallothionein (MT) promoters. We have developed a more general expression system using the promoter from the PGK1 gene (encoding murine 3-phosphoglycerate kinase) which is widely expressed in almost all cell types, including early embryonic and ES (embryonic stem) cells. Firstly, we engineered the lac repressor to include a nuclear localisation signal and placed it under control of the PGK1 promoter. Efficient nuclear localisation of the repressor was demonstrated by mobility-shift assays and immunofluorescence detection. For the target vectors, we modified the wild-type (wt) PGK1 promoter to include lac operator (lacO) sites for binding of the lac repressor and compared a number of different lacO positions and arrangements based on proximity to the native start points for transcription (tsp) and translation. In the absence of repressor, we observed reduced expression of the neo reporter gene for some placements of the lacO, but wt expression for placements near the tsp. When both target and repressor were present in the cells, we observed that the expression of neo could be strongly suppressed and reversibly regulated by induction with IPTG. In particular, for a promoter which contained two spaced lacO replacing native sequence around the major tsp, we observed 90-95% repression by the lac repressor for the neo reporter gene and up to 98% repression for the cat reporter gene. Efficient derepression by IPTG was observed in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of transcription from different RNA polymerase II promoters by high mobility group proteins 1 and 2.

High mobility group proteins (HMGs) 1 and 2 are shown to stimulate transcription in vitro from a number of RNA polymerase II promoters. Greatest effects were seen on transcription from the SV40 late promoter, then the SV40 early promoter with similar levels of transcription enhancement being seen for the human metallothionein 2A, adenovirus major late and chicken feather keratin promoters. The results indicate that HMGs 1 and 2 act to increase initiation of transcription in vitro and differential effects on the promoters are consistent with their action being in part to enhance the binding or functional activity of promoter-specific transcription factors.

High level, tissue-specific expression of a modified calcitonin/calcitonin gene-related peptide promoter in a human medullary thyroid carcinoma cell line.

The efficient and high level expression of therapeutic genes in target cells is critical for effective gene therapy. We have developed a novel promoter by utilizing tandem repeats of a tissue-specific regulatory element from the calcitonin/calcitonin gene-related peptide (CT/CGRP) gene placed in close proximity to a basal promoter, thereby removing interstitial sequences. This promoter drives expression of reporter genes at much higher levels than the natural promoter while significantly improving specificity in thyroid C cells.

Effects of DNA methylation on specific transcription by RNA polymerase II in vitro.

In vitro transcription in a HeLa cell lysate by RNA polymerase II directed by a chicken feather keratin gene promotor has been studied using unmethylated template DNA and DNA methylated in vitro by HpaII methylase. The efficiency of specific gene transcription from methylated DNA was dependent on topology of the input DNA, the most significant effect being complete inhibition of transcription from one template which contained three methylation sites, one just 5' and two greater than 500 bases 3' to the site of transcription initiation. The inhibition of transcription depends on a factor(s) which is variably present in lysate preparations and is labile on storage at -70 degrees.

Cleavage of DNA.RNA hybrids by type II restriction enzymes.

The action of a number of restriction enzymes on DNA.RNA hybrids has been examined using hybrids synthesised with RNAs of cucumber mosaic virus as templates. The enzymes EcoRI, HindII, SalI, MspI, HhaI, AluI, TaqI and HaeIII cleaved the DNA strand of the hybrids (and possible also the RNA strand) into specific fragments. For four of these enzymes, HhaI, AluI, TaqI and HaeIII, comparison of the restriction fragments produced with the known sequences of the viral RNAs confirmed that they were recognising and cleaving the DNA strand of the hybrids at their correct recognition sequences. It is likely that the ability to utilise DNA.RNA hybrids as substrates is a general property of Type II restriction enzymes.

Effects of high mobility group proteins 1 and 2 on initiation and elongation of specific transcription by RNA polymerase II in vitro.

High mobility group proteins 1 and 2 (HMGs 1 and 2) are abundant chromosomal proteins of higher eukaryotes, which have been found to be enriched in regions of active chromatin. We have previously demonstrated that they can stimulate specific transcription in vitro by RNA polymerases II and III and overcome inhibition caused by added histones. Here we study whether these effects are mediated at the level of initiation or elongation of transcription. Additions of HMGs 1 and 2 and/or histones were found to have only small or no effect on the efficiency of elongation; this was determined by comparing the relative synthesis of transcripts of different lengths, ranging from 95 to 1535 bases. The observed stimulation cannot be explained by an increased utilization of initiation complexes for multiple rounds of transcription as a similar level of stimulation by HMGs 1 and 2 was seen when RNA synthesis was limited to one round per template DNA by addition of a low level of Sarkosyl after formation of initiation complexes. The effects of HMGs 1 and 2 were principally seen on the rate of formation of effective initiation complexes. These data are consistent with the hypothesis that HMGs 1 and 2 stimulate transcription by facilitating the formation of active initiation complexes on template DNA.