The Long Noncoding RNA RP11-728F11.4 Promotes Atherosclerosis.

OBJECTIVE: Noncoding RNAs are emerging as important players in gene regulation and cardiovascular diseases. Their roles in the pathogenesis of atherosclerosis are not fully understood. The purpose of this study was to determine the role played by a previously uncharacterized long noncoding RNA, RP11-728F11.4, in the development of atherosclerosis and the mechanisms by which it acts. Approach and Results: Expression microarray analysis revealed that atherosclerotic plaques had increased expression of RP11-728F11.4 as well as the cognate gene FXYD6 (FXYD domain containing ion transport regulator 6), which encodes a modulator of Na+/K+-ATPase. In vitro experiments showed that RP11-728F11.4 interacted with the RNA-binding protein EWSR1 (Ewings sarcoma RNA binding protein-1) and upregulated FXYD6 expression. Lentivirus-induced overexpression of RP11-728F11.4 in cultured monocytes-derived macrophages resulted in higher Na+/K+-ATPase activity, intracellular cholesterol accumulation, and increased proinflammatory cytokine production. The effects of RP11-728F11.4 were enhanced by siRNA-mediated knockdown of EWSR1 and reduced by downregulation of FXYD domain containing ion transport regulator 6. In vivo experiments in apoE knockout mice fed a Western diet demonstrated that RP11-728F11.4 increased proinflammatory cytokine production and augmented atherosclerotic lesions. CONCLUSIONS: RP11-728F11.4 promotes atherosclerosis, with an influence on cholesterol homeostasis and proinflammatory molecule production, thus representing a potential therapeutic target. Graphic Abstract: A graphic abstract is available for this article.

Maternal genome-wide DNA methylation profiling in gestational diabetes shows distinctive disease-associated changes relative to matched healthy pregnancies.

Several recent reports have described associations between gestational diabetes (GDM) and changes to the epigenomic landscape where the DNA samples were derived from either cord or placental sources. We employed genome-wide 450K array analysis to determine changes to the epigenome in a unique cohort of maternal blood DNA from 11 pregnant women prior to GDM development relative to matched controls. Hierarchical clustering segregated the samples into 2 distinct clusters comprising GDM and healthy pregnancies. Screening identified 100 CpGs with a mean ß-value difference of ≥0.2 between cases and controls. Using stringent criteria, 5 CpGs (within COPS8, PIK3R5, HAAO, CCDC124, and C5orf34 genes) demonstrated potentials to be clinical biomarkers as revealed by differential methylation in 8 of 11 women who developed GDM relative to matched controls. We identified, for the first time, maternal methylation changes prior to the onset of GDM that may prove useful as biomarkers for early therapeutic intervention.

Methylation of HOXA9 and ISL1 Predicts Patient Outcome in High-Grade Non-Invasive Bladder Cancer.

INTRODUCTION: Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis. METHODS: Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases. RESULTS: The ISL1 genes' promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM). CONCLUSIONS: In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

DNA methylation profiling of synovial fluid FLS in rheumatoid arthritis reveals changes common with tissue-derived FLS.

AIM: Alterations in DNA methylation contribute to the abnormal phenotype of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). We profiled genome-wide DNA methylation in these cells from synovial fluid, a more readily accessible source of disease-associated cells. PATIENTS & METHODS: Genome-wide DNA methylation was interrogated in fluid-derived FLS from five RA and six osteoarthritis patients using Human Methylation 450 Bead Chip and bisulfite pyrosequencing. RESULTS: Array analysis identified 328 CpGs, representing 195 genes, that were differentially methylated between RA and osteoarthritis fluid-derived FLS. Comparison with the genes identified in two independent studies of tissue-derived FLS revealed 73 genes in common (~40%), of which 22 shared identity with both studies. Pyrosequencing confirmed altered methylation of these genes. CONCLUSION: Synovial fluid-derived RA FLS show methylation changes common with tissue-derived FLS, supporting the use of fluid-derived FLS for future investigations.

Methylation of the FGFR2 gene is associated with high birth weight centile in humans.

AIMS: This study examined links between DNA methylation and birth weight centile (BWC), and explored the impact of genetic variation. MATERIALS & METHODS: Using HumanMethylation450 arrays, we examined candidate gene-associated CpGs in cord blood from newborns with low (<15th centile), medium (40-60th centile) and high (>85th centile) BWC (n = 12). Candidates were examined in an investigation cohort (n = 110) using pyrosequencing and genotyping for putative methylation-associated polymorphisms performed using standard PCR. RESULTS: Array analysis identified 314 candidate genes associated with BWC extremes, four of which showed ≥ 4 BWC-linked CpGs. Of these, PM20D1 and MI886 suggested genetically determined methylation levels. However, methylation at three CpGs in FGFR2 remained significantly associated with high BWC (p = 0.004-0.027). CONCLUSION: We identified a novel biologically plausible candidate (FGFR2) for with BWC that merits further study.

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations.

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.

Epigenome-wide profiling identifies significant differences in DNA methylation between matched-pairs of T- and B-lymphocytes from healthy individuals.

Multiple reports now describe changes to the DNA methylome in rheumatoid arthritis and in many cases have analyzed methylation in mixed cell populations from whole blood. However, these approaches may preclude the identification of cell type-specific methylation, which may subsequently bias identification of disease-specific changes. To address this possibility, we conducted genome-wide DNA methylation profiling using HumanMethylation450 BeadChips to identify differences within matched pairs of T-lymphocytes and B-lymphocytes isolated from the peripheral blood of 10 healthy females. Array data were processed and differential methylation identified using NIMBL software. Validation of array data was performed by bisulfite pyrosequencing. Genome-wide DNA methylation was initially determined by analysis of LINE-1 sequences and was higher in B-lymphocytes than matched T-lymphocytes (69.8% vs. 65.2%, P ≤ 0.01). Pairwise analysis identified 679 CpGs, representing 250 genes, which were differentially methylated between T-lymphocytes and B-lymphocytes. The majority of sites (76.6%) were hypermethylated in B-lymphocytes. Pyrosequencing of selected candidates confirmed the array data in all cases. Hierarchical clustering revealed perfect segregation of samples into two distinct clusters based on cell type. Differentially methylated genes showed enrichment for biological functions/pathways associated with leukocytes and T-lymphocytes. Our work for the first time shows that T-lymphocytes and B-lymphocytes possess intrinsic differences in DNA methylation within a restricted set of functionally related genes. These data provide a foundation for investigating DNA methylation in diseases in which these cell types play important and distinct roles.

Combined influence of gene-specific cord blood methylation and maternal smoking habit on birth weight.

AIM: Evidence suggests that folic acid intake affects birth weight and that these effects may be mediated via the fetal epigenome. Our previous array data indicate that methylation in human cord blood at gene-specific CpGs is associated with birth weight percentile (BWP). Our aims were to investigate associations with BWP in specific CpGs identified by the array analysis in a significantly larger cohort and investigate the effects of other relevant factors on this association. MATERIALS & METHODS: Methylation status was examined in candidate CpGs in 129 cord blood samples using Pyrosequencing™. The effects of other potentially important factors; maternal smoking, folate-related metabolite levels and genetic variation in the MTHFR gene, were examined. Linear and logistic regression analyses were used to identify relationships between BWP and methylation levels in the context of other key factors. RESULTS: Increased cord methylation at CpGs in GSTM5 and MAP2K3 was associated with a reduced risk of having a birth weight below the 50th percentile (p = 0.010; odds ratio [OR]: 0.33 and p = 0.024; OR: 0.24, respectively) while higher methylation levels in APOB were associated with an increased risk (p = 0.023; OR: 2.56). Smoking during pregnancy modified the effect of methylation on BWP. Thus, compared with nonsmokers with a GSTM5 methylation level of >25% (median BWP: 54.7%), those who had smoked during pregnancy and whose GSTM5 methylation was <25% had the lowest median BWP (12.0%; p = 0.001). Furthermore, this latter group had the highest proportion of cases with BWPs below 50% (92.9 compared with 47.8% in nonsmokers with a GSTM5 methylation level of >25%; p = 0.013; OR: 14.2). Similar results were identified for MAP2K3, while the link with APOB reflected the inverse relationship between methylation at this locus and BWP. CONCLUSION: Our data suggest that gene-specific methylation of cord DNA is associated with BWP and this methylation provides an additional effect on BWP to that of smoking during pregnancy.

Antiepileptic drugs and the fetal epigenome.

Antiepileptic drugs (AEDs) can lower maternal folate and increase maternal homocysteine levels, which are known to affect the methyl cycle and hence DNA methylation levels. The influence of in utero exposure to AEDs on fetal DNA methylation was investigated. Genome-wide fetal epigenomic profiles were determined using the Infinium 27K BeadArray from Illumina (San Diego, CA, U.S.A.). The Infinium array measures approximately 27,000 CpG loci associated with 14,496 genes at single-nucleotide resolution. Eighteen cord blood samples (nine samples from babies exposed to AEDs and nine controls) from otherwise uncomplicated pregnancies were compared. Unsupervised hierarchic clustering was used to compare the calculated methylation profiles. A clear distinction between the methylation profiles of samples from babies exposed to AEDs in utero compared with controls was detected. These data provide evidence of an epigenetic effect associated with antenatal AED and high-dose folate supplementation during pregnancy. The differences in fetal DNA methylation of those exposed to AEDs shows that a genome-wide effect of methylation is evident. In addition, the epigenetic changes observed appear to be, in this limited sample, independent of extremes of birth weight centiles. These preliminary data highlight possible mechanisms by which AEDs might influence fetal outcomes and the potential of optimizing AED-specific folate supplementation regimens to offset these effects.

Quantitative, high-resolution epigenetic profiling of CpG loci identifies associations with cord blood plasma homocysteine and birth weight in humans.

Supplementation with folic acid during pregnancy is known to reduce the risk of neural tube defects and low birth weight. It is thought that folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. We examined the effects of folate on the human methylome using quantitative interrogation of 27,578 CpG loci associated with 14,496 genes at single-nucleotide resolution across 12 fetal cord blood samples. Consistent with previous studies, the majority of CpG dinucleotides located within CpG islands exhibited hypo-methylation while those outside CpG islands showed mid-high methylation. However, for the first time in human samples, unbiased analysis of methylation across samples revealed a significant correlation of methylation patterns with plasma homocysteine, LINE-1 methylation and birth weight centile. Additionally, CpG methylation significantly correlated with either birth weight or LINE-1 methylation were predominantly located in CpG islands. These data indicate that levels of folate-associated intermediates in cord blood reflect their influence and consequences for the fetal epigenome and potentially on pregnancy outcome. In these cases, their influence might be exerted during late gestation or reflect those present during the peri-conceptual period.

Ectoderm, endoderm, and the evolution of heterodont dentitions.

Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3beta, alpha-fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth-forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals.

Expression of Xenopus tropicalis HNF6/Onecut-1.

Onecut genes belong to a family of transcription factors that are known to be important in embryonic development. In the present study, we analyzed the pattern of expression of Onecut-1/HNF6 in Xenopus tropicalis using RT-PCR and whole mount in situ hybridization. Expression of the Xenopus tropicalis Onecut-1/HNF6 gene was found to be conserved in the neural tube, the sensory placodes and in the anterior ventral endoderm in a domain consistent with the developing liver primordium.

NKCC1 (SLC12a2) induces a secondary axis in Xenopus laevis embryos independently of its co-transporter function.

NKCC1 is a broadly expressed Na(+)-K(+)-Cl(-) co-transporter involved in regulation of ion flux across the cell membrane and in regulating cell volume. Whilst much is known about the co-transporter activity of NKCC1 and its regulation by protein kinases and phosphatases, little is known about the activities of NKCC1 that are co-transporter independent. In this report we show that over-expression of NKCC1 in embryos of Xenopus laevis induces secondary axes, independently of its co-transporter activity. In addition, over-expression of NKCC1 results in the formation of neural tissue in ectodermal explants. We also show that NKCC1 is expressed broadly but non-uniformly in embryos of Xenopus laevis and Xenopus tropicalis, with prominent expression in the notochord, nervous system and stomach. These results provide insights into an additional, previously unreported activity of NKCCl.

GATA4 and GATA5 are essential for heart and liver development in Xenopus embryos.

BACKGROUND: GATA factors 4/5/6 have been implicated in the development of the heart and endodermal derivatives in vertebrates. Work in zebrafish has indicated that GATA5 is required for normal development earlier than GATA4/6. However, the GATA5 knockout mouse has no apparent embryonic phenotype, thereby questioning the importance of the gene for vertebrate development. RESULTS: In this study we show that in Xenopus embryos GATA5 is essential for early development of heart and liver precursors. In addition, we have found that in Xenopus embryos GATA4 is important for development of heart and liver primordia following their specification, and that in this role it might interact with GATA6. CONCLUSION: Our results suggest that GATA5 acts earlier than GATA4 to regulate development of heart and liver precursors, and indicate that one early direct target of GATA5 is homeobox gene Hex.

Characterisation of the genomic canine Fgf8 locus and screen for genetic variants in 4 dogs with different face types.

We are investigating the genetic basis of morphological differences in skull shape between domestic dogs of different breeds using a candidate gene approach to identify genes involved in the genetic regulation. One such candidate is Fgf8. Fgf8 is a signalling molecule important in the embryonic development and patterning of the craniofacial region. Mice conditional null for the expression of Fgf8 after E9.5 have a short foreface and a wide skull (Trumpp et al. 1999). Using a combination of bioinformatics and PCR cloning, we have characterised the genomic loci of the canine Fgf8 gene. Like the mouse homologue, it is composed of six exons and we also predict that like the mouse, there are eight alternative isoforms that are generated by alternative splicing events. We have identified a short 200 bp sequence upstream of the Fgf8 gene that is highly conserved between species and have predicted putative transcription factor binding sites using the Transfac database. Genetic analysis of 4 dogs with different skull types identified genetic variation. None of the variants however, were predicted to have any functional significance.

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm.

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.

Characterisation of the genomic structure of chick Fgf8.

The Fgf8 gene encodes a series of secreted signalling molecules important in the normal development of the face, brain and limbs. The genomic structure of the chick Fgf8 gene has been analysed and compared to the human and mouse sequences. Divergence between the chick, human and mouse genomic structure was observed. Data indicates that the long alternatively spliced form of exon 1b observed in mouse and exon 1c observed in human and mouse do not exist in the chick Fgf8 gene. RT-PCR analysis indicates that chick Fgf8, like its mouse and human counterpart is alternatively spliced. This data along with the genomic structure data indicates that in the chick there are only two isoforms of Fgf8. This is in contrast to the human and mouse, where evidence suggests that there are 4 and 8 isoforms, respectively. Approximately 400 bp of intron 1d is highly conserved between chick, human and mouse genomic sequences. Using TRANSFAC possible conserved regulatory element binding sites within this domain were identified.

Expression of Claudin-3 during chick development.

Claudins are membrane proteins located within tight junctions. Using degenerate and gene specific primers the chick homologue of Claudin-3 was isolated. Here we show the expression of Claudin-3 transcripts in the developing chick embryo from Hamburger and Hamilton Stages (HH) 6-22. The early expression domains of Claudin 3 in the developing chick embryo include the mesoderm surrounding Hensen's node and the head fold. Between HH 9 and HH 11 expression domains include the anterior intestinal portal and otic vesicle. By HH 14, gene expression is observed in the pharyngeal endoderm and pouches, in addition to the continued expression in the otic vesicle. Expression in the more posterior pouches was also observed as development proceeded. At HH 20 expression is present in the mesonephric system and also the developing liver, lung bud and intestine.

Regionalisation of early head ectoderm is regulated by endoderm and prepatterns the orofacial epithelium.

The oral epithelium becomes regionalised proximodistally early in development, and this is reflected by the spatial expression of signalling molecules such as Fgf8 and Bmp4. This regionalisation is responsible for regulating the spatial expression of genes in the underlying mesenchyme. These genes are required for the spatial patterning of bone, cartilage orofacial development and, in mammals, teeth. The mechanism and timing of this important regionalisation during head epithelium development are not known. Using lipophilic dyes to fate map the oral epithelium in chick embryos, we show that the cells that will occupy the epithelium of the distal and the proximal mandible primordium already occupy different spatial locations in the developing head ectoderm prior to the formation of the first pharyngeal arch and neural crest migration. Moreover, the ectoderm cells fated to become proximal oral epithelium express Fgf8 and this expression requires the presence of endoderm. Thus, the first fundamental patterning process in jaw morphogenesis is controlled by the early separation of specific areas of ectoderm that are regulated by ectoderm-endoderm interactions, and does not involve neural crest cells.