Identifying the genetic associations among the psoriasis patients in eastern India.

Psoriasis is a multifactorial genetic disorder manifested by hyperproliferation and abnormal differentiation of epidermal keratinocytes, along with the infiltration of inflammatory cells into the skin. Although ~80 genetic susceptibility variants were reported in psoriasis, many loci showed population-specific associations, warranting the need for more population-specific association studies in psoriasis. We determined the association of forty single nucleotide polymorphisms (SNPs) among 2136 psoriasis patients and normal individuals from eastern India. We investigated the expression of corresponding genes and evaluated the protein structure stability for the genes with susceptible coding variants. We found fifteen SNPs significantly associated with psoriasis, while additional three SNPs showed significant association when we classified the patients based on the presence of HLA-Cw6 allele. Epistatic interaction between HLA-Cw6 and other associated loci showed significant association with the SNPs at PSORS1 region, along with other five SNPs outside PSORS1. Three genes showed significant differential expression in psoriatic tissues compared to the adjacent normal skin tissues but were not differential when classified the patients based on their genotypes. SNP rs495337 at SPATA2 (Spermatogenesis Associated 2) showed a 1.2-fold increased risk among the HLA-Cw6 patients compared to combined samples. We found significant downregulation of SPATA2 among the patients with risk genotypes and HLA-Cw6 allele compared to the non-risk genotypes. Protein structure stability analysis showed reduced structural stability for all the mutant residues caused by the associated coding variants. Our study evaluated the genetic associations of psoriasis-susceptible variants in India and evaluated the possible functional significance of these associated variants in psoriasis.

Serum miRNA profiling identified miRNAs associated with disease severity in psoriasis.

Psoriasis vulgaris is a chronic, autoimmune skin disease involving a complex interplay of epidermal keratinocytes, dermal fibroblast and infiltrating immune cells. Differential expressions of miRNAs are observed in psoriasis and the deregulated miRNAs are sometimes associated with disease severity. This study aims to identify miRNAs altered in the serum of psoriasis patients that are associated with the Psoriasis Area and Severity Index (PASI). In order to assess miRNA levels in the serum of psoriasis patients, we selected 24 differentially expressed miRNAs in the psoriatic skin are possibly derived from the skin and immune cells, as well as five miRNAs that are enriched in other tissues. We identified 16 miRNAs that exhibited significantly (p < 0.05) altered levels in the serum of psoriasis patients compared to healthy individuals. Among these, 13 miRNAs showed similar expression pattern in the serum of psoriasis patients as also observed in the psoriatic skin tissues. Ten miRNAs showed an accuracy of greater than 75% in classifying the psoriasis patients from healthy individuals. Further analysis of differential miRNA levels between the low PASI group and the high PASI group identified three miRNAs (miR-147b, miR-3614-5p, and miR-125a-5p) with significantly altered levels between the low severity and the high severity psoriasis patients. Our systematic investigation of skin and immune cell-derived miRNAs in the serum of psoriasis patients revealed alteration in miRNA levels to be associated with disease severity, which may help in monitoring the disease progression and therapeutic response.

Emerging roles of non-coding RNAs in psoriasis pathogenesis.

Psoriasis is a complex genetic skin disorder typically manifested by red, scaly, and itchy plaques most commonly over the scalp, trunk, elbows, and knees. Histopathological features include thickening of the epidermal layer due to hyper-proliferation and abnormal differentiation of epidermal keratinocytes along with infiltration of immune cells in the psoriatic skin. It is a chronic inflammatory relapsing disease, and there is currently no permanent cure for psoriasis. Proper medications can reduce the severity of the disease and improve the quality of life of the patients. While the genetic components of psoriasis pathogenesis are well explored, the full understanding of its epigenetic component remains elusive. Non-coding RNAs (ncRNAs) are documented to regulate various epigenetic processes that lead to the pathogenesis of different diseases including psoriasis. In this review, we have discussed the molecular interplay of different ncRNAs in psoriasis pathogenesis. The roles of microRNAs (miRNAs) in psoriasis are pretty well studied, whereas the roles of long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are emerging. This review provides ideas covering some of the latest findings of different modes of functions played by those different ncRNAs documented in the literature. As an ever-evolving topic, some works are still ongoing as well as there are several fields that need rigorous scientific ventures. We have proposed the areas which claim more explorations to better understand the roles played by the ncRNAs in psoriasis pathogenesis.

Effect of deletions in the α-globin gene on the phenotype severity of ß-thalassemia.

Thalassemia is the most common inherited hemoglobinopathy worldwide. Variation of clinical symptoms in this hemoglobinopathy entails differences in disease-onset and transfusion requirements. The aim of this study was to investigate the role of α-globin gene deletions in modulating the clinical heterogeneity of ß-thalassemia (ß-thal) syndromes. A total number 270 ß-thal subjects were enrolled. Hematological parameters were recorded. ß-Globin mutations were determined by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR), gap-PCR and Sanger sequencing. α-Globin gene deletions were determined by multiplex PCR. Out of 270 ß-thal subjects, 19 carried ß+/ß+, 74 had ß0/ß0 and 177 had the ß0/ß+ genotype. When we determined the severity of the different ß-thal subjects in coinherited with the α gene deletion, it was revealed that, 84.2% ß+/ß+ subjects carried a non severe phenotype and did not have an α gene deletion. Of the ß0/ß0 individuals, 95.9% presented a severe phenotype, irrespective of α-globin gene deletions. In cases with the ß0/ß+ genotype, 19.2% subjects also carried a deletion on the α gene. Of these, 61.8% presented a non severe phenotype and 38.2% were severely affected. Only in the ß0/ß+ category did α gene deletions make a significant contribution (p < 0.001) toward alleviation of clinical severity. Therefore, it can be stated that α-globin gene deletions play a role in ameliorating the phenotype in patients with a ß+/ß0 genotype.

Metabolic impairment in response to early induction of C/EBPß leads to compromised cardiac function during pathological hypertrophy.

Chronic pressure overload-induced left ventricular hypertrophy in heart is preceded by a metabolic perturbation that prefers glucose over lipid as substrate for energy requirement. Here, we establish C/EBPß (CCAAT/enhancer-binding protein ß) as an early marker of the metabolic derangement that triggers the imbalance in fatty acid (FA) oxidation and glucose uptake with increased lipid accumulation in cardiomyocytes during pathological hypertrophy, leading to contractile dysfunction and endoplasmic reticulum (ER) stress. This is the first study that shows that myocardium-targeted C/EBPß knockdown prevents the impaired cardiac function during cardiac hypertrophy led by maladaptive metabolic response with persistent hypertrophic stimuli, whereas its targeted overexpression in control increases lipid accumulation significantly compared to control hearts. A new observation from this study was the dual and opposite transcriptional regulation of the alpha and gamma isoforms of Peroxisomal proliferator activated receptors (PPARα and PPARγ) by C/EBPß in hypertrophied cardiomyocytes. Before the functional and structural remodeling sets in the diseased myocardium, C/EBPß aggravates lipid accumulation with the aid of the increased FA uptake involving induced PPARγ expression and decreased fatty acid oxidation (FAO) by suppressing PPARα expression. Glucose uptake into cardiomyocytes was greatly increased by C/EBPß via PPARα suppression. The activation of mammalian target of rapamycin complex-1 (mTORC1) during increased workload in presence of glucose as the only substrate was prevented by C/EBPß knockdown, thereby abating contractile dysfunction in cardiomyocytes. Our study thus suggests that C/EBPß may be considered as a novel cellular marker for deranged metabolic milieu before the heart pathologically remodels itself during hypertrophy.

The exosome encapsulated microRNAs as circulating diagnostic marker for hepatocellular carcinoma with low alpha-fetoprotein.

Diagnosis of hepatocellular carcinoma (HCC) remains challenging to clinicians, particularly in a patient with low alpha-fetoprotein. Here, in silico, ex vivo and in vitro data were combined to identify liver-specific exosomal miRNAs as an early diagnostic marker for HCC. Transcriptome profiling for mRNA and small RNA in same HCV-HCC and normal liver tissues followed by cross-validation of 41 deregulated miRNAs (log2 FoldChange > 1.5, Padj < .1) with GEO/TCGA datasets of HCV/HBV related HCC vs normal/adjacent tissue revealed three miRNAs were commonly deregulated (miR-10b/miR-21/miR-182) among all HCC irrespective of viral etiology. Targets of top deregulated miRNAs were identified by TargetScan/miRwalk and validated in mRNA transcriptome data followed by Panther/Gene Ontology enrichment/Cytoscape analysis suggested that targets were mostly from carcinogenesis pathways. Hence, those miRNAs were validated in normal and HCV-HCC tissues by qRT-PCR and subsequently in plasma-derived-exosomes of both HBV/HCV infected non-HCC (chronic hepatitis [CH]/liver cirrhosis [LC]) and HCC samples, and in liver-specific Anti-Asgr2 immuno-enriched exosomes. Exosomes were verified using Nanosight/TEM/immune-blotting with anti-Alix/anti-GRP78/anti-Asgr2. Along with miR-21-5p, miR-10b-5p/miR-221-3p/miR-223-3p was found significantly upregulated in the exosome of HCC patients than CH/non-HCC. The comparable expression pattern was seen in anti-Asgr2 immuno-precipitated exosomes. Interestingly, the AFP level was found below 250 ng/mL in about 94% of HCV-HCC and 62% of HBV-HCC patients. ROC analysis showed that miR-10b-5p + miR-221-3p + miR-223-3p + miR-21-5p could differentiate CH/non-HCC(CH + LC) from HCC with AUROC: 0.86 (97.5% CI: 0.77-0.94)/0.80 (97.5% CI: 0.70-0.89), sensitivity: 74%/58% and specificity: 86%/95% while miR-10b-5p + miR-221-3p + miR-223-3p showed AUROC: 0.84 (97.5% CI: 0.74-0.94)/0.74 (97.5% CI: 0.63-0.84), sensitivity: 86%/86% and specificity:66%/53% for low AFP-HCC vs CH/non-HCC, respectively, having better sensitivity than the combination of four miRNAs. Multivariate analysis further revealed low Albumin and high miR-21-5p as probable independent risk factor for HCC.

A novel microRNA boosts hyper-ß-oxidation of fatty acids in liver by impeding CEP350-mediated sequestration of PPARα and thus restricts chronic hepatitis C.

Imbalance in lipid metabolism induces steatosis in liver during Chronic hepatitis C (CHC). Contribution of microRNAs in regulating lipid homoeostasis and liver disease progression is well established using small RNA-transcriptome data. Owing to the complexity in the development of liver diseases, the existence and functional importance of yet undiscovered regulatory miRNAs in disease pathogenesis was explored in this study using the unmapped sequences of the transcriptome data of HCV-HCC liver tissues following miRDeep2.pl pipeline. MicroRNA-c12 derived from the first intron of LGR5 of chromosome 12 was identified as one of the miRNA like sequences retrieved in this analysis that showed human specific origin. Northern blot hybridization has proved its existence in the hepatic cell line. Enrichment of premiR-c12 in dicer-deficient cells and miR-c12 in Ago2-RISC complex clearly suggested that it followed canonical miRNA biogenesis pathway and accomplished its regulatory function. Expression of this miRNA was quite low in CHC tissues than normal liver implying HCV-proteins might be regulating its biogenesis. Promoter scanning and ChIP analysis further revealed that under expression of p53 and hyper-methylation of STAT3 binding site upon HCV infection restricted its expression in CHC tissues. Centrosomal protein 350 (CEP350), which sequestered PPARα, was identified as one of the targets of miR-c12 using Miranda and validated by luciferase assay/western blot analysis. Furthermore, reduced triglyceride accumulation and enhanced PPARα mediated transcription of ß-oxidation genes upon restoration of miR-c12 in liver cells suggested its role in lipid catabolism. Thus this study is reporting miR-c12 for the first time and showed its' protective role during chronic HCV infection.

A powerful method to integrate genotype and gene expression data for dissecting the genetic architecture of a disease.

To decipher the genetic architecture of human disease, various types of omics data are generated. Two common omics data are genotypes and gene expression. Often genotype data for a large number of individuals and gene expression data for a few individuals are generated due to biological and technical reasons, leading to unequal sample sizes for different omics data. Unavailability of standard statistical procedure for integrating such datasets motivates us to propose a two-step multi-locus association method using latent variables. Our method is powerful than single/separate omics data analysis and it unravels comprehensively deep-seated signals through a single statistical model. Extensive simulation confirms that it is robust to various genetic models as its power increases with sample size and number of associated loci. It provides p-values very fast. Application to real dataset on psoriasis identifies 17 novel SNPs, functionally related to psoriasis-associated genes, at much smaller sample size than standard GWAS.

p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

Oral submucous fibrosis: A global challenge. Rising incidence, risk factors, management, and research priorities.

Oral submucous fibrosis is a potentially malignant disorder of the oral cavity, with a high rate of malignant transformation. It is very common among habitual areca nut chewers. The pathogenesis of oral submucous fibrosis is not well established, but it is believed to be a disease of multifactorial origin, including areca nut chewing, ingestion of chilies, genetic factors, immunologic processes and nutritional deficiencies. Genetically susceptible individuals when exposed to areca nut chewing develop this disease over a variable period of time. Oral submucous fibrosis is considered to be a disease of collagen metabolism. Several genetic factors are reported but there is no consensus about the exact mechanism of disease initiation. Variations in histopathological presentation are noted among oral submucous fibrosis patients with habitual areca nut chewing in different forms and other additive agents, eg betel quid, pan masala and gutkha, together with a variety of tobacco habits. The role of epigenetic modifications, such as miRNA regulation, and DNA methylation is also being reported as part of the pathogenesis of oral submucous fibrosis. A combined approach, including analysis of genetic and epigenetic regulations with different habits, might be helpful to better understand the contributory factors and pathogenesis of this serious disorder.

Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington's Disease.

Altered expression levels of protein-coding genes and microRNAs have been implicated in the pathogenesis of Huntington's disease (HD). The involvement of other ncRNAs, especially long ncRNAs (lncRNA), is being realized recently and the related knowledge is still rudimentary. Using small RNA sequencing and PCR arrays we observed perturbations in the levels of 12 ncRNAs in HD mouse brain, eight of which had human homologs. Of these, Meg3, Neat1, and Xist showed a consistent and significant increase in HD cell and animal models. Transient knock-down of Meg3 and Neat1 in cell models of HD led to a significant decrease of aggregates formed by mutant huntingtin and downregulation of the endogenous Tp53 expression. Understanding Meg3 and Neat1 functions in the context of HD pathogenesis is likely to open up new strategies to control the disease.

Dominant bacterial phyla in caves and their predicted functional roles in C and N cycle.

BACKGROUND: Bacteria present in cave often survive by modifying their metabolic pathway or other mechanism. Understanding these adopted bacteria and their survival strategy inside the cave is an important aspect of microbial ecology. Present study focuses on the bacterial community and geochemistry in five caves of Mizoram, Northeast India. The objective of this study was to explore the taxonomic composition and presumed functional diversity of cave sediment metagenomes using paired end Illumina sequencing using V3 region of 16S rRNA gene and bioinformatics pipeline. RESULTS: Actinobacteria, Proteobacteria, Verrucomicrobia and Acidobacteria were the major phyla in all the five cave sediment samples. Among the five caves the highest diversity is found in Lamsialpuk with a Shannon index 12.5 and the lowest in Bukpuk (Shannon index 8.22). In addition, imputed metagenomic approach was used to predict the functional role of microbial community in biogeochemical cycling in the cave environments. Functional module showed high representation of genes involved in Amino Acid Metabolism in (20.9%) and Carbohydrate Metabolism (20.4%) in the KEGG pathways. Genes responsible for carbon degradation, carbon fixation, methane metabolism, nitrification, nitrate reduction and ammonia assimilation were also predicted in the present study. CONCLUSION: The cave sediments of the biodiversity hotspot region possessing a oligotrophic environment harbours high phylogenetic diversity dominated by Actinobacteria and Proteobacteria. Among the geochemical factors, ferric oxide was correlated with increased microbial diversity. In-silico analysis detected genes involved in carbon, nitrogen, methane metabolism and complex metabolic pathways responsible for the survival of the bacterial community in nutrient limited cave environments. Present study with Paired end Illumina sequencing along with bioinformatics analysis revealed the essential ecological role of the cave bacterial communities. These results will be useful in documenting the biospeleology of this region and systematic understanding of bacterial communities in natural sediment environments as well.

Association of IL12B risk haplotype and lack of interaction with HLA-Cw6 among the psoriasis patients in India.

Psoriasis is a complex multifactorial chronic inflammatory skin disorder involving both genetic and environmental susceptibility factors. It is strongly associated with HLA-Cw6, but several studies suggested that further genetic factors may confer additional risk. We investigated the association of two single-nucleotide polymorphisms (SNPs), rs3212227 at the 3'-untranslated region and rs7709212 located at ~6.7 kb upstream from the transcription start site of IL12B gene in a case-control study comprising 1702 individuals from India. We found both SNPs were significantly associated with psoriasis (rs7709212: odds ratio (OR)=1.37, P-value=1.09 × 10-5; rs3212227: OR=1.38, P-value=8.88 × 10-6). IL12B gene was significantly upregulated in involved skin of psoriasis patients with risk genotype carriers (rs7709212_TT and rs3212227_TT) compared with non-risk genotype carriers (rs7709212_CC and rs3212227_GG). Significantly higher serum protein concentration of IL12 was also observed among risk allele carriers compared with non-risk allele carriers irrespective of the presence of HLA-Cw6 allele. Haplotype analysis suggested significant increased risk (OR=1.50, P-value=5.01 × 10-8) to the disease when both risk alleles of IL12B were present. IL12 serum protein concentration of risk haplotype (TT-TT) carriers showed significant upregulation compared with the non-risk carriers independent of HLA-Cw6 alleles. Our data suggested the association of IL12B with the psoriasis, however no evidence was observed for the epistatic effect of IL12B with HLA-Cw6 among the psoriasis patients in India.

Hepatic miR-126 is a potential plasma biomarker for detection of hepatitis B virus infected hepatocellular carcinoma.

Controversies about the origin of circulating miRNAs have encouraged us to identify organ specific circulating miRNAs as disease biomarkers. To identify liver-specific miRNAs for hepatocellular carcinoma (HCC), global expression profiling of miRNAs in liver tissue of HBV-HCC and HBV-control with no or mild fibrosis was evaluated. A total of 40 differentially expressed miRNAs were identified in HCC. Among ten highly altered miRNAs, six miRNAs were successfully validated in tissues, whereas only two miRNAs, miR-126 and miR-142-3p showed increased expression in plasma of HBV-HCC compared to HBV-non-HCC patients. Subsequently, ROC curve analysis revealed that neither miR-126 nor miR-142-3p performed better than AFP in discriminating HCC from non-HCC while combination of each with AFP showed significantly higher efficiency rather than AFP alone (AUC: 0.922, 0.908 vs. 0.88; sensitivity: 0.84, 0.86 vs. 0.82 and specificity: 0.92, 0.94 vs. 0.86 respectively). Interestingly, triple combination of markers (miR-126 + miR-142-3p + AFP) showed no additive effect on efficiency (AUC: 0.925) over the dual combination. Again, the expression of only miR-126 was noticed significantly higher in HBV-HCC patients with low-AFP [<250 ng/ml] compared to either non-HCC or liver cirrhosis (AUC: 0.77, 0.64, respectively). Furthermore, no alteration in expression of mir-126 in HCV-HCC or non-viral-HCC revealed that miR-126 + AFP might be specific to HBV-HCC. To understand the physiological role of these two miRNAs in hepato-carcinogenesis, target genes related to cancer pathways (APAF1, APC2, CDKN2A, IRS1, CRKL, LIFR, EGR2) were verified. Thus, combination of circulating miR-126 + AFP is a promising noninvasive diagnostic biomarker for HBV-HCC and may be useful in the management of HCC patients.

CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo.

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.

Predisposition to cancer caused by genetic and functional defects of mammalian Atad5.

ATAD5, the human ortholog of yeast Elg1, plays a role in PCNA deubiquitination. Since PCNA modification is important to regulate DNA damage bypass, ATAD5 may be important for suppression of genomic instability in mammals in vivo. To test this hypothesis, we generated heterozygous (Atad5(+/m)) mice that were haploinsuffficient for Atad5. Atad5(+/m) mice displayed high levels of genomic instability in vivo, and Atad5(+/m) mouse embryonic fibroblasts (MEFs) exhibited molecular defects in PCNA deubiquitination in response to DNA damage, as well as DNA damage hypersensitivity and high levels of genomic instability, apoptosis, and aneuploidy. Importantly, 90% of haploinsufficient Atad5(+/m) mice developed tumors, including sarcomas, carcinomas, and adenocarcinomas, between 11 and 20 months of age. High levels of genomic alterations were evident in tumors that arose in the Atad5(+/m) mice. Consistent with a role for Atad5 in suppressing tumorigenesis, we also identified somatic mutations of ATAD5 in 4.6% of sporadic human endometrial tumors, including two nonsense mutations that resulted in loss of proper ATAD5 function. Taken together, our findings indicate that loss-of-function mutations in mammalian Atad5 are sufficient to cause genomic instability and tumorigenesis.

CpG methylation recruits sequence specific transcription factors essential for tissue specific gene expression.

CG methylation is an epigenetically inherited chemical modification of DNA found in plants and animals. In mammals it is essential for accurate regulation of gene expression and normal development. Mammalian genomes are depleted for the CG dinucleotide, a result of the chemical deamination of methyl-cytosine in CG resulting in TpG. Most CG dinucleotides are methylated, but ~15% are unmethylated. Five percent of CGs cluster into ~20,000 regions termed CG islands (CGI) which are generally unmethylated. About half of CGIs are associated with housekeeping genes. In contrast, the gene body, repeats and transposable elements in which CGs are generally methylated. Unraveling the epigenetic machinery operating in normal cells is important for understanding the epigenetic aberrations that are involved in human diseases including cancer. With the advent of high-throughput sequencing technologies, it is possible to identify the CG methylation status of all 30million unique CGs in the human genome, and monitor differences in distinct cell types during differentiation and development. Here we summarize the present understanding of DNA methylation in normal cells and discuss recent observations that CG methylation can have an effect on tissue specific gene expression. We also discuss how aberrant CG methylation can lead to cancer. This article is part of a Special Issue entitled: Chromatin in time and space.

Contribution of nucleosome binding preferences and co-occurring DNA sequences to transcription factor binding.

BACKGROUND: Chromatin plays a critical role in regulating transcription factors (TFs) binding to their canonical transcription factor binding sites (TFBS). Recent studies in vertebrates show that many TFs preferentially bind to genomic regions that are well bound by nucleosomes in vitro. Co-occurring secondary motifs sometimes correlated with functional TFBS. RESULTS: We used a logistic regression to evaluate how well the propensity for nucleosome binding and co-occurrence of a secondary motif identify which canonical motifs are bound in vivo. We used ChIP-seq data for three transcription factors binding to their canonical motifs: c-Jun binding the AP-1 motif (TGA(C)/(G)TCA), GR (glucocorticoid receptor) binding the GR motif (G-ACA---(T)/(C)GT-C), and Hoxa2 (homeobox a2) binding the Pbx (Pre-B-cell leukemia homeobox) motif (TGATTGAT). For all canonical TFBS in the mouse genome, we calculated intrinsic nucleosome occupancy scores (INOS) for its surrounding 150-bps DNA and examined the relationship with in vivo TF binding. In mouse mammary 3134 cells, c-Jun and GR proteins preferentially bound regions calculated to be well-bound by nucleosomes in vitro with the canonical AP-1 and GR motifs themselves contributing to the high INOS. Functional GR motifs are enriched for AP-1 motifs if they are within a nucleosome-sized 150-bps region. GR and Hoxa2 also bind motifs with low INOS, perhaps indicating a different mechanism of action. CONCLUSION: Our analysis quantified the contribution of INOS and co-occurring sequence to the identification of functional canonical motifs in the genome. This analysis revealed an inherent competition between some TFs and nucleosomes for binding canonical TFBS. GR and c-Jun cooperate if they are within 150-bps. Binding of Hoxa2 and a fraction of GR to motifs with low INOS values suggesting they are not in competition with nucleosomes and may function using different mechanisms.

CpG methylation of half-CRE sequences creates C/EBPalpha binding sites that activate some tissue-specific genes.

DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBPα transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBPα activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBPα and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBPα binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBPα. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBPα mediated activation. In primary dermal fibroblasts, C/EBPα activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBPα that are needed to activate tissue-specific genes.