Convenient expression, purification and quantitative liquid chromatography-tandem mass spectrometry-based analysis of TET2 5-methylcytosine demethylase.

5-Methylcytosine within CpG islands in DNA plays a crucial role in epigenetic transcriptional regulation during metazoan development. Recently, it has been established that the Ten-Eleven Translocation (TET) family, Fe(II)- and 2-oxoglutarate (2OG/αKG)-dependent oxygenases initiate 5-methylcytosine demethylation by iterative oxidation reactions. Mutations in the TET2 gene are frequently detected in patients with myeloid malignancies. Here, we describe the cloning of untagged human TET2 demethylase using Gateway technology and its efficient expression in E. coli. The untagged TET2 enzyme was purified using cation exchange and heparin sepharose chromatography. In addition, a reliable quantitative liquid chromatography-tandem mass spectrometry-based assay was utilized to analyze the activity of TET2 oxygenase. This assay was further used to analyze the activity of a number of clinical TET2 variants with mutations in the 2OG binding sites. Our results demonstrate that the activity of one TET2 mutant, TET2-R1896S, can be restored using an excess of 2OG in the reaction mixture. These studies suggest that dietary 2OG supplements, which are commonly used for several other conditions, may be used to treat some patients with myeloid malignancies harboring TET2-R1896S mutation. Results described in this paper serve as a foundation for better characterization of wild type as well as mutant TET2 demethylases.

Prohexadione, a plant growth regulator, inhibits histone lysine demethylases and modulates epigenetics.

BACKGROUND: Epigenetic modifications, particularly DNA methylation and posttranslational histone modifications regulate heritable changes in transcription without changes in the DNA sequence. Despite a number of studies showing clear links between environmental factors and DNA methylation, little is known about the effect of environmental factors on the recently identified histone lysine methylation. Since their identification numerous studies have establish critical role played by these enzymes in mammalian development. OBJECTIVES: Identification of the Jumonji (Jmj) domain containing histone lysine demethylase have added a new dimension to epigenetic control of gene expression by dynamic regulation of histone methylation marks. The objective of our study was to evaluate the effect of prohexadione and trinexapac, widely used plant growth regulators of the acylcyclohexanediones class, on the enzymatic activity of histone lysine demethylases and histone modifications during the neural stem/progenitor cell differentiation. METHODS: Here we show that prohexadione, but not trinexapac, directly inhibits non-heme iron (II), 2-oxoglutarate-dependent histone lysine demethylase such as Jmjd2a. We used molecular modeling to show binding of prohexadione to Jmjd2a. We also performed in vitro demethylation assays to show the inhibitory effect of prohexadione on Jmjd2a. Further we tested this molecule in cell culture model of mouse hippocampal neural stem/progenitor cells to demonstrate its effect toward neuronal proliferation and differentiation. RESULTS: Molecular modeling studies suggest that prohexadione binds to the 2-oxoglutarate binding site of Jmjd2a demethylase. Treatment of primary neural stem/progenitor cells with prohexadione showed a concentration dependent reduction in their proliferation. Further, the prohexadione treated neurospheres were induced toward neurogenic lineage upon differentiation. CONCLUSIONS: Our results describe an important chemico-biological interaction of prohexadione, in light of critical roles played by histone lysine demethylases in human health and diseases.

Hypoxia mediated expression of stem cell markers in VHL-associated hemangioblastomas.

Hemangioblastomas of the retina, central nervous system, and kidney are observed in patients with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Mutations in the VHL lead to constitutive activation of hypoxia-inducible-factor (HIF) pathway. HIF-mediated expression of pro-angiogenic genes causes extensive pathological neovascularization in hemangioblastomas. A number of studies have shown coexistence of pro-angiogenic and stem cell markers in 'tumorlet-like stromal cells' in the retinal and optic nerve hemangioblastomas, leading to suggestions that hemangioblastomas originate from developmentally arrested stem cells or embryonic progenitors. Since recent studies have shown that the HIF pathway also plays a role in the maintenance/de-differentiation of normal and cancerous stem cells, we evaluated the role of the HIF pathway in the expression of stem cell markers in VHL-/- renal cell carcinoma cells under normoxia or VHL+/+ retinal pigment epithelial cells under hypoxia. Here we show that the expression of stem cell markers in hemangioblastomas is due to activation of the HIF pathway. Further, we show that honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources, blocks the expression of stem cell markers. Our results show the mechanism for the cytological origin of neoplastic stromal cells in hemangioblastomas, and suggest that inhibition of the HIF pathway is an attractive strategy for the treatment of hemangioblastomas.

Relationship of APOA5, PPARγ and HL gene variants with serial changes in childhood body mass index and coronary artery disease risk factors in young adulthood.

BACKGROUND: Triglycerides is an independent risk factor for coronary artery disease (CAD) and is especially important in Indians because of high prevalence of hypertriglyceridemia in this population. Both genetic and environmental factors determine triglyceride levels. In a birth cohort from India, hypertriglyceridemia was found in 41% of men and 11% of women. Subjects who had high triglycerides had more rapid body mass index (BMI) or weight gain than rest of the cohort throughout infancy, childhood and adolescence. We analysed polymorphisms in APOA5, hepatic lipase and PPARγ genes and investigated their association with birth weight and serial changes in BMI. RESULTS: Polymorphisms in APOA5 (-1131T > C, S19W), PPARγ (Pro12Ala) and hepatic lipase (-514C > T) were studied by polymerase chain reaction (PCR) followed by restriction digestion in 1492 subjects from the New Delhi Birth Cohort (NDBC). We assessed whether these polymorphisms influence lipid and other variables and serial changes in BMI, both individually and together.The risk allele of APOA5 (-1131C) resulted in 23.6 mg/dl higher triglycerides as compared to normal allele (P < 0.001). Risk allele of HL (-514T) was associated with significantly higher HDL2 levels (P = 0.002). Except for the marginal association of PPARγ Pro12Ala variation with a lower conditional weight at 6 months, (P = 0.020) and APOA5 S19W with a higher conditional BMI at 11 yrs of age (P = 0.030), none of the other associations between the gene polymorphisms and serial changes in body mass index from birth to young adulthood were significant. CONCLUSION: The promoter polymorphism in APOA5 was associated with raised serum triglycerides and that of HL with raised HDL2 levels. None of the polymorphisms had any significant relationship with birth weight or serial changes in anthropometry from birth to adulthood in this cohort.

Impact of common variants of PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, CDKN2A, IGF2BP2, and CDKAL1 on the risk of type 2 diabetes in 5,164 Indians.

OBJECTIVE: Common variants in PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, CDKN2A, IGF2BP2, and CDKAL1 genes have been shown to be associated with type 2 diabetes in European populations by genome-wide association studies. We have studied the association of common variants in these eight genes with type 2 diabetes and related traits in Indians by combining the data from two independent case-control studies. RESEARCH DESIGN AND METHODS: We genotyped eight single nucleotide polymorphisms (PPARG-rs1801282, KCNJ11-rs5219, TCF7L2-rs7903146, SLC30A8-rs13266634, HHEX-rs1111875, CDKN2A-rs10811661, IGF2BP2-rs4402960, and CDKAL1-rs10946398) in 5,164 unrelated Indians of Indo-European ethnicity, including 2,486 type 2 diabetic patients and 2,678 ethnically matched control subjects. RESULTS: We confirmed the association of all eight loci with type 2 diabetes with odds ratio (OR) ranging from 1.18 to 1.89 (P = 1.6 x 10(-3) to 4.6 x 10(-34)). The strongest association with the highest effect size was observed for TCF7L2 (OR 1.89 [95% CI 1.71-2.09], P = 4.6 x 10(-34)). We also found significant association of PPARG and TCF7L2 with homeostasis model assessment of beta-cell function (P = 6.9 x 10(-8) and 3 x 10(-4), respectively), which looked consistent with recessive and under-dominant models, respectively. CONCLUSIONS: Our study replicates the association of well-established common variants with type 2 diabetes in Indians and shows larger effect size for most of them than those reported in Europeans.

TCF7L2 gene polymorphisms do not predict susceptibility to diabetes in tropical calcific pancreatitis but may interact with SPINK1 and CTSB mutations in predicting diabetes.

BACKGROUND: Tropical calcific pancreatitis (TCP) is a type of chronic pancreatitis unique to developing countries in tropical regions and one of its important features is invariable progression to diabetes, a condition called fibro-calculous pancreatic diabetes (FCPD), but the nature of diabetes in TCP is controversial. We analysed the recently reported type 2 diabetes (T2D) associated polymorphisms in the TCF7L2 gene using a case-control approach, under the hypothesis that TCF7L2 variants should show similar association if diabetes in FCPD is similar to T2D. We also investigated the interaction between the TCF7L2 variants and N34S SPINK1 and L26V CTSB mutations, since they are strong predictors of risk for TCP. METHODS: Two polymorphisms rs7903146 and rs12255372 in the TCF7L2 gene were analyzed by direct sequencing in 478 well-characterized TCP patients and 661 healthy controls of Dravidian and Indo-European ethnicities. Their association with TCP with diabetes (FCPD) and without diabetes was tested in both populations independently using chi-square test. Finally, a meta analysis was performed on all the cases and controls for assessing the overall significance irrespective of ethnicity. We dichotomized the whole cohort based on the presence or absence of N34S SPINK1 and L26V CTSB mutations and further subdivided them into TCP and FCPD patients and compared the distribution of TCF7L2 variants between them. RESULTS: The allelic and genotypic frequencies for both TCF7L2 polymorphisms, did not differ significantly between TCP patients and controls belonging to either of the ethnic groups or taken together. No statistically significant association of the SNPs was observed with TCP or FCPD or between carriers and non-carriers of N34S SPINK1 and L26V CTSB mutations. The minor allele frequency for rs7903146 was different between TCP and FCPD patients carrying the N34S SPINK1 variant but did not reach statistical significance (OR = 1.59, 95% CI = 0.93-2.70, P = 0.09), while, TCF7L2variant showed a statistically significant association between TCP and FCPD patients carrying the 26V allele (OR = 1.69, 95% CI = 1.11-2.56, P = 0.013). CONCLUSION: Type 2 diabetes associated TCF7L2 variants are not associated with diabetes in TCP. Since, TCF7L2 is a major susceptibility gene for T2D, it may be hypothesized that the diabetes in TCP patients may not be similar to T2D. Our data also suggests that co-existence of TCF7L2 variants and the SPINK1 and CTSB mutations, that predict susceptibility to exocrine damage, may interact to determine the onset of diabetes in TCP patients.

Phylogeography of mtDNA haplogroup R7 in the Indian peninsula.

BACKGROUND: Human genetic diversity observed in Indian subcontinent is second only to that of Africa. This implies an early settlement and demographic growth soon after the first 'Out-of-Africa' dispersal of anatomically modern humans in Late Pleistocene. In contrast to this perspective, linguistic diversity in India has been thought to derive from more recent population movements and episodes of contact. With the exception of Dravidian, which origin and relatedness to other language phyla is obscure, all the language families in India can be linked to language families spoken in different regions of Eurasia. Mitochondrial DNA and Y chromosome evidence has supported largely local evolution of the genetic lineages of the majority of Dravidian and Indo-European speaking populations, but there is no consensus yet on the question of whether the Munda (Austro-Asiatic) speaking populations originated in India or derive from a relatively recent migration from further East. RESULTS: Here, we report the analysis of 35 novel complete mtDNA sequences from India which refine the structure of Indian-specific varieties of haplogroup R. Detailed analysis of haplogroup R7, coupled with a survey of approximately 12,000 mtDNAs from caste and tribal groups over the entire Indian subcontinent, reveals that one of its more recently derived branches (R7a1), is particularly frequent among Munda-speaking tribal groups. This branch is nested within diverse R7 lineages found among Dravidian and Indo-European speakers of India. We have inferred from this that a subset of Munda-speaking groups have acquired R7 relatively recently. Furthermore, we find that the distribution of R7a1 within the Munda-speakers is largely restricted to one of the sub-branches (Kherwari) of northern Munda languages. This evidence does not support the hypothesis that the Austro-Asiatic speakers are the primary source of the R7 variation. Statistical analyses suggest a significant correlation between genetic variation and geography, rather than between genes and languages. CONCLUSION: Our high-resolution phylogeographic study, involving diverse linguistic groups in India, suggests that the high frequency of mtDNA haplogroup R7 among Munda speaking populations of India can be explained best by gene flow from linguistically different populations of Indian subcontinent. The conclusion is based on the observation that among Indo-Europeans, and particularly in Dravidians, the haplogroup is, despite its lower frequency, phylogenetically more divergent, while among the Munda speakers only one sub-clade of R7, i.e. R7a1, can be observed. It is noteworthy that though R7 is autochthonous to India, and arises from the root of hg R, its distribution and phylogeography in India is not uniform. This suggests the more ancient establishment of an autochthonous matrilineal genetic structure, and that isolation in the Pleistocene, lineage loss through drift, and endogamy of prehistoric and historic groups have greatly inhibited genetic homogenization and geographical uniformity.