Tissue-specific characterisation of DNA methylation in the gonad-specific proto-oncogene, c-mos, in the male laboratory mouse

The proto-oncogene, c-mos, which is expressed only in the germ cells of both testis and ovary, plays an important role in meiotic maturation of these cells. In this research, the methylation status of several CpG sites, present both upstream and within the coding region of the c-mos gene, has been studied. The HpaII and HhaI sites examined in the 5' half of the coding region were unmethylated in both the c-mos expressing and non-expressing tissues. A HhaI site, h3, present 380bp downstream of the transcription start site, was unmethylated in germ cells, but was partially methylated in the somatic tissues, inversely correlating with the expression status of the gene. In contrast to these tissues, in the mouse fibroblast cell line L929, all the analysed sites were completely methylated

Characterisation of developmentally regulated chromatin structure in the coding region of the proto-oncogene, c-fos,in the male laboratory mouse

In mouse, tissue-specific developmental de novo methylation of the proto-oncogene c-fos,which is abundantly expressed during embryonic stages, occurs perinatally (between the day of birth to 20 dpp) and is maintained in the adult. In liver, where c-fos is only active up to the day of birth, the gene has more sites methylated than in brain, where it is expressed until about day 5 post-partum. We have studied chromatin organisation of c-fos and compared this to DNA methylation in the fetal and adult brain and liver. Purified nuclei of these tissues from fetus as well as adult were digested with the restriction enzyme MspI. DNA was extracted from the MspI digested chromatin and probed with two DNA segments covering the major part of the body of the gene (from distal part of second exon to major part of fourth exon). Southern hybridisation studies revealed that in the fetus, in both liver and brain, the chromatin in the coding region was sensitive to MspI digestion and the extent of sensitivity was nearly the same between the two. In the adult tissues, however, chromatin from brain was almost as sensitive as in the fetus, but in the liver it was highly resistant to MspI. We suggest that a shift from the undermethylated state in the fetus to the heavy methylated state in the adult causes a corresponding change in the organisation of chromatin of c-fos in the coding region. Furthermore, the difference in the tissuespecificity in the methylation induced chromatin compaction could be due to differences in the transcription levels of c-fos and de novo methylation during early neonatal development

A clinical study to evaluate the effect of Lekhana Vasti in the management of Sthaulya

Sthaulya is one of the Santarpanotta Vyadhi. It is considered as one among the Ashta Ninditeeya Purusha. It is a Rasa Nimittija and Medo Pradoshaja Vyadhi Avyayama, Athiasana, Athishayana and so on are some of the Nidanas of Sthaulya. Most of the world’s population live in countries where overweight and obesity kills more people than underweight. Apatarpana Chikitsa is main line of treatment in Sthaulya. When there is involvement of Kapha Medas and pathogenesis of Avarana, the condition needs Teekshna Basti with Lekhana properties. The Varanadi Gana Kwatha which is having similar action is selected as Kalka and Kwatha Dravya in the present study. To increase the Teekshnata, the Amla Kanji as Avapa Dravya has been selected. Objectives - Hence, the following study was taken to evaluate the effect of Lekhana Basti in management of Sthaulya. Methodology - 20 patients were taken in the study and were administered. Varanadi Lekhana Basti in Kala Basti format. Matra Basti with Murchita Tila Taila was given. Patients were assessed for weight BMI, biomarkers, bodily circumferences before and after treatment and after follow up

The IG-DMR and the MEG3-DMR at Human Chromosome 14q32.2: Hierarchical Interaction and Distinct Functional Properties as Imprinting Control Centers

Human chromosome 14q32.2 harbors the germline-derived primary DLK1-MEG3 intergenic differentially methylated region (IG-DMR) and the postfertilization-derived secondary MEG3-DMR, together with multiple imprinted genes. Although previous studies in cases with microdeletions and epimutations affecting both DMRs and paternal/maternal uniparental disomy 14-like phenotypes argue for a critical regulatory function of the two DMRs for the 14q32.2 imprinted region, the precise role of the individual DMR remains to be clarified. We studied an infant with upd(14)pat body and placental phenotypes and a heterozygous microdeletion involving the IG-DMR alone (patient 1) and a neonate with upd(14)pat body, but no placental phenotype and a heterozygous microdeletion involving the MEG3-DMR alone (patient 2). The results generated from the analysis of these two patients imply that the IG-DMR and the MEG3-DMR function as imprinting control centers in the placenta and the body, respectively, with a hierarchical interaction for the methylation pattern in the body governed by the IG-DMR. To our knowledge, this is the first study demonstrating an essential long-range imprinting regulatory function for the secondary DMR

The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer

BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions.Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates.The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation

Distinct Methylation Changes at the IGF2-H19 Locus in Congenital Growth Disorders and Cancer

Background: Differentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown. Methodology/Principal Findings: We analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC

Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function