Browsing by Author "Manisha Sachan"

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Development of a multiplex MethyLight assay for he detection of DAPK1 and SOX1 methylation in epithelial ovarian cancer in a north Indian population
(2016) Manpreet Kaur; Alka Singh; Manisha Sachan; Kiran Singh; Sameer Gupta
Ovarian cancer is the fourth most common cancer in women worldwide. It is very heterogeneous at the clinical, histopathological and molecular levels and is caused by the accumulation of genetic and epigenetic changes in regulatory genes. More than 90% of ovarian cancers are epithelial in origin. Ovarian cancer is typically asymptomatic in its early stages, and due to difficulties in early detection, most ovarian cancers are diagnosed at an advanced stage. The positive predictive value of CA-125, a routinely used serum protein marker is < 30%; therefore, for effective screening, there is a need to develop a marker with high sensitivity for early detection. Development of blood-based biomarkers that detect DNA methylation in cellfree tumor-specific DNA is now being considered as a potential approach for the early diagnosis of cancer. Our objective in this study was to develop an absolute quantitative method, the MethyLight assay, to detect the promoter methylation status of two tumor suppressor genes. We analyzed the methylation level of the promoter regions of these genes in 42 tumor samples using the MethyLight assay. SOX1 promoter methylation was significantly higher in cancer samples than in normal samples (P = 0.011), whereas this difference between cancer and normal samples was not significant for DAPK1 promoter methylation (P = 0.18), when analyzed separately in a singleplex assay, whereas the detection frequency and significance level increased several-fold when these genes were analyzed together in a multiplex assay (P = 0.0004).The sensitivity was found to be 62% and 83% for DAPK1 and SOX1, respectively, when analyzed separately in the singleplex assay, but increased to 90% in the multiplex assay when either the SOX1 or the DAPK1 gene promoters showed methylation. © 2016, The Genetics Society of Japan. All rights reserved.
Developmental methylation of the coding region of c-fos occurs perinatally, stepwise and sequentially in the liver of laboratory mouse
(2008) Manisha Sachan; Rajiva Raman
We have studied the dynamics of de novo DNA methylation of 16 contiguous CpGs in the non-CpG island-coding region of the proto-oncogene c-fos during mouse development by Na-bisulfite sequencing. Methylation commences from 16.5 dpc and occurs in stepwise-manner. In liver 7 sites are methylated between 16.5 dpc and day 5 after birth, but all the sites are completely methylated on 20 dpp and remain so in the adult liver. The present study provides evidence that (1) pattern of methylation of c-fos is distinct from those DNA sequences which methylate pre- and post-implantation, both in terms of the timing and spreading, and (2) spacing of CpGs is an important factor in determining the course of methylation. We suggest that there could be other isoforms of Dnmtases for the c-fos like embryonic genes, not only because they methylate later in development but also because of the difference in kinetics of the reaction, and that the nucleation of certain methylated sites facilitate methylation of neighbouring sites and their maintenance in subsequent cell generations. © 2008 Elsevier B.V. All rights reserved.
Developmental methylation of the regulatory region of HoxB5 gene in mouse correlates with its tissue-specific expression
(2006) Manisha Sachan; Rajiva Raman
We have studied the dynamics of de novo CpG methylation in the regulatory region of one of the homeobox gene HoxB5 during mouse development by sodium bisulfite sequencing. Methylation pattern was examined at embryonic day 18.5 and adult in kidney and spleen while in the liver the same exercise has been done in 11.5 dpc, 18.5 dpc, 5 dpp and in adult. In the liver at 11.5 dpc, all the 47 contiguous sites (including a CpG island from 2035 to 2330 bp) at 5' regulatory region of HoxB5 were unmethylated. Random methylation commences from 18.5 dpc and continues in 5 dpp and in the adult. In the kidney at 18.5 dpc, 26 CpGs were examined (excluding the CpG island region) and all of them were unmethylated but the fetal spleen had at least a few sites considerably methylated. In the adult there was a low level methylation in the kidney, on the other hand, in the spleen, all the CpGs were methylated except a few sites and certain sites were totally methylated. Thus in the adult, the level of methylation was much higher than in the fetal stage. On the other hand semi-quantitative RT-PCR revealed that the extent of expression of HoxB5 was higher in embryonic stages than in the adult. Thus HoxB5 is a good paradigm to support that the developmental methylation of HoxB5 and its expression pattern show an inverse correlation. © 2006 Elsevier B.V. All rights reserved.
Heterogeneous pattern of DNA methylation in developmentally important genes correlates with its chromatin conformation
(BioMed Central Ltd., 2017) Puja Sinha; Kiran Singh; Manisha Sachan
Background: DNA methylation is a major epigenetic modification, playing a crucial role in the development and differentiation of higher organisms. DNA methylation is also known to regulate transcription by gene repression. Various developmental genes such as c-mos, HoxB5, Sox11, and Sry show tissue-specific gene expression that was shown to be regulated by promoter DNA methylation. The aim of the present study is to investigate the establishment of chromatin marks (active or repressive) in relation to heterogeneous methylation in the promoter regions of these developmentally important genes. Results: Chromatin-immunoprecipitation (ChIP) assays were performed to immuno-precipitate chromatin by antibodies against both active (H3K4me3) and repressive (H3K9me3) chromatin regions. The analysis of ChIP results showed that both the percentage input and fold enrichment of activated chromatin was higher in tissues expressing the respective genes as compared to the tissues not expressing the same set of genes. This was true for all the genes selected for the study (c-mos, HoxB5, Sox11, and Sry). These findings illustrate that inconsistent DNA methylation patterns (sporadic, mosaic and heterogeneous) may also influence gene regulation, thereby resulting in the modulation of chromatin conformation. Conclusions: These findings illustrate that various patterns of DNA methylation (asynchronous, mosaic and heterogeneous) correlates with chromatin modification, resulting in the gene regulation. © 2017 The Author(s).
High resolution methylation analysis of the HoxA5 regulatory region in different somatic tissues of laboratory mouse during development
(Elsevier B.V., 2017) Puja Sinha; Kiran Singh; Manisha Sachan
Homeobox genes encode a group of DNA binding regulatory proteins whose key function occurs in the spatial-temporal organization of genome during embryonic development and differentiation. The role of these Hox genes during ontogenesis makes it an important model for research. HoxA5 is a member of Hox gene family playing a central role during axial body patterning and morphogenesis. DNA modification studies have shown that the function of Hox genes is partly governed by the methylation-mediated gene expression regulation. Therefore the study aimed to investigate the role of epigenetic events in regulation of tissue-specific expression pattern of HoxA5 gene during mammalian development. The methodology adopted were sodium bisulfite genomic DNA sequencing, quantitative real-time PCR and chromatin-immunoprecipitation (ChIP). Methylation profiling of HoxA5 gene promoter shows higher methylation in adult as compared to fetus in various somatic tissues of mouse being highest in adult spleen. However q-PCR results show higher expression during fetal stages being highest in fetal intestine followed by brain, liver and spleen. These results clearly indicate a strict correlation between DNA methylation and tissue-specific gene expression. The findings of chromatin-immunoprecipitation (ChIP) have also reinforced that epigenetic event like DNA methylation plays important role in the regulation of tissue specific expression of HoxA5. © 2017
Silicon tackles butachlor toxicity in rice seedlings by regulating anatomical characteristics, ascorbate-glutathione cycle, proline metabolism and levels of nutrients
(Nature Research, 2020) Durgesh Kumar Tripthi; Rishi Kumar Varma; Swati Singh; Manisha Sachan; Gea Guerriero; Bishwajit Kumar Kushwaha; Shruti Bhardwaj; Naleeni Ramawat; Shivesh Sharma; Vijay Pratap Singh; Sheo Mohan Prasad; Devendra Kumar Chauhan; Nawal Kishore Dubey; Shivendra Sahi
Reckless use of herbicides like butachlor (Buta) in the fields represents a serious threat to crop plants, and hence to their productivity. Silicon (Si) is well known for its implication in the alleviation of the effects of abiotic stresses; however, its role in mitigating Buta toxicity is not yet known. Therefore, this study was carried out to explore the role of Si (10 µM) in regulating Buta (4 µM) toxicity in rice seedlings. Buta reduced growth and photosynthesis, altered nitric oxide (NO) level and leaf and root anatomy, inhibited enzyme activities of the ascorbate-glutathione cycle (while transcripts of associated enzymes, increased except OsMDHAR), as well as its metabolites (ascorbate and glutathione) and uptake of nutrients (Mg, P, K, S, Ca, Fe, etc. except Na), while addition of Si reversed Buta-induced alterations. Buta stimulated the expression of Si channel and efflux transporter genes- Lsi1 and Lsi2 while the addition of Si further greatly induced their expression under Buta toxicity. Buta increased free proline accumulation by inducing the activity of Δ1-pyrroline-5-carboxylate synthetase (P5CS) and decreasing proline dehydrogenase (PDH) activity, while Si reversed these effects caused by Buta. Our results suggest that Si-governed mitigation of Buta toxicity is linked with favorable modifications in energy flux parameters of photosynthesis and leaf and root anatomy, up-regulation of Si channel and transporter genes, ascorbate-glutathione cycle and nutrient uptake, and lowering in oxidative stress. We additionally demonstrate that NO might have a crucial role in these responses. © 2020, The Author(s).