https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/184 2026-04-27T15:42:57Z 2026-04-27T15:42:57Z Madhulika, Srivastava Uddin, Faizan https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1274 2025-12-05T12:45:04Z 2021-01-01T00:00:00Z

Title: Characterization of transcripts emanating from enhancer Eb of the murine TCRb locus Authors: Madhulika, Srivastava; Uddin, Faizan Abstract: Enhancers are well established as critical regulators of gene expression, but the mechanisms underlying the molecular basis of their specificity and activity are only partly understood. One of the most exciting recent observations is the discovery of enhancer RNA (eRNA), a class of noncoding RNAs derived from enhancer regions. Transcription of developmentally regulated enhancers has been observed to be associated with their active state. The nature of transcripts (eRNA) and their functional attributes are diverse and context dependent. The majority of eRNA are nonpolyadenylated and present in low abundance owing to their low stability, and may represent transcriptional noise. However, some eRNAs have been reported to be reasonably long and stable, are enriched in nuclei, exhibit tissue-specific expression and may contribute to enhancer function. Transcription of enhancers has been postulated to mediate enhancer function through either the act of transcription or via the transcribed RNA per se and is a useful feature to be analysed to understand mechanisms underlying enhancer activity. Enhancer Eβ at the murine TCRβ locus has been reported to exhibit enhanced occupancy of RNA polymerase II in developing thymocytes. Here, we investigated the transcriptional potential of Eβ in developing thymocytes and detected overlapping bidirectional transcripts at Eβ ranging between 0.7 and 1.7 kb. These noncoding transcripts are capped, polyadenylated, nuclear and expressed specifically in thymocytes. Delineation of these characteristics is important to further investigate their functional roles in mediating enhancer activity.

2021-01-01T00:00:00Z Srivastava, Madhulika Uddin, Faizan https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/1091 2025-12-05T12:43:22Z 2020-01-01T00:00:00Z

Title: Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA Authors: Srivastava, Madhulika; Uddin, Faizan Abstract: Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

2020-01-01T00:00:00Z Srivastava, Madhulika https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/479 2025-12-05T12:42:31Z 2012-01-01T00:00:00Z

Title: An ectopic CTCF-dependent transcriptional insulator influences the choice of Vβ gene segments for VDJ recombination at TCRβ locus Authors: Srivastava, Madhulika Abstract: Insulators regulate transcription as they modulate the interactions between enhancers and promoters by organizing the chromatin into distinct domains. To gain better understanding of the nature of chromatin domains defined by insulators, we analyzed the ability of an insulator to interfere in VDJ recombination, a process that is critically dependent on long-range interactions between diverse types of cis-acting DNA elements. A well-established CTCF-dependent transcriptional insulator, H19 imprint control region (H19-ICR), was inserted in the mouse TCRβ locus by genetic manipulation. Analysis of the mutant mice demonstrated that the insulator retains its CTCF and position-dependent enhancer-blocking potential in this heterologous context in vivo. Remarkably, the inserted H19-ICR appears to have the ability to modulate cis-DNA interactions between recombination signal sequence elements of the TCRβ locus leading to a dramatically altered usage of Vβ segments for Vβ-to-DβJβ recombination in the mutant mice. This reveals a novel ability of CTCF to govern long range cis-DNA interactions other than enhancer-promoter interactions and suggests that CTCF-dependent insulators may play a diverse and complex role in genome organization beyond transcriptional control. Our functional analysis of mutated TCRβ locus supports the emerging role of CTCF in governing VDJ recombination.

2012-01-01T00:00:00Z Srivastava, Madhulika https://dspace.nii.res.in//https://dspace.nii.res.in/handle/123456789/478 2025-12-05T12:41:24Z 2008-01-01T00:00:00Z

Title: Enhancer blocking activity of the insulator at H19-ICR is independent of chromatin barrier establishment Authors: Srivastava, Madhulika Abstract: Transcriptional insulators are cis regulatory elements that organize chromatin into independently regulated domains. At the imprinted murine Igf2/H19 locus, the H19-ICR insulator prevents the activation of the Igf2 promoter on the maternal allele by enhancers that activate H19 on the same chromosome. Given the well-demonstrated role of H19-ICR as an enhancer blocker, we investigated its ability to define a chromatin barrier, as the two activities are coincident on several insulators and may act in concert to define a functional chromatin boundary between adjacent genes with distinct transcriptional profiles. Allele-specific association of posttranslationally modified histones, reflecting the presence of active or inactive chromatin, was analyzed in the region encompassing H19-ICR using chromatin immunoprecipitation. The existence of differential histone modifications upstream and downstream of H19-ICR specifically on the maternal chromosome was observed, which is suggestive of a chromatin barrier formation. However, H19-ICR deletion analysis indicated that distinct chromatin states exist despite the absence of an intervening "barrier." Also, the enhancers can activate the Igf2 promoter despite some parts of the intervening chromatin being in the silent state. Hence, H19-ICR insulator activity is not dependent on preventing the enhancer-mediated alteration of the histone modifications in the region between the Igf2 promoter and the cognate enhancers.

2008-01-01T00:00:00Z