Dr. Madhulika Srivastava
Nuclear processes depend on interactions between cis acting regulatory elements and trans-acting factors and are intricately regulated by higher order chromatin organization. Additionally, intranuclear positions of genes with respect to various components of the nucleus are non-random and altered during development concordant with their activation status. Misregulation of the dynamic nuclear and chromatin organization is associated with various developmental disorders, cancers and other diseases. Various aspects underlying this intriguing organization, relevant for regulation of transcription and VDJ recombination in lymphocytes, are being deciphered by chromosome conformation capture analyses and other biochemical assays as well as direct visualization of developmentally regulated loci within the nucleus in wild type and genetically manipulated mouse mutants.
Keywords : Genome architecture and dynamics, Chromatin organization, VDJ recombination, Epigenetics, Mammalian gene regulation
Role of enhancers in genome organization and function: Enhancers are the vanguards of tissue specific gene regulation but the mechanisms underlying their ability to activate specific cognate promoters are diverse and not completely understood. Our recent studies have demonstrated an unambiguous role of enhancer Eb in defining TCRb location relative to chromosome territory. The ability to reposition the target locus relative to chromosome territory highlights a novel aspect pertaining to activity of enhancers which may contribute to their ability to regulate gene expression and adds to the diversity of mechanisms underlying enhancer activity. Further, enhancers are well known as critical regulators of chromatin accessibility. Our observations support the idea that enhancers also influence intranuclear movements of loci relative to chromosome territory. This has implications for understanding the role of enhancers in three-dimensional genome organization and function and provides a useful start point to gain insights in understanding how intranuclear positions of genes change during development.
Role of CTCF in chromatin organization : CTCF, a DNA binding protein with multifunctional attributes, is an important contributor to chromatin organization. Global analysis by ChIP-Seq and ChIA-PET has demonstrated the presence of several thousand CTCF binding sites in the human and mouse genomes and emphasized their importance in defining chromatin looping patterns critical for specific regulation of genes. Understanding about the role and mechanisms of CTCF based higher order chromatin organization has emerged based on quest to understand transcriptional regulation. In addition, higher order chromatin organization is also critically important for VDJ recombination at antigen receptor loci (Igh, Igk, TCRa/d and TCRb) in developing B-cells and T-cells and each of these loci have several CTCF binding sites. Considering the diverse and multifunctional attributes of CTCF, current efforts in the lab focus on understanding different aspects underlying CTCF based regulation of transcription and VDJ recombination. Our chromosome conformation capture analysis indicated that CTCF plays an important role in VDJ recombination at TCRb locus. It mediates long-range chromatin looping interactions important for establishment of recombination center as well as facilitates the spatial proximity of TCRb variable gene segments to the recombination center. Our analysis also suggests that even mild changes in the CTCF-based chromatin organization, that may arise due to mutations or epigenetic aberrations, can have profound functional consequences for gene expression.
Monika Yadav, Sarojini Minj, Kevla Nand and Bhan Singh
- Yadav M, Jalan M, Srivastava M (2022) Enhancer dependent repositioning of TCRb locus with respect to the chromosome territory. Journal of Molecular Biology, volume 434(7):167509 doi: 10.1016/j.jmb.2022.167509
- Uddin F, Srivastava M (2021) Characterization of transcripts emanating from enhancer Eb of the murine TCRb locus. FEBS Open Bio. volume 11(4):1014-1028
- Uddin F and Srivastava M (2020) Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA Biotechniques 69 issue 2, 141-147
- Rawat P, Jalan M, Sadhu A, Kanaujia A, Srivastava M (2017) Chromatin Domain Organization of TCRb locus and its perturbation by ectopic CTCF binding Molecular and Cellular Biology, 37, Issue 9, e00557-16
- Varma G, Rawat P, Jalan M, Vinayak M, Srivastava M (2015) Influence of a CTCF dependent insulator on multiple aspects of enhancer mediated chromatin organization Molecular and Cellular Biology,35, 3504-3516
- Shrimali S, Srivastava S, Varma G, Grinberg A, Pfeifer K andSrivastava M(2012) An ectopic CTCF dependent transcriptional insulator influences the choice of Vb gene segments for VDJ recombination at TCRb locus Nucleic Acids Research 40: 7753-7765.
- Singh V and Srivastava M (2008) Enhancer blocking activity of the insulator at H19- ICR is independent of chromatin barrier establishment Molecular and Cellular Biology 28: 3767-3775.
- Srivastava M, Frolova E, Rottinghaus B, Boe S, Grinberg A, Lee E, Love PE, Pfeifer K(2003) Imprint control element mediated secondary methylation imprints at the Igf2/H19 locus Journal for Biological Chemistry 278: 5977-5983
- Kaffer CR, Srivastava M, ParkKye-Yoon, Ives E, Hsieh S, Batlle J, Grinberg A, Huang SP, and Pfeifer K (2000) A transcriptional insulator at the imprinted H19/Igf2 locus Genes and Development 14: 1908-1919.
- Srivastava M, Hsieh S, Grinberg A, William Simons L, Huang SP, Pfeifer K (2000) H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting region upstream of H19 Genes and Development 14: 1186-1195.