Biology subjects

Rishi, V.

Publications and source records attributed to Rishi, V..

6 recordsLinked to original sources

bioRxiv2025

Mapping the dimerization specificity of bZIP transcription factors in bread wheat

Dimerization plays a pivotal role in target-binding interactions among various transcription factors (TFs), including bZIPs, bHLHs, MYBs, and zinc finger TFs. The bZIP family is defined by an -helix class of TFs containing a basic region that binds to the major groove of double-stranded DNA (dsDNA), followed by a leucine zipper motif that mediates dimerization through coiled-coil structures, forming homo- or heterodimers. In polyploid bread wheat (Triticum aestivum cv.), bZIPs and their homeologs interact through the dimerization motifs to form specific bZIP pairs with distinct regulatory functions. The dimerization pattern of bZIPs is critical for gene regulation, however, the mechanisms underlying their specificity remain poorly understood. Here, we focus on the dimerization specificity of coiled-coil - helical bZIP proteins in bread wheat, highlighting the leucine zipper region as a key dimerization motif. We identified 265 bZIPs containing the bZIP motif, following dimerization criteria established in humans, Drosophila, and Arabidopsis based on the presence of specific amino acids at the a, d, e, and g positions in heptads. Eight key bZIP TFs were cloned, expressed, and purified for the structural analysis. This study not only provides the dimerization trend exhibited by bZIPs during thermal stability analysis but also provides the reversibility pattern followed by the thermal denaturation exposure at 85{degrees}C. Our study also points towards that how amino acid substitutions and the length of the leucine zipper influence the formation of coiled-coil structures among bZIP homeologs of polyploid wheat. Overall, the study advances our understanding of the role of leucine zippers, in determining the specificity and stability of bZIP dimers, which in turn governs DNA binding and gene regulation in plants.

biochemistry↗

bioRxiv2024

WGBS of Differentiating Adipocytes Reveals Variations in DMRs and Context-Dependent Gene Expression

Obesity, characterised by the accumulation of excess fat, is a complex condition resulting from the combination of genetic and epigenetic factors. Recent studies have found correspondence between DNA methylation and cell differentiation, suggesting a role of the former in cell fate determination. There is a lack of comprehensive understanding concerning the underpinnings of preadipocyte differentiation, specifically when cells are undergoing terminal differentiation (TD). To gain insight into dynamic genome-wide methylation, 3T3 L1 preadipocyte cells were differentiated by a hormone cocktail. The genomic DNA was isolated from undifferentiated cells and 4 hrs (4H), 2 days (2D) post-differentiated cells, and 15 days (15D) TD cells. We employed whole-genome bisulfite sequencing (WGBS) to ascertain global genomic DNA methylation alterations at single base resolution as preadipocyte cells differentiate. The genome-wide distribution of DNA methylation showed similar overall patterns in pre- and post- and terminally differentiated adipocytes, according to WGBS analysis. DNA methylation decreases at 4H after differentiation initiation, followed by methylation gain as cells approach TD. Studies revealed novel differentially methylated regions (DMRs) associated with adipogenesis. DMR analysis suggested that though DNA methylation is global, noticeable changes are observed at specific sites known as hotspots. Hotspots are genomic regions rich in transcription factor (TF) binding sites and exhibit methylation-dependent TF binding. Subsequent analysis indicated hotspots as part of DMRs. The gene expression profile of key adipogenic genes in differentiating adipocytes is context-dependent, as we found a direct and inverse relationship between promoter DNA methylation and gene expression.

genomics↗

bioRxiv2024

Identifying the genetic and epigenetic basis for asymmetric bZIP expression in temperature-stressed bread wheat

Asymmetric expression in the bread wheat (Triticum aestivum) genome refers to the differential expression of genes from A, B, and D parental genomes. Bread wheat is a hexaploid crop with six copies of each chromosome. This complexity can result in unequal expression of genes from each parental genome, leading to asymmetry in gene expression. In other polyploid crops like cotton, transcription factors (TF) exhibit genome-biased expression; however, there are no comparable studies for bread wheat. One of the most prominent TFs families in plants is the basic Leucine Zippers (bZIP) which are eukaryote-specific proteins and regulate various biological processes, including stress-related responses. bZIP proteins are dimeric and several heptads long. They exhibit typical coiled-coil structures with strategically placed amino acids in each heptad, responsible for their stability and specificity. Here, we aim to decipher the structural basis of the asymmetric expression of the bZIP TFs in wheat under low and high-temperature conditions. Furthermore, 19 highly expressed stress-related TabZIP TFs were analysed for their asymmetric expression profiles as plants were exposed to temperature-stress conditions. Two benchmarks were used to analyse the asymmetric gene expression of bZIPs, i.e., a) the promoters occupancy by the epigenetic marker histones, namely, H3K4me3, H3k9ac (active) and H3K27me3 (repressive), b) density and diversity of cis-regulatory elements in the promoters. Notably, the genetic basis of the differences in protein sequences of bZIP triads was explored, which may impart structural stability to a specific homeolog enabling the plant to endure the stress conditions better.

plant biology↗

bioRxiv2021

Synergistic regulation of bZIP53 and dimerizing partners results in abnormal seed phenotype in Arabidopsis: Use of a designed dominant negative protein A-ZIP53

In Arabidopsis, basic leucine zipper (bZIP) family of transcription factors (TFs) are key proteins to regulate the expression of seed maturation (MAT) genes. bZIPs are functionally redundant and their DNA-binding activity is dependent on dimerization partner. The intervention of loss of function mutation is inadequate to understand and regulate the redundant behavior of TFs and one such example is bZIP53, which is known as a key regulator of seed maturation phenomena. Here, to examine the consequences of hindering the function of bZIP53 and its known and unknown heterodimerizing partners, a transgenic Arabidopsis constitutively expressing a novel dominant negative (DN) protein A-ZIP53 was raised. Transgenic plants demonstrated a delayed growth and retarded seed phenotype. The in vivo inhibition of DNA binding of bZIP53, bZIP10, and bZIP25 to the G-box demonstrated the efficacy of A-ZIP53 protein. In first generation, majority of plants failed to survive beyond four weeks suggesting a pleiotropic nature of bZIP53. Plants expressing A-ZIP53 have small flower, shorter siliques, and small-seeded phenotype. RNA seq analysis of the transgenic lines revealed the reduced expression of target genes of bZIP53 and its heterodimerizing partners. Furthermore, immunoprecipitation followed by mass spectrometry (IP-MS/MS) of transgenic plants helped to identify the additional heterodimerizing partners of the A-ZIP53. The interactions were subsequently confirmed with the transient transfection experiments. Unlike other gene knock out technologies, DN protein can inhibit the function of members of the same group of bZIP TFs.

plant biology↗

bioRxiv2020

Transient delivery of A-C/EBP protein perturbs differentiation of 3T3-L1 cells and induces preadipocyte marker genes

Transformation of committed 3T3-L1 preadipocytes to lipid-laden adipocytes involves timely appearance of numerous transcription factors (TFs), foremost among them C/EBP{beta}, is expressed during early phases of differentiation. Here we describe liposome-mediated protein transfection approach to rapidly downregulate C/EBP{beta} by A-C/EBP protein inhibitor. Signals from tagged A-C/EBP were observed in 3T3-L1 cells within 2hrs of protein inhibitor transfections whereas for gene transfection signals appeared in 48hrs. Following transient transfections, expression profiles of 24 marker genes belonging to pro- and anti-adipogenic, cell cycle, and preadipocytes pathways was analyzed. mRNA and protein expression profiles of adipocyte-marker genes showed lower expression in both A-C/EBP protein and gene transfected samples. Interestingly, for preadipocytes and cell fate determinant genes, striking differences were observed between protein and gene transfected samples. Preadipocyte differentiation factors Stat5a and Creb were downregulated in A-C/EBP protein samples. Five preadipocyte markers, namely, Pdgfr, Pdgfr{beta}, Ly6A, CD34 Itgb1 showed high expression in protein samples whereas only Ly6A and CD34 were expressed in gene transfected samples. Pdgfr and Pdgfr{beta}, two known cell fate markers were expressed in protein transfected samples 5-days post-differentiation suggesting a possible reversal of differentiation. Our study provides evidences for the robust and efficient knockdown of C/EBP{beta} protein to understand time-dependent gene regulation during adipogenesis.

molecular biology↗

bioRxiv2019

Wheat inositol pyrophosphate kinase (TaVIH2-3B) interacts with Fasciclin-like arabinogalactan (FLA6) protein and alters the plant cell-wall composition

BackgroundInositol pyrophosphates (PP-InsPs) are high-energy cellular molecules involved in different signalling and regulatory responses. Two distinct classes of inositol phosphate kinases responsible for the synthesis of PP-InsPs have been identified in Arabidopsis i.e. ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were shown to control pathogenic defence and phosphate response signals. However, other roles offered by plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. ResultsHere, we characterized two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2, for their physiological functions. We demonstrated that wheat VIH proteins could utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Overexpression of TaVIH2-3B homolog in Arabidopsis conferred tolerance to drought stress and rescued the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B transgenic lines of Arabidopsis showed a genome-wide reprogramming with remarkable effects on cell-wall biosynthesis genes with enhanced the accumulation of polysaccharides (arabinogalactan, cellulose and arabinoxylan). ConclusionsOverall, this work identifies a novel function of VIH proteins, implying their roles in modulating cell-wall homeostasis genes and providing water-deficit stress tolerance. This work suggests that the plant VIH enzymes could be linked to drought tolerance and also opens up investigations to address the roles of plant VIH derived products in generating drought resistant plants.

plant biology↗