Biology subjects

Basu, U.

Publications and source records attributed to Basu, U..

7 recordsLinked to original sources

bioRxiv2026

Circadian Clock Programming of Anticipatory Antiviral Immunity Gates Enteric Virus Infection Susceptibility

Susceptibility to viral infection varies widely but is not fully explained by genetics, immune status, or exposure level. We show that time of day strongly influences infection outcome, with up to 100-fold differences in enteric viral burden depending on infection timing. This temporal gating is abolished in mice lacking a functional circadian clock. We identify the antiviral transcription factor IRF1 as a direct target of the circadian transcription factor BMAL1, resulting in rhythmic expression of a basal antiviral gene program prior to infection. Loss of IRF1 eliminates this program and abrogates time-of-day-dependent differences in viral replication. This circuit operates within intestinal myeloid cells, establishing a preexisting antiviral state. These findings indicate that the circadian clock programs host susceptibility in the intestine, before infection occurs.

immunology↗

bioRxiv2022

Yeast mtDNA transcription initiation in single nucleotide addition steps

Transcription initiation catalyzed by the RNA polymerase is a multistep process involving promoter binding, transcription bubble formation, abortive RNA synthesis, and transition into elongation following promoter escape. We report cryo-EM structures of yeast mitochondrial RNA polymerase initiation complexes (ICs) with transcription factor MTF1 catalyzing RNA synthesis from de novo initiation to 6-mer synthesis at single-nucleotide steps on fully-resolved transcription bubbles. The growing RNA:DNA hybrid is accommodated by continuous scrunching of the template strand while the non-template and MTF1 C-tail in the polymerase cleft are structurally reorganized. Each nucleotide addition accumulates stress energy, which drives abortive RNA synthesis during early transcription initiation steps and promoter release later. The non-template scrunches as loops in IC2/IC3, and unscrunching assists abortive synthesis of 2-/3-mer RNAs. Subsequently, in IC5 and IC6, the non-template strand assumes a stable structure by stacking its bases into a spiral staircase-like structure that supports processive synthesis. In IC6, the template scrunches to the maximum and places the -1 nucleotide in a pocket near the thumb domain. Subsequently, the -1 nucleotide acts as a pivot point for promoter escape ushering the IC into the elongation phase. The structural snapshots visualize the interplay between abortive and productive synthesis regulating transcription initiation.

biochemistry↗

bioRxiv2020

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

biochemistry↗

bioRxiv2020

Auto-inhibitory regulation of DNA binding by the C-terminal tails of the mitochondrial transcription factors Mtf1 and TFB2M

The structurally homologous Mtf1 and TFB2M proteins serve as transcription initiation factors of the Saccharomyces cerevisiae and human mitochondrial RNA polymerases, respectively. These transcription factors directly interact with the non-template strand of the transcription bubble to drive promoter melting. Given the key roles of Mtf1 and TFB2M in promoter-specific transcription initiation, it is expected that the DNA binding activity of the mitochondrial transcription factors would be regulated to prevent DNA binding at inappropriate times. However, there is little information on how mitochondrial DNA transcription is regulated. While studying the C-tail deletion mutants of Mtf1 and TFB2M, we stumbled upon a new finding that suggested that the flexible C-tail region of these factors autoregulates their DNA binding activity. Quantitative DNA binding studies with fluorescence anisotropy-based titrations show that Mtf1 with an intact C-tail has no affinity for the DNA but the deletion of C-tail greatly increases the DNA binding affinity. Similar observations were made with TFB2M, although autoinhibition by the C-tail of TFB2M was not as absolute as in Mtf1. Analysis of available TFB2M structures show that the C-tail makes intramolecular interactions with the DNA binding groove in the free factor, which we propose masks the DNA binding activity. Further studies show that the RNA polymerase relieves autoinhibition by interacting with the C-tail and engaging it in complex formation. Thus, our biochemical and structural analysis identify previously unknown autoinhibitory and activation mechanisms of mitochondrial transcription factors that regulate the DNA binding activity and aid in specific assembly of the initiation complexes.

biochemistry↗

bioRxiv2019

The Dynamic Landscape of Transcription Initiation in Yeast Mitochondria

Controlling efficiency and fidelity in the early stage of mitochondrial DNA transcription is crucial for regulating cellular energy metabolism. Studies of bacteriophage and bacterial systems have revealed that transcription occurs through a series of conformational transitions during the initiation and elongation stages; however, how the conformational dynamics progress throughout these stages remains unknown. Here, we used single-molecule fluorescence resonance energy transfer techniques to examine the conformational dynamics of the two-component transcription system of yeast mitochondria with single-base resolution. We show that, unlike its single-component homologue in bacteriophages, the yeast mitochondrial transcription initiation complex dynamically transitions between closed, open, and scrunched conformations throughout the initiation stage, and then makes a sharp irreversible transition to an unbent conformation by promoter release at position +8. Remarkably, stalling the initiation complex revealed unscrunching dynamics without dissociating the RNA transcript, manifesting the existence of backtracking transitions with possible regulatory roles. The dynamic landscape of transcription initiation revealed here suggests a kinetically driven regulation of mitochondrial transcription.

biophysics↗

bioRxiv2019

CD8+ T-cell-mediated immunoediting influences genomic evolution and immune evasion in murine gliomas

Cancer immunoediting shapes tumor progression by the selection of tumor cell variants that can evade immune recognition. Given the immune evasion and intra-tumor heterogeneity intrinsic to gliomas, we hypothesized that CD8+ T-cells mediate immunoediting in these tumors. We evaluated glioma progression in the absence of CD8+ T-cells by depleting this immune cell population in transgenic murine gliomas. Upon transplantation, gliomas that developed in the absence of CD8+ T-cells engrafted poorly in recipients with intact immunity but engrafted well in those with CD8+ T-cell depletion. Gliomas developed in absence of CD8+ T-cells exhibited increased chromosomal instability, MAPK signaling, gene fusions, and macrophage/microglial infiltration. MAPK activation correlated with macrophage/microglial recruitment in this model and in the human disease. Our results indicate that CD8+ T-cells mediate immunoediting during gliomagenesis, influencing the genomic stability of glioma and its microenvironment, leading to immune evasion.\n\nSignificanceImmune evasion renders cancer resistant to anti-tumoral immunity. Therapeutic intervention often fails for gliomas because of the plasticity of tumor cell variants that resist immune surveillance. Our results demonstrate a mechanism of immune evasion in gliomas that derives from CD8+ T-cells during the development and progression of this disease.

cancer biology↗

bioRxiv2018

Mitochondrial RNA capping: highly efficient 5’-RNA capping with NAD+ and NADH by yeast and human mitochondrial RNA polymerase

Bacterial and eukaryotic nuclear RNA polymerases (RNAPs) cap RNA with the oxidized and reduced forms of the metabolic effector nicotinamide adenine dinucleotide, NAD+ and NADH, using NAD+ and NADH as non-canonical initiating nucleotides for transcription initiation. Here, we show that mitochondrial RNAPs (mtRNAPs) cap RNA with NAD+ and NADH, and do so more efficiently than nuclear RNAPs. Direct quantitation of NAD+- and NADH-capped RNA demonstrates remarkably high levels of capping in vivo: up to ~60% NAD+ and NADH capping of yeast mitochondrial transcripts, and up to ~10% NAD+ capping of human mitochondrial transcripts. The capping efficiency is determined by promoter sequence at, and upstream of, the transcription start site and, in yeast and human cells, by intracellular NAD+ and NADH levels. Our findings indicate mtRNAPs serve as both sensors and actuators in coupling cellular metabolism to mitochondrial gene expression, sensing NAD+ and NADH levels and adjusting transcriptional outputs accordingly.

biochemistry↗