Biology subjects

Choudhary, C.

Publications and source records attributed to Choudhary, C..

15 recordsLinked to original sources

bioRxiv2025

The SPATA5-SPATA5L1-CINP-C1ORF109 complex is essential for cytoplasmic pre-60S ribosome maturation

The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein-protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5-SPATA5L1-CINP-C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5s ATPase activity is more important than SPATA5L1s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCCs role in pre-60S maturation.

biochemistry↗

bioRxiv2024

Mammalian enhancers and GWASs act proximally and seldom skip active genes

Enhancers play a critical role in regulating transcription. Nearly 90% of human genetic variants identified in genome-wide association studies (GWAS) are located in distal regions, underscoring the importance of enhancers in human development, diseases, and traits. It is widely suggested that mammalian enhancers frequently skip active genes, and thus, linear proximity is a poor predictor of their targets. A key unresolved question is how often mammalian enhancers skip proximal active genes to specifically target distal genes. Genome-wide enhancer-promoter mapping shows that enhancers frequently bypass active genes, while ultra-deep locus-specific analyses reveal extensive multi-way interactions between enhancers and promoters, forming nested microcompartments. The functional significance of these seemingly contrasting phenomena remains unclear. Here, we compared hundreds of enhancer-target gene pairs identified using enhancer-promoter chromatin contact maps, enhancer-promoter RNA interaction data, and genome-scale CRISPR interference (CRISPRi) perturbations. Our findings reveal limited overlap between active gene-skipping enhancer-gene pairs identified through physical interaction mapping and CRISPRi. Additionally, promoters involved in multi-way enhancer interactions are not co-regulated by shared coactivators. Notably, gene-skipping and non-skipping enhancers identified via CRISPRi differ fundamentally in chromatin features, gene activation strength, false discovery rates, target gene distance, coactivator requirements, and cell-type specificity of target genes. These results suggest that gene-skipping enhancer-promoter interactions observed in chromatin and RNA-based analyses do not reliably predict functional enhancer-gene relationships. We propose that linear enhancer-promoter proximity and coactivator dependency offer a simple, scalable, and cost-effective method for genome-wide prediction of enhancer and GWAS targets, with accuracy comparable to state-of-the-art experimental techniques. While enhancers can skip active genes, such deliberate skipping appears to be the exception rather than the rule.

genomics↗

bioRxiv2024

A unified model of gene expression control by cohesin and CTCF

Cohesin and CTCF fold vertebrate genomes into loops and topologically associating domains (TADs). The genome folding by cohesin and CTCF is considered crucial for enhancer-promoter communication and gene regulation. However, the extent of cohesin and CTCF-dependent looping in global enhancer function and their potential non-architectural roles in gene activation have remained unclear. Here, we demonstrate that the acute removal of cohesin or CTCF in mouse cells dysregulates hundreds of genes, albeit subtly. Among various possible enhancer types, cohesin almost exclusively facilitates gene activation by one enhancer type--the CBP/p300-dependent enhancers. Interestingly, CTCF plays a dual role, operating both with and without cohesin, and promoting gene activation both in an enhancer-dependent and independent manner. By anchoring cohesin loops, CTCF directs enhancers to specific target genes and prevents their mistargeting. Independently of cohesin and enhancers, CTCF acts as a transcriptional activator or repressor, depending on its precise binding position near promoters. Acting as a canonical transcription activator, CTCF directly activates hundreds of housekeeping genes, including those essential for mammalian cell proliferation. Mechanistically, promoter-bound CTCF controls DNA accessibility and RNA polymerase II recruitment. The transcriptional activator function of CTCF appears unique to vertebrates and is shared by its vertebrate-specific paralog, CTCFL, despite CTCFLs inability to anchor cohesin loops. These findings reveal the scope of cohesin and CTCF in gene regulation, delineate their shared and unique functions, define a specific class of enhancers using cohesin-mediated looping, and establish a crucial function of CTCF in gene regulation independent of its architectural role. The work reconciles conflicting views and provides a unified model for gene regulation by cohesin and CTCF.

genomics↗

bioRxiv2024

TSSK homologue regulates the expression of protamine and mosquito testes specific genes in Anopheles stephensi

The testis-specific serine/threonine-protein kinase (tssk) gene is known to play an important role in spermiogenesis in mammals. This study identifies tssk homologues, As_tssk3 and Aea_tssk1 in Anopheles stephensi and Aedes aegypti, respectively. Both As_tssk3 and Aea_tssk1 were found to express in a male-specific manner throughout the development. Efficient knockdown in the expression of As_tssk3 gene was observed upon feeding An. stephensi larvae with target dsRNA. As_tssk3 knockdown led to a reduced sperm reservoir in male adults. Additionally, the knockdown of As_tssk3 resulted in the reduced expression of two male specifically expressed genes, protamine & mosquito testes specific (mts- a sperm-specific marker) homologues of An. stephensi. In-silico interaction analysis suggests that As_TSSK3 protein interacts with the Nucleosome Assembly Protein1 like-4 (NAP1like-4), a putative transcription factor. We hypothesize that As_TSSK3 regulates the transcription of protamine and mts genes via NAP, as both NAP and protamine have previously been shown to function in the establishment and maintenance of chromatin dynamics, in independent studies. The study presented here is the first report of characterisation of tssk homologues in any mosquito species. The knockout studies and phosphorylation assays would provide more insights to explore the mechanism by which As_tssk3 regulates protamine and mts genes in Anopheles stephensi. HighlightsO_LIThis study identifies the tssk homologues in Ae. aegypti and An. stephensi and showed their male-specific expression in both the mosquito species. C_LIO_LIdsRNA mediated knockdown of As_TSSK3 resulted into the males with a reduced size of sperm reservoir. C_LIO_LIdsRNA mediated knockdown of As_TSSK3 led to the reduction in the expression of As_prot and As_mts, homologues of protamine and mosquito testis specific, genes in An. stephensi. C_LIO_LIIn-silico studies suggest that As_TSSK3 regulates the expression of As_prot and As_mts genes via NAP. C_LI

genetics↗

bioRxiv2024

Modeling nascent transcription from chromatin landscape and structure

BackgroundDifferent cell types and their associated functionalities emerge from a single genomic sequence when certain regions are expressed while others remain silenced. Modeling gene expression and its potential malfunctioning in different cellular contexts is hence pivotal to understand both development and disease. ResultsWe present the Chromatin Landscape and Structure to Expression Regressor (CLASTER), an epigenetic-based deep neural network that can integrate different data modalities describing the chromatin landscape and its 3D structure. CLASTER effectively translates them into nascent transcription levels measured by EU-seq at a kilobasepair resolution. Our predictions reached a Pearson correlation with targets above r=0.86 at both bin and gene levels, without relying on DNA sequence nor explicitly extracted chromatin features. The model mostly used the information found within 10 kbp of the predicted locus, even when a wide genomic region of 1 Mbp was available. Explicit modeling of long-range interactions using multi-headed attention and high-resolution chromatin contact maps had little impact on model performance, despite the model correctly identifying elements in these inputs influencing nascent transcription. The trained model served then as a platform to predict the transcriptional impact of simulated epigenetic silencing perturbations. ConclusionsOur results point towards a rather local, integrative and combinatorial paradigm of gene regulation, where changes in the chromatin environment surrounding a gene shape its context-specific transcription. We conclude that the predominant locality and limitations of current machine learning approaches might emerge as a genuine signature of genomic organization, having broad implications for future modeling approaches.

bioinformatics↗

bioRxiv2024

Genome assembly and annotation of the red flour beetle (Tribolium castaneum) from India

The largest insect order, Coleoptera, includes several economically important beetles that also serve as major model species for biological research. Perhaps foremost among these is the red flour beetle, a global pest of stored grains and flour whose genome was sequenced in 2008. However, the currently available reference genome (Tcas5.2) is incomplete, fragmented and contains many gaps, and the Y chromosome is not assembled. Here we present inTcas1, an updated genome assembly and annotation of T. castaneum collected from India, assembled using both short and long read sequencing, and annotated using two transcriptome datasets. We report that inTcas1 has fewer gaps, less fragmentation, and many new genes and new isoforms of previously annotated genes. This new resource provides a useful update, comparison, and reference for new beetle genome assemblies. The first Y chromosome assembly for this species also provides critical data to study the evolution of insect sex chromosomes and sex determination systems. SIGNIFICANCEWe present here an improved T. castaneum genome assembly (inTcas1) for beetles sampled from India, including the first Y chromosome of this important pest and laboratory model species. The new genome should facilitate more comprehensive analysis of Coleoptera genome and transcriptome datasets, especially beetle populations from Asia - the previously available genome, Tcas5.2, was assembled using the GA2 strain from USA. The updated genome should also facilitate analyses of genome evolution, including sex determination and sex chromosome dynamics; and the new gene annotations can expand the genetic toolkit for this beetle.

genomics↗

bioRxiv2023

A systems-scale, integrated view of the ubiquitylation site occupancy and dynamics

Ubiquitylation regulates virtually all proteins and biological processes in a cell. However, the global site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have never been quantified. Here, we present the first integrated picture of ubiquitylation site occupancy and half-life. Ubiquitylation occupancy spans four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and the regulation of sites by proteasome inhibitors show strong interrelationships. These properties can discriminate signaling-relevant sites from the sites involved in proteasomal degradation. The sites strongly upregulated by proteasome inhibitors have a longer half-life, and the half-life increases with increasing protein length. Importantly, a previously unknown surveillance mechanism rapidly deubiquitylates all ubiquitin-specific E1 and E2 enzymes and protects them against bystander ubiquitylation accumulation. This work reveals general principles of ubiquitylation-dependent governance and offers conceptual insights into the dynamic regulation of the cell. HighlightsO_LIUbiquitylation site occupancy is 3 orders of magnitude lower than phosphorylation C_LIO_LIThe highest 80% and the lowest 20% occupancy sites have distinct properties C_LIO_LIHigh occupancy sites are concentrated in the cytoplasmic domains of SLC proteins C_LIO_LIA dedicated mechanism prevevents ubiquitylation accumulation in E1s and E2s C_LI

biochemistry↗

bioRxiv2023

A sex-specific homologue of snake waprin is required for embryonic development in the Red Flour Beetle, Tribolium castaneum

1.Waprin, an extracellular secretory protein, is widely known for its antibacterial activity. Through the analysis of sex-specific transcriptomes of adult Tribolium castaneum, we identified a homologue of snake waprin (Tc_WapF) with significantly higher level of its transcripts in females. Developmental- and tissue-specific profiling revealed the widespread expression of Tc_WapF in adult female tissues. Interestingly, Tc_WapF expression is not regulated by the classical sex-determination cascade of T. castaneum, as we fail to get any attenuation in Tc_WapF transcript levels in Tcdsx and Tctra (key players of sex determination cascade of T. castaneum) knockdown females. The eggs laid by Tc_WapF knockdown females never hatched, indicating the involvement of Tc_WapF in the embryonic development in T. castaneum. This is the first report of identification a sex-specific waprin homologue in an insect and its involvement as a novel candidate in the embryonic development. Future investigations on the functional conservation of insect waprins and their mechanistic role in embryonic development can be exploited for improving pest management strategies. Summary statementTribolium castaneum possesses a female-specific homologue of snake Waprin (Tc_WapF) which is essential for its embryonic development. Expression of Tc_WapF is independent of Tribolium sex-determination cascade.

molecular biology↗

bioRxiv2023

The BOD1L subunit of the mammalian SETD1A complex sustains the expression of DNA damage repair genes despite restraining H3K4 trimethylation

SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, the Setd1a mouse knock-out dies in early embryogenesis at the epiblast stage and mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that ESC death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central alpha-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of DNA repair gene expression, the converse changes in H3K4 methylation after loss of SETD1A or BOD1L implies that H3K4 methylation is not essential for expression of the target DNA repair genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex, and BOD1L in particular, are key nodes for the DNA damage repair network.

cell biology↗

bioRxiv2023

TOBF1 modulates mouse embryonic stem cell fate through co-transcriptional regulation of alternative splicing

Embryonic stem (ES) cells retain the ability to undergo lineage-specific differentiation that can eventually give rise to different cell types that constitute an organism. Although stem cell specific biological networks of transcription factors and epigenetic modifiers are well established, how the ES cell specific transcriptional and alternative splicing (AS) machinery regulate their expression has not been sufficiently explored. In this study, we show that the lncRNA associated protein TOBF1 regulates the co-transcriptional alternative splicing of transcripts necessary for maintaining stem cell identity in mouse ES cells. Overlaying information derived from TOBF1 chromatin occupancy, the distribution of its pluripotency-associated OCT-SOX binding motifs, and transcripts undergoing differential expression and alternative splicing upon its disruption unmasked local nuclear territories where these distinct events converge, ultimately leading to the maintenance of mouse ES cell identity.

developmental biology↗

bioRxiv2022

The logic of native enhancer-promoter compatibility and cell-type-specific gene expression variation

Cis-regulatory enhancers are essential for differential expression of developmental and housekeeping genes. However, the specificity of native mammalian enhancers and how it shapes cell-type-specific gene expression landscapes remain largely unknown. We show that endogenous enhancers are broadly compatible with the promoters of developmental and housekeeping genes. Broad enhancer compatibility affords retrofitting new regulatory capabilities to housekeeping genes that evolved before the advent of enhancers. This enables cell-type-specific tuning of ubiquitously expressed genes. Segregation between enhancer-dependent and -independent type regulation is blurred. Within the same cell type, a single promoter can be activated by enhancers and non-enhancer promoter-regulatory elements (PREs). It is the tunable and integrated strengths of enhancers and PREs that quantitatively shape gene expression landscapes, within and across cell types. Our findings have broad implications for understanding cell-type-specific quantitative gene expression variation, as well as the emergence and rewiring of gene regulatory networks in disease and organismal evolution.

genomics↗

bioRxiv2022

A unique H2B acetylation signature marks active enhancers and predicts their target genes

Chromatin features are widely used for genome-scale mapping of enhancers. However, discriminating active enhancers from other cis-regulatory elements, predicting enhancer strength, and identifying their target genes remains challenging. Here we establish histone H2B N-terminus multisite lysine acetylation (H2BNTac) as a genuine signature of active enhancers. H2BNTac prominently marks candidate active enhancers and their target promoters and discriminates them from ubiquitously active promoters. Two mechanisms afford the distinct H2BNTac specificity. (1) Unlike H3K27ac, H2BNTac is specifically catalyzed by CBP/p300. (2) H2A-H2B, but not H3-H4, are rapidly exchanged through transcription-induced nucleosome remodeling. H2BNTac-positive candidate enhancers show a high validation rate in orthogonal enhancer activity assays, and a vast majority of endogenously active enhancers are marked by H2BNTac and H3K27ac. Notably, H2BNTac intensity predicts enhancer strength and outperforms the current state-of-the-art models in predicting enhancer target genes. These findings have broad implications for generating fine-grained enhancer maps and modeling enhancer-dependent gene regulation.

genomics↗

bioRxiv2020

Histone deacetylase inhibitors butyrate and bufexamac inhibit de novo HIV-1 infection in CD4 T-cells

While current combined antiretroviral therapy (cART) allows control of HIV replication in patients and effectively suppresses plasma viral loads, it is unable to target latent reservoirs, which are responsible for virus rebound after discontinuation of therapy. Several histone deacetylase inhibitors (HDACIs) have been shown to target reservoirs and to reactivate latent HIV. While this effect is highly desired, it carries the risk that HIV-1 may be reactivated in tissue compartments were cART concentrations are insufficient and thus leading to de novo infections in this sites. To address this concern, we evaluated the effect of different HDACIs for their ability to reverse HIV latency and to modulate de novo infections. Two of the inhibitors, sodium butyrate and bufexamac, significantly inhibited de novo HIV-1 infection in activated CD4+ T-cells. Transcriptome and proteome analysis indicated global changes of protein abundancies, exhibited reduced proliferation of CD4+ T-cells, and revealed butyrate-based proteasomal degradation of EP300, an important factor for HIV-1 replication. Our results disclose new potential treatment strategies and minimizes the concern of potential reservoir reseeding by HDACIs.

microbiology↗

bioRxiv2019

The dynamic equilibrium of nascent and parental MCMs safeguards replicating genomes

The MCM2-7 (minichromosome maintenance) protein complex is a DNA unwinding motor required for the eukaryotic genome duplication1. Although a huge excess of MCM2-7 is loaded onto chromatin in G1 phase to form pre-replication complexes (pre-RCs), only 5-10 percent are converted into a productive CDC45-MCM-GINS (CMG) helicase in S phase - a perplexing phenomenon often referred to as the MCM paradox2. Remaining pre-RCs stay dormant but can be activated under replication stress (RS)3. Remarkably, even a mild reduction in MCM pool results in genome instability4, 5, underscoring the critical requirement for high-level MCM maintenance to safeguard genome integrity across generations of dividing cells. How this is achieved remains unknown. Here, we show that for daughter cells to sustain error-free DNA replication, their mothers build up a stable nuclear pool of MCMs both by recycling of chromatin-bound MCMs (referred to as parental pool) and synthesizing new MCMs (referred to as nascent pool). We find that MCMBP, a distant MCM paralog6, ensures the influx of nascent MCMs to the declining recycled pool, and thereby sustains critical levels of MCMs. MCMBP promotes nuclear translocation of nascent MCM3-7 (but not MCM2), which averts accelerated MCM proteolysis in the cytoplasm, and thereby fosters assembly of licensing-competent nascent MCM2-7 units. Consequently, lack of MCMBP leads to reduction of nascent MCM3-7 subunits in mother cells, which translates to poor MCM inheritance and grossly reduced pre-RCs formation in daughter cells. Unexpectedly, whereas the pre-RC paucity caused by MCMBP deficiency does not alter the overall bulk DNA synthesis, it escalates the speed and asymmetry of individual replisomes. This in turn increases endogenous replication stress and renders cells hypersensitive to replication perturbations. Thus, we propose that surplus of MCMs is required to safeguard replicating genomes by modulating physiological dynamics of fork progression through chromatin marked by licensed but inactive MCM2-7 complexes.

cell biology↗

bioRxiv2017

Higher eukaryote-specific APC/C composition is a determinant of spindle assembly checkpoint importance

The multisubunit ubiquitin ligase APC/C (anaphase promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. Proper timing of APC/C activation is regulated by the spindle assembly checkpoint (SAC), which is initiated by the kinase MPS1 and implemented by MAD2-dependent inhibition of the APC/C. Here we analysed the contribution of the higher eukaryote-specific APC/C subunits APC7 and APC16 to APC/C composition, function and regulation. APC16 is required for APC7 assembly into the APC/C, while APC16 assembles independently of APC7. {Delta}APC7 and {Delta}APC16 cells display no major defects in mitotic progression, cyclin B1 degradation or SAC response. Strikingly, however, deletion of either APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. {Delta}APC7{Delta}MAD2 cells display an accelerated mitosis and require SAC-independent MPS1 function for maintaining their genome stability. Overall, these results show how human APC/C composition critically influences the cellular fate upon loss of SAC activity.

cell biology↗