Biology subjects

Sivaramakrishnan, P.

Publications and source records attributed to Sivaramakrishnan, P..

8 recordsLinked to original sources

bioRxiv2026

Distinct roles for partially redundant transcription factors in Caenorhabditis elegans mesoderm lineage development

Developmental transcription factors often have overlapping functions, making it difficult to define the distinct roles of individual factors during lineage specification. We investigated the partially redundant transcription factors TBX-35 and CEH-51 in the Caenorhabditis elegans embryonic MS mesodermal lineage using 4D lineage tracing, reporter imaging, genetics, and single-cell RNA sequencing. In tbx-35 mutants, MS descendants showed progressively slower cell cycles and a division pattern that increasingly resembled the cousin C lineage. Fate-regulator expression also shifted toward C-like features, including ectopic pal-1 and expanded HLH-1 expression, although mutant cells did not simply adopt normal C-lineage positions. Loss of tbx-35 also impaired a later MS-dependent Notch induction in the AB lineage while leaving an earlier induction intact. CEH-51 showed a different pattern of activity whereby its protein became enriched in anterior MS daughters, and ceh-51 mutants produced later, more restricted lineage defects that were strongest in descendants of cells with higher CEH-51 levels. Single-cell profiling identified overlapping but nonidentical sets of genes dependent on the two factors. TBX-35-dependent changes were strongest at earlier stages, whereas CEH-51-dependent genes became more prominent later and were enriched in anterior MS sublineages. Finally, temperature-shift experiments determined that the severity and onset of tbx-35 mutant phenotypes depend on the maternal temperature environment and cannot be explained by differences in residual CEH-51 expression. These findings reveal that TBX-35 and CEH-51 contribute differently across the MS lineage and that reliable mesoderm development is supported by overlapping zygotic and maternal regulatory inputs.

developmental biology↗

bioRxiv2026

Targeted modulation of IGFBP5/IGF1, THPO, and P38 MAPK signaling are potent therapeutic strategies generalizable for mitochondrial respiratory chain disease and osteosarcoma

Primary mitochondrial diseases (PMD) have limited disease-modifying therapies, currently applicable to only 3 of over 400 discrete gene disorders. Cycloheximide (CHX) is a global cytosolic translation inhibitor we previously reported to rescue PMD preclinical models, although its toxicity precluded clinical development. To identify specific mediators underlying CHX treatment benefit in PMD, SOMAscan-based proteomics was performed in complex I deficient and genetic disease fibroblast cell line models grown in galactose. Thrombopoietin (THPO) and insulin-like growth factor binding protein 5 (IGFBP5) were the only two differentially regulated proteins, together with ERK/MAPK pathway dysregulation, identified upon CHX treatment in PMD versus healthy control cells. THPO inhibition by siRNA or pharmacologic approaches rescued stress-induced viability loss in patient fibroblasts having diverse PMD gene etiologies, and significantly improved mitochondrial stress, linear growth, and neuromuscular function in a classical ndufs2-/- C. elegans model. IGFBP5 overexpression by lentiviral or mRNA approaches rescued cell viability across distinct PMD gene etiologies, as did IGF1 pharmacologic inhibition across both PMD mutant and C. elegans models. MAPK pharmacologic inhibition rescued multiple distinct complex I disease cells survival, as well as mitochondrial stress in SLC25A46-/- C. elegans. Combination therapies targeting multiple of these glucose signaling pathway proteins, together with glucose and N-acetylcysteine, yielded superior therapeutic benefit in complex I disease cell and C. elegans models. Additionally, single or combined pharmacologic inhibition of THPO or IGF1 significantly enhanced primary and metastatic osteosarcoma cell death. Collectively, targeted small molecule and genetic modulation of THPO, IGF1, or MAPK recapitulated the significant therapeutic benefit of CHX in PMD, while avoiding global translation inhibition. These novel PMD therapies likely confer benefit by attenuating MAPK-driven autophagy and potentially promoting noncanonical glucose uptake, improving cellular energy balance. Overall, these glucose signaling cellular pathway targets hold broad therapeutic promise for PMD patients, warranting further clinical research development.

genetics↗

bioRxiv2022

Modulation of RNA polymerase processivity affects double-strand break repair in the presence of a DNA-end binding protein

Homologous recombination is the predominant double-strand DNA break repair pathway in Escherichia coli. A recent report of non-homologous end joining involving the Ku-like Gam protein of bacteriophage Mu (MuGam) has sparked interest in understanding the functions of DNA end-binding proteins in bacteria. MuGam binds to DNA ends, but how it interferes with DNA repair or transcription in live bacteria remains elusive. In E. coli, RNA polymerase secondary channel interactors, such as the Gre factors, play a role in coordinating transcription with DNA replication and break repair. Here we show that MuGam inhibits break repair by slowing down DNA resection and impeding recombination in living cells. Loss of GreA restores break repair in the presence of MuGam by allowing for increased DNA resection due to a potential release of MuGam from the DNA. Using MuGam as a DNA break sensor, we found that breaks are generated when translation is inhibited, more so in the presence of GreA, supporting the model where transcription-translation uncoupling increases transcription/replication collisions. Significantly, this work reveals that modulation of RNA polymerase can impact DNA break repair in presence of a Ku-like protein.

molecular biology↗

bioRxiv2021

Transcript accumulation rates in the early C. elegans embryo

Dynamic changes in transcription are widespread in developing embryos, where cell cycles are rapid and cell fate decisions need to be made quickly, often before the next cell division. Some fate decisions in the early Caenorhabditis elegans embryo overcome these constraints through the rapid production of high absolute levels of transcription factor mRNAs. Single cell accumulation rates for a small subset of developmental genes are known, but genome-scale measurements are lacking. Furthermore, how different aspects of transcription kinetics are fine-tuned for different genes to achieve the appropriate RNA levels is still being worked out. We describe a novel strategy to analyze single cell RNA sequencing data from the early C. elegans embryo. We estimate the absolute accumulation rates of zygotic genes up to the 16-cell stage and calibrate predicted rates with single molecule transcript imaging. We show that rapid transcript accumulation is common across different cell types and lineages and rates are the highest soon after zygotic transcription begins. High-rate transcription is a characteristic of genes encoding transcription factors with functions in cell fate specification. These genes share common genomic features and are more likely to have undergone recent duplication. We identify core promoter motifs that might drive high absolute RNA accumulation rates. We measured the contributions of core promoter elements to accumulation rate for one rapidly accumulating gene, ceh-51, which is required for mesoderm development. We find that mutating individual motifs modestly decreases the accumulation rate of ceh-51 mRNA, suggesting multifactorial control of transcript accumulation rates. These results are a step towards estimating absolute transcription kinetics during embryonic fate specification and understanding how transcript dosage drives developmental decisions.

developmental biology↗

bioRxiv2021

The anterior Hox gene ceh-13 and elt-1/GATA activate the posterior Hox genes nob-1 and php-3 to specify posterior lineages in the C. elegans embryo

Hox transcription factors play a conserved role in specifying positional identity during animal development, with posterior Hox genes typically repressing the expression of more anterior Hox genes. Here, we dissect the regulation of the posterior Hox genes nob-1 and php-3 in the nematode C. elegans. We show that nob-1 and php-3 are co-expressed in gastrulation-stage embryos in cells that previously expressed the anterior Hox gene ceh-13. This expression is controlled by several partially redundant transcriptional enhancers. These enhancers require ceh-13 for expression, providing a striking example of an anterior Hox gene positively regulating a posterior Hox gene. Several other regulators also act positively through nob-1/php-3 enhancers, including elt-1/GATA, ceh-20/ceh-40/Pbx, unc-62/Meis, pop-1/TCF, ceh-36/Otx and unc-30/Pitx. We identified defects in both cell position and cell division patterns in ceh-13 and nob-1;php-3 mutants, suggesting that these factors regulate lineage identity in addition to positional identity. Together, our results highlight the complexity and flexibility of Hox gene regulation and function and the ability of developmental transcription factors to regulate different targets in different stages of development. Author SummaryHox genes are critical for head-to-tail patterning during embryonic development in all animals. Here we examine the factors that are necessary to turn on two posterior Hox genes, nob-1 and php-3, in the nematode worm, C. elegans. We identified six new transcription factors and three enhancer regions of DNA that can activate expression of nob-1/php-3. Unexpectedly, these activating transcription factors included ceh-13, an anterior Hox gene, and elt-1, a regulator of skin development that is briefly expressed in many cells that do not adopt skin fates, including the cells that express nob-1. Furthermore, the cellular defects we observed in ceh-13 and nob- 1;php-3 mutant embryos indicate that the early embryonic functions of these Hox genes help determine the identity of cells as well as their position within the embryo. Our findings identify new roles for Hox genes in C. elegans and emphasize the ability of transcription factors to contribute to the diversification of cell types and the adoption of specific cell types at different phases of embryonic development.

genetics↗

bioRxiv2019

Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans

The relationship of genotypes to phenotypes can be modified by environmental inputs. Such crucial environmental inputs include metabolic cues derived from microbes living together with animals. Thus the analysis of genetic effects on animals physiology can be confounded by variations in the metabolic profile of microbes. Caenorhabditis elegans exposed to distinct bacterial strains and species exhibit phenotypes different at cellular, developmental and behavioral levels. Here we reported metabolomic profiles of three Escherichia coli strains, B strain OP50, K-12 strain MG1655, and B-K-12 hybrid strain HB101, and also different mitochondrial and fat storage phenotypes of C. elegans exposed to MG1655 and HB101 versus OP50. We found that these metabolic phenotypes of C. elegans are not correlated with overall metabolic patterning of bacterial strains, but their specific metabolites. In particular, the fat storage phenotype is traced to the betaine level in different bacterial strains. HT115 is another K-12 E. coli strain that is commonly utilized to elicit an RNA interference response, and we showed that C. elegans exposed to OP50 and HT115 exhibit differences in mitochondrial morphology and fat storage levels. We thus generated an RNA interference competent OP50 (iOP50) strain that can robustly and consistently knockdown endogenous C. elegans genes in different tissues. Together, these studies suggest the importance of specific bacterial metabolites in regulating the hosts physiology, and provide a tool to prevent confounding effects when analyzing genotype-phenotype interactions under different bacterial backgrounds.

genetics↗

bioRxiv2019

H3K9me2 orchestrates inheritance of spatial positioning of peripheral heterochromatin through mitosis

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

cell biology↗

bioRxiv2019

A lineage-resolved molecular atlas of C. elegans embryogenesis at single cell resolution

C. elegans is an animal with few cells, but a striking diversity of cell types. Here, we characterize the molecular basis for their specification by profiling the transcriptomes of 84,625 single embryonic cells. We identify 284 terminal and pre-terminal cell types, mapping most single cell transcriptomes to their exact position in C. elegans invariant lineage. We use these annotations to perform the first quantitative analysis of the relationship between lineage and the transcriptome for a whole organism. We find that a strong lineage-transcriptome correlation in the early embryo breaks down in the final two cell divisions as cells adopt their terminal fates and that most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state. Users can explore our data with a graphical application "VisCello".

genomics↗