Biology subjects

Sridharan, R.

Publications and source records attributed to Sridharan, R..

14 recordsLinked to original sources

bioRxiv2026

dCas9 metabolic enzyme fusions modulate global and locus specific gene expression

Central metabolites function as essential co-substrates for chromatin-modifying enzymes, directly linking cellular metabolism to chromatin regulation. Accordingly, whole-cell fluctuations in co-substrate availabilities have been shown to promote diverse phenotypes through chromatin-dependent mechanisms. There is emerging evidence that metabolic enzymes producing co-substrates for chromatin modifying enzymes can exist in the nucleus, suggesting that nucleus-specific metabolite availability regulates chromatin state. Here, we developed CRISPRm (CRISPR metabolite) to assess how nucleus-specific metabolic perturbations influence chromatin function. Five dCas9-metabolic enzyme fusions (i.e., dCas9-ACSS2, -NMNAT1, -MAT2A, -GDH, and -AHCY) were used to modulate nuclear levels of essential co-substrates involved in histone (de)acetylation and (de)methylation reactions. Transient expression of all dCas9 fusions in HEK293T cells induced distinct global changes in gene expression patterns, with dCas9-ACSS2 (acetyl-CoA producing) and NMNAT1 (NAD+ producing) eliciting large opposing changes in gene expression, suggesting transcriptional responses to nuclear acetyl-CoA and NAD+ production may be directly facilitated by acetylation or deacetylation reactions, respectively. Targeting dCas9-ACSS2 and -NMNAT1 to promoters of select candidate genes revealed enhanced transcriptional modulation. dCas9-ACSS2 upregulated, and dCas9-NMNAT1 downregulated genes showed basal enrichment of H3K9ac, H3K18ac, H3K27ac, H3K4me3, and p300, suggesting these genomic loci reside within epigenetic environments susceptible to fluctuations in acetyl-CoA and NAD+ availability. Of significant genes altered, dCas9-MAT2A (SAM producing) increased expression of 72% whereas dCAS9-GDH (alpha-ketoglutarate producing) decreased expression of 79%. Surprisingly, dCAS9-AHCY (SAH hydrolysis) led to down-regulation of shared genes up-regulated by dCas9-MAT2A. The observations amongst the methylation-specific enzymes revealed unexpected and unique gene-regulatory sensitivities to SAM, SAH and alpha-ketoglutarate. Together, these results demonstrate the utility of CRISPRm in studying nuclear metabolic regulation of transcription and provide strong evidence that perturbations in nuclear co-substrates do not lead to a large mass- action changes in chromatin acetylation/methylation but rather to modulation of select chromatin-modifying enzymes with targeted transcription responses. HighlightsO_LICRISPRm is a novel, modular dCas9-effector platform that enables interrogation of the metabolism-epigenome axis C_LIO_LIdCas9-ACSS2, -NMNAT1, -MAT2A, -AHCY, and -GDH induce distinct transcriptional programs. C_LIO_LITargeting CRISPRm to promoters enhances transcriptional responses. C_LIO_LIdCas9-ACSS2 and -NMNAT1 sensitive gene promoters exhibit unique enrichment of chromatin features, including H3K9ac, H3K18ac, H3K27ac, H3K4me3 and p300. C_LI

biochemistry↗

bioRxiv2025

DOT1L induces RNAPII accumulation independent of its catalytic activity in pluripotent stem cells

Pluripotent stem cells can be propagated in vitro from embryos in specific culture conditions that capture subtle developmental transitions. Naive pluripotent mouse embryonic stem cells (ESCs) can exist in two distinct epigenetic states--the metastable state in serum/LIF conditions and the ground state with pharmacological inhibition of differentiation-inducing pathways in the 2i/LIF conditions, which better resembles the in vivo blastocyst. Here, we acutely induced one feature of 2i/LIF ESCs, an increase in the H3K79 methyltransferase, DOT1L, in serum/LIF ESCs to determine its effects on metastable pluripotency. We find that DOT1L induction causes an increase in RNA Polymerase II (RNAPII) accumulation at the transcription start site, irrespective of catalytic activity, mimicking 2i/LIF RNAPII pattern. However, the pulse of DOT1L and consequent RNAPII accumulation is insufficient to induce immediate changes in steady-state or nascent RNA expression. Genes with higher transcription and elongation rates exhibit moderate changes in RNAPII accumulation, while lowly transcribed genes separate into two distinct groups, with one group showing the strongest RNAPII accumulation in response to DOT1L induction and the other showing the weakest. This differential accumulation is reduced at H3K27me3-enriched and bivalent genes. We also find that cells that sustain DOT1L expression have a homogenous NANOG protein profile without affecting Nanog transcription. Taken together, we find that a pulse of DOT1L in serum/LIF ESCs is sufficient to partially recapitulate certain features of ground state pluripotency, reinforcing its importance in this state of the pluripotency continuum.

molecular biology↗

bioRxiv2025

Uncovering Functional Gene Regulatory Networks in Bulk and Single-Cell Data through Robust Transcription Factor Activity Estimation and Model-Guided Experimental Validation

Reconstructing genome-scale gene regulatory networks (GRNs) remains a difficult problem in systems biology, and many experimental and computational methods have been developed to address this problem. Recent computational methods have aimed to more accurately model GRNs by estimating the hidden Transcription Factor Activity (TFA) from prior knowledge of TF target regulatory connections, encoded as an input directed graph, to relax the assumption that mRNA level of the regulator correlates with the protein activity of the regulator. However, the noise in the prior knowledge can adversely affect the estimated TFA levels and the quality of the downstream inferred GRNs. Here, we present a new approach, MERLIN+P+TFA, that uses prior knowledge-guided sparsity regularization to robustly and accurately estimate TFA and downstream GRNs. We apply our method to simulated and real expression data in yeast and mammalian systems and show improved quality of inferred GRNs for both bulk and single-cell datasets. Regularized TFA offers benefits to a variety of other GRN inference algorithms, including those that have traditionally been used with expression alone, in both bulk and scRNA-seq settings. We used the inferred GRN to prioritize key regulators for the mouse Embryonic Stem Cell (mESC) state and validate 58 regulators experimentally. We identified both known and novel regulators of the mESC state and further validate the targets of 4 known and novel regulators. Our validation experiments suggest that computationally inferred networks can capture functional targets of TFs with higher precision than estimated in current benchmarks, however, it is important to generate context-specific gold standards.

bioinformatics↗

bioRxiv2023

KDM3A and KDM3B Maintain Naive Pluripotency Through the Regulation of Alternative Splicing

Histone modifying enzymes play a central role in maintaining cell identity by establishing a conducive chromatin environment for lineage specific transcription factor activity. Pluripotent embryonic stem cell (ESC) identity is characterized by a lower abundance of gene repression associated histone modifications that enables rapid response to differentiation cues. The KDM3 family of histone demethylases removes the repressive histone H3 lysine 9 dimethylation (H3K9me2). Here we uncover a surprising role for the KDM3 proteins in the maintenance of the pluripotent state through post-transcriptional regulation. We find that KDM3A and KDM3B interact with RNA processing factors such as EFTUD2 and PRMT5. Acute selective degradation of the endogenous KDM3A and KDM3B proteins resulted in altered splicing independent of H3K9me2 status or catalytic activity. These splicing changes partially resemble the splicing pattern of the more blastocyst-like ground state of pluripotency and occurred in important chromatin and transcription factors such as Dnmt3b, Tbx3 and Tcf12. Our findings reveal non-canonical roles of histone demethylating enzymes in splicing to regulate cell identity.

cell biology↗

bioRxiv2022

Antagonistic H3K79me-H3K9ac crosstalk determines elongation at housekeeping genes to promote pluripotency

Pluripotent embryonic stem cells (ESCs) have a transcriptionally permissive chromatin environment enriched for gene activation-associated histone modifications as compared to somatic cells. A striking exception is DOT1L-mediated H3K79 methylation that is considered a positive regulator of transcription. Here we find that ESCs maintain low H3K79 methylation to facilitate RNA polymerase II (RNAPII) elongation for greater nascent transcription. Inhibiting DOT1L during the reprogramming of somatic to induced pluripotent stem cells (iPSCs) enables ESC-like RNAPII and transcriptional status. Mechanistically, DOT1L inhibition causes a local gain of histone acetylation at genes that lose the most H3K79me, which unexpectedly are ubiquitously expressed genes that perform essential functions in every cell, rather than lineage specifying genes. Maintenance of this elevated histone acetylation is required for the enhanced conversion to iPSCs upon DOT1L inhibition. Remarkably, increasing global DOT1L or site-specific tethering of DOT1L is sufficient to decrease H3K9ac in ESCs. We discover a high H3ac-low H3K79me epigenetic mechanism that promotes transcription elongation at ubiquitously expressed genes to enforce pluripotent cell identity.

developmental biology↗

bioRxiv2022

Inference of cell type-specific gene regulatory networks on cell lineages from single cell omic datasets

Cell type-specific gene expression patterns are outputs of transcriptional gene regulatory networks (GRNs) that connect transcription factors and signaling proteins to target genes. Single-cell technologies such as single cell RNA-sequencing (scRNA-seq) and single cell Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq), can examine cell-type specific gene regulation at unprecedented detail. However, current approaches to infer cell type-specific GRNs are limited in their ability to integrate scRNA-seq and scATAC-seq measurements and to model network dynamics on a cell lineage. To address this challenge, we have developed single-cell Multi-Task Network Inference (scMTNI), a multi-task learning framework to infer the GRN for each cell type on a lineage from scRNA-seq and scATAC-seq data. Using simulated and real datasets, we show that scMTNI is a broadly applicable framework for linear and branching lineages that accurately infers GRN dynamics and identifies key regulators of fate transitions for diverse processes such as cellular reprogramming and differentiation.

bioinformatics↗

bioRxiv2020

Dot1L interaction partner AF10 safeguards cell identity during the acquisition of pluripotency

Histone 3 Lysine 79 methylation (H3K79me) is enriched on gene bodies proportional to gene expression levels, and serves as a strong barrier for reprogramming of somatic cells to induced pluripotent stem cells (iPSCs). DOT1L is the sole histone methyltransferase that deposits all three orders - mono, di, and tri methylation - at H3K79. Here we leverage genetic and chemical approaches to parse the specific functions of the higher orders of H3K79me in maintaining cell identity. DOT1L interacts with AF10 (Mllt10) which recognizes unmodified H3K27 and boosts higher order H3K79 methylation. AF10 deletion evicts higher order H3K79me2/3, and reorganizes H3K79me1 to the transcription start site, to facilitate iPSC formation in the absence of steady state transcriptional changes. Instead, AF10 loss redistributes RNA polymerase II to a uniquely pluripotent pattern at highly expressed, rapidly transcribed housekeeping genes. Taken together, we reveal a mechanism for higher order histone methylation located at the gene body in reinforcing cell identity.

genetics↗

bioRxiv2020

Dynamic regulatory module networks for inference of cell type specific transcriptional networks

Multi-omic datasets with parallel transcriptomic and epigenomic measurements across time or cell types are becoming increasingly common. However, integrating these data to infer regulatory network dynamics is a major challenge. We present Dynamic Regulatory Module Networks (DRMNs), a novel approach that uses multi-task learning to infer cell type-specific cis-regulatory networks dynamics. Compared to existing approaches, DRMN integrates expression, chromatin state and accessibility, accurately predicts cis-regulators of context-specific expression and models network dynamics across linearly and hierarchically related contexts. We apply DRMN to three dynamic processes of different experimental designs and predict known and novel regulators driving cell type-specific expression patterns.

bioinformatics↗

bioRxiv2020

The Requirement For US28 During Cytomegalovirus Latency Is Independent Of US27 And US29 Gene Expression

The ability to establish a latent infection with periodic reactivation events ensures herpesviruses, like human cytomegalovirus (HCMV), lifelong infection and serial passage. The host-pathogen relationship throughout HCMV latency is complex, though both cellular and viral factors influence the equilibrium between latent and lytic infection. We and others have shown one of the viral-encoded G protein-coupled receptors, US28, is required for HCMV latency. US28 potentiates signals both constitutively and in response to ligand binding, and we previously showed deletion of the ligand binding domain or mutation of the G protein-coupling domain results in the failure to maintain latency similar to deletion of the entire US28 open reading frame (ORF). Interestingly, a recent publication detailed an altered phenotype from that previously reported, showing US28 is required for viral reactivation rather than latency, suggesting the US28 ORF deletion impacts transcription of the surrounding genes. Here, we show an independently generated US28-stop mutant, like the US28 ORF deletion mutant, fails to maintain latency in hematopoietic cells. Further, we found US27 and US29 transcription in each of these mutants was comparable to their expression during wild type infection, suggesting neither US28 mutant alters mRNA levels of the surrounding genes. Finally, infection with a US28 ORF deletion virus expressed US27 protein comparable to its expression following wild type infection. In sum, our new data strongly support previous findings from our lab and others, detailing a requirement for US28 during HCMV latent infection.

microbiology↗

bioRxiv2019

Depletion of H3K79 methylation specifically enhances reprogramming to pluripotency but not transdifferentiation

The ability of pluripotent stem cells to be poised to differentiate into any somatic cell type is partly derived from a unique chromatin structure that is depleted for transcriptional elongation associated epigenetic modifications, primarily H3K79 methylation. Inhibiting the H3K79 methyltransferase, Dot1L, increases the efficiency of reprogramming somatic cells to induced pluripotent stem cells (iPSCs) most potently at the mid-point of the process. Surprisingly, despite the enrichment of H3K79me2 on thousands of actively transcribed genes, Dot1L inhibition (Dot1Li) results in few changes in steady state mRNA levels during reprogramming. Dot1Li spuriously upregulates genes not involved in pluripotency and does not shutdown the somatic program. Depletion of the few genes that are downregulated, such as Nfix, enhances reprogramming efficiency in cooperation with Dot1Li. Contrary to the prevalent view, Dot1Li promotes iPSC generation beyond early phases of reprogramming such as the mesenchymal to epithelial transition and from already epithelial cell types including keratinocytes. Significantly, Dot1L inhibition does not enhance lineage conversion to neurons or muscle cells. Taken together, our results indicate that H3K79me is not a universal barrier of cell fate transitions but specifically protects somatic cells from reverting to the pluripotent state.

developmental biology↗

bioRxiv2019

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate cellular epigenetic states. This metabolism-epigenome link enables the sensitization of chromatin methylation to altered SAM abundance. However, a chromatin-wide understanding of the adaptive/responsive mechanisms that allow cells to actively protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear de novo mono-methylation of H3 Lys 9 (H3K9) at the expense of global losses in histone di- and tri-methylation. Under SAM-depleted conditions, de novo H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide the first evidence for active epigenetic adaptation and persistence to metabolic stress.

biochemistry↗

bioRxiv2019

Integrated Approach On Degaradtion Of Azo Dyes Using Laccase Enzyme And Nanoparticle With Its Interaction By In Silco Analysis

Azo dyes, released by the textile industries causes severe damage to the environment and living organisms. The degradation of azo dyes is widely studied using enzymatic methods. Laccase, is a copper containing enzyme that degrades the azo dyes into less toxic compounds. In this work, Laccase enzyme produced by the alkaliphile Pseudomonas mendocina in the degradation of mixed azo dye showed 0.386 U/Ml activity at pH 8.5. Combination of enzymatic and green synthesised nanoparticle were used in the degradation mixed azo dye. Laccase used in the degradation of mixed azo dyes showed 58.46% in 72 hours while the photocatalytic degradation of mixed azo dyes showed 15.98%. The degradation of azo dyes using copper iodide nanoparticle resulted in 15.835% of mixed azo dye degradation. But it was noticed that combined method removed 62.35% of mixed azo dyes in 60 minutes. Interaction of laccase enzyme with azo dyes using in silico analysis predicted the binding energy of RR (-7.19 kcal/mol), RB (-8.57 kcal/mol) and RBL (-9.17 kcal/mol).

microbiology↗

bioRxiv2019

White Matter Hyperintensity Quantification in Large-Scale Clinical Acute Ischemic Stroke Cohorts - The MRI-GENIE Study

White matter hyperintensity (WMH) burden is a critically important cerebrovascular phenotype linked to prediction of diagnosis and prognosis of diseases, such as acute ischemic stroke (AIS). However, current approaches to its quantification on clinical MRI often rely on time intensive manual delineation of the disease on T2 fluid attenuated inverse recovery (FLAIR), which hinders high-throughput analyses such as genetic discovery. In this work, we present a fully automated pipeline for quantification of WMH in clinical large-scale studies of AIS. The pipeline incorporates automated brain extraction, intensity normalization and WMH segmentation using spatial priors. We first propose a brain extraction algorithm based on a fully convolutional deep learning architecture, specifically designed for clinical FLAIR images. We demonstrate that our method for brain extraction outperforms two commonly used and publicly available methods on clinical quality images in a set of 144 subject scans across 12 acquisition centers, based on dice coefficient (median 0.95; inter-quartile range 0.94-0.95) and Pearson correlation of total brain volume (r=0.90). Subsequently, we apply it to the large-scale clinical multi-site MRI-GENIE study (N=2783) and identify a decrease in total brain volume of -2.4cc/year. Additionally, we show that the resulting total brain volumes can successfully be used for quality control of image preprocessing. Finally, we obtain WMH volumes by building on an existing automatic WMH segmentation algorithm that delineates and distinguishes between different cerebrovascular pathologies. The learning method mimics expert knowledge of the spatial distribution of the WMH burden using a convolutional auto-encoder. This enables successful computation of WMH volumes of 2,533 clinical AIS patients. We utilize these results to demonstrate the increase of WMH burden with age (0.950 cc/year) and show that single site estimates can be biased by the number of subjects recruited.

neuroscience↗

bioRxiv2019

Defining reprogramming checkpoints from single-cell analysis of induced pluripotency

Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a novel combination of epigenetic and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress towards iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play novel functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenetic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.

developmental biology↗