Biology subjects

Kainth, A. S.

Publications and source records attributed to Kainth, A. S..

7 recordsLinked to original sources

bioRxiv2026

Large RNA polymerase II condensates are promoter-centric assemblies associated with early stages of transcription at all expressed genes

Transcriptional condensates concentrate the machinery required for RNA polymerase II mediated transcription. These structures range from numerous small, short-lived species, to a handful of larger, stable assemblages. Large condensates have been implicated in driving potent transcription of several super-enhancer regulated genes, yet the underlying mechanisms and the range of their client genes remain unclear. Here, we developed a biochemical approach which combines density gradient centrifugation and affinity purification to partially purify large transcriptional condensates from nuclei, allowing systematic characterization of their nucleic acid components. We find that transcriptional condensate isolates engage thousands of gene promoters and harbor the nascent transcriptome, but do not stably co-purify with distal enhancers. Binding patterns of RNA polymerase II within condensates suggest these structures could facilitate promoter escape and promoter-proximal pause release. Together, our work supports a promoter-centric condensate organization and paves the way towards understanding the functional link between condensate architecture and nascent transcription.

molecular biology↗

bioRxiv2026

Promiscuous RNA binding by WDR5 remodels the KMT2A (MLL1) histone methyltransferase complex to an inactive state

Chromatin-modifying complexes are critical in gene regulation, yet their proposed interactions with RNA remain poorly understood. Here, we show that WDR5, an essential subunit of the MLL1 (KMT2A) histone methyltransferase complex, binds RNA with high affinity but without sequence specificity. Using a stringent approach, we demonstrate that WDR5 directly engages a diverse pool of RNAs in cells, predominantly as a function of RNA abundance rather than specific motifs. Equilibrium binding assays further show that RNA length, rather than sequence, dictates WDR5 affinity. We map multiple RNA-binding regions on WDR5, some of which overlap with surfaces used in MLL1 complex subunit interfaces. Strikingly, we find that RNA binding disrupts the MLL1 complex by competitively displacing WDR5 from these critical protein-protein interactions, leading to a marked inhibition of MLL1 catalytic activity. This regulatory mechanism provides a layer of control over histone methylation, potentially integrating transcriptional activity with chromatin state.

molecular biology↗

bioRxiv2024

A critical role for Pol II CTD phosphorylation in heterochromatic gene activation

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription. Here we demonstrate that such activation occurs in a substantial fraction of cells, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, CTD phosphorylation at Ser2, Ser5 and Ser7 is abundant at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated in ctk1{Delta} bur1 cells when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene - an activity that may help supplant the need for histone epigenetic modifications.

genetics↗

bioRxiv2022

Merging short and stranded long reads improves transcript assembly

Long-read RNA sequencing has arisen as a counterpart to short-read sequencing, with the potential to capture full-length isoforms, albeit at the cost of lower depth. Yet this potential is not fully realized due to inherent limitations of current long-read assembly methods and underdeveloped approaches to integrate short-read data. Here, we critically compare the existing methods and develop a new integrative approach to characterize a particularly challenging pool of low-abundance long noncoding RNA (lncRNA) transcripts from short- and long-read sequencing in two distinct cell lines. Our analysis reveals severe limitations in each of the sequencing platforms. For short-read assemblies, coverage declines at transcript termini resulting in ambiguous ends, and uneven low-coverage results in segmentation of a single transcript into multiple transcripts. Conversely, long-read transcript assembly lacks strand-of-origin information and depth, culminating in erroneous assembly and quantitation of transcripts. We also discover a cDNA synthesis artifact in long-read datasets that markedly impacts the identity and quantitation of assembled transcripts. Towards remediating these problems, we develop a computational pipeline to "strand" long-read cDNA libraries that rectifies inaccurate mapping and assembly of long-read transcripts. Leveraging the strengths of each platform and our computational stranding, we present and benchmark a hybrid assembly approach that drastically increases the sensitivity and accuracy of full-length transcript assembly on the correct strand and improves detection of biological features of the transcriptome. When applied to a challenging set of under-annotated and cell-type variable lncRNA, our method resolves the segmentation problem of short-read sequencing and the depth problem of long-read sequencing, resulting in the assembly of coherent transcripts with precise 5 and 3 ends. Our workflow can be applied to existing datasets for superior demarcation of transcript ends and refined isoform structure, which can enable better differential gene expression analyses and molecular manipulations of transcripts.

bioinformatics↗

bioRxiv2021

The cardiac lncRNA Chantico directly regulates Cxcl1 chemokine transcription

Withdrawal StatementThe corresponding author has withdrawn this preprint owing to inability to reproduce some of the data, instances of inappropriate data exclusion, and loss of much of the primary experimental records/data. Specifically, Figures 2B,E,H; 3; 4; 5A,B,D; 6; S2A,C; S4A; S5; and S6A,B and attendant text contain analyses for which the primary record and/or raw data no longer exist; the analyses, where still available, suffer from inappropriate data exclusion and thus should not be construed to be an accurate reflection of the experiments. Attempts by others in the lab to repeat several of the experiments in these indicated panels have failed reproduce the presented effects, despite showing much greater precision. Therefore the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

molecular biology↗

bioRxiv2021

Inducible transcriptional condensates drive 3D genome reorganization in the heat shock response

Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved, capable of stress-inducible gene activation or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor Heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, while an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become mammalian-like: constitutively active without intergenic coalescence. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.

molecular biology↗

bioRxiv2018

Heat Shock Factor 1 Dynamically Restructures the Yeast Genome

Transcriptional induction of Heat Shock Protein (HSP) genes in yeast is accompanied by dynamic changes in their 3D structure and spatial organization, yet the molecular basis for these phenomena remains unknown. Using chromosome conformation capture and single cell imaging, we show that genes transcriptionally activated by Heat Shock Factor 1 (Hsf1) specifically interact across chromosomes and coalesce into diffraction-limited intranuclear foci. Genes activated by the alternative stress regulators Msn2 and Msn4, in contrast, do not interact among themselves nor with Hsf1 targets. Likewise, constitutively expressed genes, even those interposed between HSP genes, show no detectable interaction. Hsf1 forms discrete subnuclear puncta when stress-activated, and these puncta dissolve in concert with transcriptional attenuation, paralleling the kinetics of HSP gene coalescence and dissolution. Nuclear Hsf1 and RNA Pol II are both necessary for intergenic HSP gene interactions, while DNA-bound Hsf1 is necessary and sufficient to drive coalescence of a heterologous gene. Our findings demonstrate that Hsf1 can dynamically restructure the yeast genome.

genetics↗