Biology subjects

Vihervaara, A.

Publications and source records attributed to Vihervaara, A..

12 recordsLinked to original sources

bioRxiv2026

BAF perturbation augments cancer cell dependence on CDK12-driven transcription elongation by RNA polymerase II

CDK12 facilitates transcriptional elongation and processivity by RNA polymerase II (Pol II). Although it has emerged as a promising actor and target in cancer, better understanding of CDK12 gene transcription control could inform the design of novel anti-cancer strategies. Here, we identify a co-dependency between CDK12 and the BAF chromatin-remodeling complex in triple-negative breast cancer (TNBC). Genome-scale CRISPR interference screening revealed multiple BAF subunits as strong dependencies upon CDK12 inhibition. In turn, pharmacological co-targeting of CDK12 and BAF ATPase synergistically reduced the viability of multiple TNBC models. Mechanistically, the co-inhibition attenuated di-phosphorylation of the Pol II C-terminal domain at Serine-2 and Serine-5 and depleted canonical pre-mRNA 3'-end processing factors from chromatin, driving accumulation of chromatin-associated Pol II, upstream of apoptosis. Whereas BAF inhibition rapidly reduced transcription-coupled DNA accessibility, CDK12 inhibition imposed a gene length-biased transcriptional defect exacerbated by BAF deficiency. In turn, co-inhibited cells, marked by synergistic induction of MYC and repression of long cell-cycle and mitotic genes, showed a failure of DNA synthesis and mitotic entry, culminating in MYC-mediated apoptosis. Together, our findings reveal a regulatory axis in which impaired Pol II elongation creates a heightened requirement for BAF-dependent chromatin remodeling. This discovered co-dependency provides a rationale for co-targeting the CDK12-BAF axis in transcriptionally addicted cancers.

molecular biology↗

bioRxiv2025

Transcriptional responses to proteotoxic stressors are profoundly diverse and tissue-specific

Cells counteract proteotoxic conditions by launching transcriptional stress responses. While synthesis of Heat shock proteins (HSPs) upon acute stress is well-characterized, how distinct proteotoxic conditions reshape the transcriptome remains poorly understood. Here, we analyse polyA+ RNA expression under heat shock, HSP90 inhibition, and polyglutamine (polyQ) aggregation. We find fundamentally distinct transcriptional responses to proteotoxic stressors, and a systemic deficiency of mice under chronic stress to launch acute responses. While heat shock and HSP90 inhibition induce chaperones, polyQ aggregation increases RNAs linked to transcription repression, chromatin remodeling, and autophagy. Analysing wildtype and Huntingtons Disease (HD) mice reveals tissue-specific transcriptional adaptations to polyQ, including repressed cell-type specific functions and altered energy metabolism. Despite profound reprogramming, remarkably few RNAs are consistently induced (Acy3, Abdh1, Tmc3) or reduced (Fos) across HD brain regions. These results emphasize cellular background in disease manifestation, and support energy metabolism and detoxifying enzymes as therapeutic targets in late-stage HD. Moreover, the systemic deficiency of chronically stressed mice to launch responses challenges strategies that rely on induced transcription. Altogether, we characterize transcription signatures to proteotoxic stresses, identify key trans-activators driving proteotoxic stress responses, provide an interactive gene-by-gene viewer of global changes, and delineate tissue-specific transcription programs in HD mice.

cell biology↗

bioRxiv2025

Transcriptional architecture and Pol II regulation at promoters, enhancers, and enhancer clusters in Canis lupus familiaris

Domestic dog exhibits remarkable phenotypic diversity and provides versatile models for genomics, evolution, and complex traits. DNA sequences and stable RNAs have revealed regulatory regions in the dog genome. However, transcriptional activity, regulatory architecture, and control of RNA Polymerase II (Pol II) across genes and enhancers remain uncharacterized. Here, we track transcription at nucleotide-resolution, measure RNA expression and stability, and analyse mechanisms of Pol II regulation in golden retriever macrophages. We report the precise architectures of promoters, enhancers, and enhancer clusters, and quantify Pol II progression from the initiation, through the pause-region, into elongation and termination. Triggering transcriptional change by heat stress reveals instant reprogramming of genes via promoter-proximal pause-regulation and enhancers via initiation. Enhancers within a cluster mount a unified response. This study identifies functional genomic regions de novo, characterizes transcriptional architectures of genes, enhancers, and enhancer clusters, quantifies RNA synthesis and stability, and reveals mechanisms of transcription in Canis lupus familiaris.

genomics↗

bioRxiv2024

Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

Transcriptional reprogramming drives differentiation and coordinates cellular responses. While mRNA expression in distinct cell types has been extensively analyzed, the mechanisms that control RNA synthesis upon lineage specifications remain unclear. Here, we induce erythroid differentiation in human cells, track transcription and its regulation at nucleotide-resolution, and identify molecular mechanisms that orchestrate gene and enhancer activity during erythroid specification. We uncover waves of transcription and reveal that a brief differentiation signal launches sustained and propagating changes in RNA synthesis and mRNA expression over cell divisions. NRF2, a strong trans-activator upon oxidative stress, drives erythroid differentiation without a detectable increase in reactive oxygen species. In erythroid precursors, NRF2 induces architecturally primed, differentiation-linked enhancers, and genes encoding globin and antioxidant proteins. Projecting signal-induced transcription to DNA accessibility and mRNA expression in single human bone marrow cells, reveals ordered activation of myeloid (GABPA) and erythroid (GATA1, TAL1 and HEMGN) factors in lineage-specification, followed by NRF2-triggered antioxidant response in the late erythroid cells. This study establishes molecular mechanisms that prime, execute, and temporally coordinate RNA synthesis during erythroid differentiation. Furthermore, we show that master regulators of differentiation and stress co-orchestrate erythropoiesis and produce the antioxidant machinery before erythroid cells mature to oxygen transporting enucleated erythrocytes.

genomics↗

bioRxiv2023

Characterization of gene regulatory elements and dynamic antimicrobial immune responses in mosquito cells using PRO-seq

Aedes aegypti mosquitoes are the principal vectors for epidemic arboviruses, including dengue, Zika, and chikungunya viruses. In these insect vectors, the concerted action of immune pathways shapes virus replication and transmission; yet, mechanistic and dynamic understanding of the transcriptional immune responses in mosquitoes remain limited. To address this knowledge gap, we profiled nascent transcription in mosquito cells and midgut tissue using Precision Run-On sequencing (PRO-seq). We identified exact transcription start nucleotides (TSNs), characterized promoter architectures, and generated the first genome-wide list of putative enhancers in Aedes aegypti, substantially expanding the regulatory annotation of this non-model organism. To investigate dynamic immune responses, we stimulated Aag2 cells with heat-inactivated E. coli and measured RNA synthesis and mRNA levels from matched samples. Bacterial stimulation induced rapid and temporally organized transcriptional responses associated with distinct biological functions and transcription factor motifs. Integration with mRNA-seq revealed that temporal patterns of mRNA accumulation were shaped by RNA synthesis, gene length, and stability, indicating multilayered regulation of mosquito immune responses. Implementing PRO-seq for mosquito midgut enabled characterization of tissue-specific transcription and TSN usage. Importantly, we demonstrate for the first time that PRO-seq captures in vivo microbial transcription within an organism, co-profiling host and microbiome nascent RNA synthesis. Altogether, this study substantially expands the regulatory annotation and functional genomic landscape of Aedes aegypti, reveals multilayered regulation of mosquito immune responses, and extends transcriptional profiling to host and microbiome activity in vivo. These insights advance understanding of gene regulation and immunity in this important arbovirus vector.

molecular biology↗

bioRxiv2023

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in heat shock protein (hsp) transcription in fly ovaries, but the mechanisms remain unclear. Here we demonstrate that in mammalian cells, MRTFs are dispensable for hsp gene induction. However, the widely used small molecule inhibitors of MRTF/SRF transcription pathway, derived from CCG-1423, efficiently inhibit hsp gene transcription in both fly and mammalian cells also in absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF/SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

molecular biology↗

bioRxiv2023

PRO-IP-seq Tracks Molecular Modifications of Engaged Pol II Complexesat Nucleotide Resolution

RNA Polymerase II (Pol II) is a multi-subunit complex that undergoes covalent modifications as transcription proceeds through genes and enhancers. Rate-limiting steps of transcription control Pol II recruitment, site and degree of initiation, pausing duration, productive elongation, nascent transcript processing, transcription termination, and Pol II recycling. Here, we developed Precision Run-On coupled to Immuno-Precipitation sequencing (PRO-IP-seq) and tracked phosphorylation of Pol II C-terminal domain (CTD) at nucleotide-resolution. We uncovered precise positional control of Pol II CTD phosphorylation as transcription proceeds from the initiating nucleotide, through early and late promoter-proximal pause, and into productive elongation. Pol II CTD was predominantly unphosphorylated in the early pause-region, whereas serine-2- and serine-5-phosphorylations occurred preferentially in the later pause-region. Serine-7-phosphorylation dominated after the pause-release in a region where Pol II accelerates to its full elongational speed. Interestingly, tracking transcription upon heat-induced reprogramming demonstrated that Pol II with phosphorylated CTD remains paused on heat-repressed genes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/527107v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1cd08c7org.highwire.dtl.DTLVardef@1fb7507org.highwire.dtl.DTLVardef@1c05c1borg.highwire.dtl.DTLVardef@1deffdf_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2021

HSFs drive stress type-specific transcription of genes and enhancers

Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 and HSF2 trans-activate genes independently of each other, demonstrating, for the first time, that HSF2 is a bona fide transcription factor. Taken together, we show that HSFs function as multi-stress-responsive factors that activate specific genes and enhancers when encountering changes in temperature and redox state.

molecular biology↗

bioRxiv2021

Quantifying RNA Synthesis at Rate-Limiting Steps of Transcription Using Nascent RNA-Sequencing Data

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide data-analytical steps to 1) identify transcribed regulatory elements de novo genome-wide, 2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies and termination windows, and 3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol follows RNA synthesis and genome-regulation in mammals, as demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara, et al., 2021. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=128 HEIGHT=200 SRC="FIGDIR/small/454856v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@18301b2org.highwire.dtl.DTLVardef@1e4636forg.highwire.dtl.DTLVardef@13c1fd8org.highwire.dtl.DTLVardef@4eb935_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2019

Stress-Induced Transcriptional Memory Accelerates Promoter-Proximal Pause-Release and Decelerates Termination over Mitotic Divisions

Heat shock triggers an instant reprogramming of gene and enhancer transcription, but whether cells encode a memory to stress, at the level of nascent transcription, has remained unknown. Here, we measured transcriptional response to acute heat stress in unconditioned cells and in daughters of cells that had been exposed to a single or multiple heat shocks. Tracking RNA Polymerase II (Pol II) genome-wide at nucleotide-resolution revealed that cells precisely remember their transcriptional identity throughout stress, restoring Pol II distribution at gene bodies and enhancers upon recovery. However, single heat shock primed faster gene-induction in the daughter cells by increasing promoter-proximal Pol II pausing, and accelerating the pause-release. In repeatedly stressed cells, both basal and inducible transcription was refined, and pre-mRNA processing decelerated, which retained transcripts on chromatin and reduced recycling of the transcription machinery. These results mechanistically uncovered how the steps of pause-release and termination maintain transcriptional memory over mitosis.\n\nHighlights-Cell type-specific transcription precisely recovers after heat-induced reprogramming\n-Single heat shock primes genes for accelerated induction over mitotic divisions via increased promoter-proximal Pol II pausing and faster pause-release\n-Multiple heat shocks refine basal and inducible transcription over mitotic divisions to support survival of the daughter cells\n-Decelerated termination at active genes reduces recycling of Pol II to heat-activated promoters and enhancers\n-HSF1 increases the rate of promoter-proximal pause-release via distal and proximal regulatory elements

molecular biology↗

bioRxiv2019

Chromatin conformation remains stable upon extensive transcriptional changes driven by heat shock

Heat shock (HS) initiates rapid, extensive, and evolutionarily conserved changes in transcription that are accompanied by chromatin decondensation and nucleosome loss at heat shock loci. Here we have employed in situ Hi-C to determine how heat stress affects long-range chromatin conformation in human and Drosophila cells. We found that compartments, topologically-associated domains (TADs), and looping interactions all remain unchanged by an acute HS. Knockdown of Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the HS response, identified HSF1-dependent genes and revealed that up-regulation is often mediated by distal HSF1 bound enhancers. HSF1-dependent genes were usually found in the same TAD as the nearest HSF1 binding site. However, the HSF1 binding sites and the target promoters did not exhibit a focal increase in contact frequencies compared with surrounding regions, nor did we find evidence of increased contact frequency following HS. Integrating information about HSF1 binding strength, RNA polymerase abundance at the enhancer, and contact frequency with a target promoter in the non-heat shock (NHS) condition accurately predicted which up-regulated genes were direct targets of HSF1 during HS. Our results suggest that the chromatin conformation necessary for a robust HS response is pre-established in normal (uninduced) cells of diverse metazoan species.

molecular biology↗

bioRxiv2018

CBP/EP300-dependent acetylation and stabilization of HSF2 are compromised in the rare disorder, Rubinstein-Taybi syndrome

Cells respond to protein-damaging insults by activating heat shock factors (HSFs), key transcription factors of proteostasis. Abnormal HSF protein levels occur in cancer and neurodegenerative disorders, highlighting the importance of the tight control of HSF expression. HSF2 is a short-lived protein, but it is abundant in the prenatal brain cortex and required for brain development. Here, we reveal that HSF2 is acetylated and co-localized with the lysine-acetyl transferases CBP and EP300 in human brain organoids. Using unbiased, biochemical, cell-imaging, and in silico approaches, we show that CBP/EP300 acetylates HSF2 at specific lysine residues, which promotes HSF2 stabilization, whereas the lysine deacetylase HDAC1 catalyzes its proteasomal degradation. The CBP KIX domain and KlX-recognizing motifs in HSF2 are critical for its interaction with acetylating enzymes. The functional importance of acetylated HSF2 is evidenced in Rubinstein-Taybi syndrome (RSTS), characterized by mutated CBP or EP300. We show that RSTS patient cells exhibit decreased HSF2 levels and impaired heat shock response. The dysregulated HSF pathway in RSTS opens new avenues for understanding the molecular basis of this multifaceted pathology.

developmental biology↗