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DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage., (© 2024 Ajit et al.; Published by Cold Spring Harbor Laboratory Press.)

Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

DNA is constantly exposed to endogenous and exogenous damage. Various types of DNA repair counteract highly toxic DNA double-strand breaks (DSBs) to maintain genome stability. Recent findings suggest that the human DNA damage response (DDR) utilizes small RNA species, which are produced as long non-coding (nc)RNA precursors and promote recognition of DSBs. However, regulatory principles that control production of such transcripts remain largely elusive. Here we show that the Abelson tyrosine kinase c-Abl/ABL1 causes formation of RNA polymerase II (RNAPII) foci, predominantly phosphorylated at carboxy-terminal domain (CTD) residue Tyr1, at DSBs. CTD Tyr1-phosphorylated RNAPII (CTD Y1P) synthetizes strand-specific, damage-responsive transcripts (DARTs), which trigger formation of double-stranded (ds)RNA intermediates via DNA-RNA hybrid intermediates to promote recruitment of p53-binding protein 1 (53BP1) and Mediator of DNA damage checkpoint 1 (MDC1) to endogenous DSBs. Interference with transcription, c-Abl activity, DNA-RNA hybrid formation or dsRNA processing impairs CTD Y1P foci formation, attenuates DART synthesis and delays recruitment of DDR factors and DSB signalling. Collectively, our data provide novel insight in RNA-dependent DDR by coupling DSB-induced c-Abl activity on RNAPII to generate DARTs for consequent DSB recognition., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates transcription and coordinates it with pre-mRNA processing. We show here that Rct1, a nuclear multidomain cyclophilin from Schizosaccharomyces pombe, is encoded by an essential gene that interacts with the CTD and regulates its phosphorylation in vivo. Downregulation of Rct1 levels results in increased phosphorylation of the CTD at both Ser2 and Ser5 and in a commensurate decrease in RNAP II transcription. In contrast, overexpression of Rct1 decreases phosphorylation on both sites. The close association of Rct1 with transcriptionally active chromatin suggests a role in regulation of RNAP II transcriptional activity. These data, together with the pleiotropic phenotype upon Rct1 deregulation, suggest that this multidomain cyclophilin is an important player in maintaining the correct phosphorylation code of the CTD and thereby regulating CTD function.

AtCyp59 and its orthologs from different organisms belong to a family of modular proteins consisting of a peptidyl-prolyl cis-trans isomerase (PPIase) domain, followed by an RNA recognition motif (RRM), and a C-terminal domain enriched in charged amino acids. AtCyp59 was identified in a yeast two-hybrid screen as an interacting partner of the Arabidopsis SR protein SCL33/SR33. The interaction with SCL33/SR33 and with a majority of Arabidopsis SR proteins was confirmed by in vitro pull-down assays. Consistent with these interactions, AtCyp59 localizes to the cell nucleus, but it does not significantly colocalize with SR proteins in nuclear speckles. Rather, it shows a punctuate localization pattern resembling transcription sites. Indeed, by using yeast two-hybrid, in vitro pull-down, and immunoprecipitation assays, we found that AtCyp59 interacts with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. Ectopic expression of the tagged protein in Arabidopsis cell suspension resulted in highly reduced growth that is most probably due to reduced phosphorylation of the CTD. Together our data suggest a possible function of AtCyp59 in activities connecting transcription and pre-mRNA processing. We discuss our data in the context of a dynamic interplay between transcription and pre-mRNA processing.

Genotoxic stress resulting from DNA damage is resolved through a signaling cascade known as the DNA Damage Response (DDR). The repair of damaged DNA is essential for cell survival, often requiring the DDR to attenuate other cellular processes such as the cell cycle, DNA replication, and transcription of genes not involved in DDR. The complex relationship between DDR and transcription has only recently been investigated. Transcription can facilitate the DDR in response to double-strand breaks (DSBs) and stimulate nucleotide excision repair (NER). However, transcription may need to be reduced to prevent potential interference with the repair machinery. In this review, we discuss various mechanisms that regulate transcription repression in response to different types of DNA damage, categorizing them by their range and duration of effect. Finally, we explore various models of transcription recovery following DNA damage-induced repression. [ABSTRACT FROM AUTHOR]