The GATAD2B-NuRD complex drives DNA:RNA hybrid-dependent chromatin boundary formation upon DNA damage.

Double-strand breaks (DSBs) are the most lethal form of DNA damage. Transcriptional activity at DSBs, as well as transcriptional repression around DSBs, are both required for efficient DNA repair. The chromatin landscape defines and coordinates these two opposing events. However, how the open and condensed chromatin architecture is regulated remains unclear. Here, we show that the GATAD2B-NuRD complex associates with DSBs in a transcription- and DNA:RNA hybrid-dependent manner, to promote histone deacetylation and chromatin condensation. This activity establishes a spatio-temporal boundary between open and closed chromatin, which is necessary for the correct termination of DNA end resection. The lack of the GATAD2B-NuRD complex leads to chromatin hyperrelaxation and extended DNA end resection, resulting in homologous recombination (HR) repair failure. Our results suggest that the GATAD2B-NuRD complex is a key coordinator of the dynamic interplay between transcription and the chromatin landscape, underscoring its biological significance in the RNA-dependent DNA damage response.

Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.

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.

Age-related changes in gonadotropin-releasing hormone (GnRH) splice variants in mouse brain.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of the mammalian reproductive axis. We investigated the spatiotemporal expression of GnRH splice variants (V1, V2, and V3) and splicing factors (Srsf7, Srsf9, and Tra-2) in the male mice brain. Further, using in silico tools, we predicted protein structure and the reason for the low translational efficiency of V2 and V3. Messenger RNA levels of GnRH variants and splicing factors were quantified using real-time reverse transcription-polymerase chain reaction at different age groups. Our data show that expression of almost all the variants alters with aging in all the brain regions studied; even in comparison to the hypothalamus, several brain areas were found to have higher expression of these variants. Hypothalamic expression of splicing factors such as Srsf7, Srsf9, and Tra-2 also change with aging. Computational studies have translation repressors site on the V3, which probably reduces its translation efficiency. Also, V2 is an intrinsically disordered protein that might have a regulatory or signaling function. In conclusion, this study provides novel crucial information and multiple starting points for future analysis of GnRH splice variants in the brain.

Lactate-Dependent Cross-Talk Between Astrocyte and GnRH-I Neurons in Hypothalamus of Aged Brain: Decreased GnRH-I Transcription.

GnRH-I produced by hypothalamic neurosecretory cells is considered a master regulator of mammalian reproduction. Although GnRH-I transcription is well studied, the effect of ageing on transcriptional regulation of GnRH-I has not yet been explored. Here, we elucidate the effects of ageing on the metabolic environment like lactate level and TNF-α and how these affect GnRH-I transcription. Using pathway analysis of transcriptomic data, we found that lactate is upregulated in ageing astrocytes due to the downregulation of cellular respiration pathways possibly resulting in greater pyruvate concentration for lactate production. This lactate could then be shuttled into neurons where it would affect GnRH-I transcription. We showed that supra-physiological level of lactate in young mouse brain can mimic metabolic disturbances in the old brain and cause downregulation in GnRH-I transcription at a young age. In particular, we found upregulation of GnRH-I repressors in the young brain treated with high levels of lactate similar to old brain. Hence, this confirmed that aged metabolic environment can affect GnRH-I transcription even in the young brain. Further downstream analysis using the TRUST database showed NF-Kb signalling which lies downstream of both lactate and TNF-α as being capable of upregulating GnRH-I repressors. Since NF-Kb signalling has been shown in our study as well as others to be induced by TNF-α during ageing, it is likely that GnRH-I transcriptional regulation is mediated through these pathways. Thus, we formed a model for explaining the downregulation of GnRH-I transcription during ageing through differential expression of its TFs in an aged metabolic environment.

Elucidating evolutionarily conserved mechanisms of diapause regulation using an in silico approach.

Embryonic diapause is an enigmatic phenomenon that appears in diverse species. Although regulatory mechanisms have been established, there is much to be discovered. Herein, we have made the first comprehensive attempt to elucidate diapause regulatory mechanisms using a computational approach. We found transcription factors unique to promoters of genes in diapause species. From pathway analysis and STRING PPI networks, the signaling pathways regulated by these unique transcription factors were identified. The pathways were then consolidated into a model to combine various known mechanisms of diapause regulation. This work also highlighted certain transcription factors that may act as 'master transcription factors' to regulate the phenomenon. Promoter analysis further suggested evidence for independent evolution for some of regulatory elements involved in diapause.