Mediator links transcription and DNA repair by facilitating Rad2/XPG recruitment.

Mediator is a large multiprotein complex conserved in all eukaryotes. The crucial function of Mediator in transcription is now largely established. However, we found that this complex also plays an important role by connecting transcription with DNA repair. We identified a functional contact between the Med17 Mediator subunit and Rad2/XPG, the 3' endonuclease involved in nucleotide excision DNA repair. Genome-wide location analyses revealed that Rad2 is associated with RNA polymerase II (Pol II)- and Pol III-transcribed genes and telomeric regions in the absence of exogenous genotoxic stress. Rad2 occupancy of Pol II-transcribed genes is transcription-dependent. Genome-wide Rad2 occupancy of class II gene promoters is well correlated with that of Mediator. Furthermore, UV sensitivity of med17 mutants is correlated with reduced Rad2 occupancy of class II genes and concomitant decrease of Mediator interaction with Rad2 protein. Our results suggest that Mediator is involved in DNA repair by facilitating Rad2 recruitment to transcribed genes.

Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons.

The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting ß-cells and neurons and underline the existence of trans­nosology pathways in diabetes and its co-morbidities.

Natural variation of histone modification and its impact on gene expression in the rat genome.

Histone modifications are epigenetic marks that play fundamental roles in many biological processes including the control of chromatin-mediated regulation of gene expression. Little is known about interindividual variability of histone modification levels across the genome and to what extent they are influenced by genetic variation. We annotated the rat genome with histone modification maps, identified differences in histone trimethyl-lysine levels among strains, and described their underlying genetic basis at the genome-wide scale using ChIP-seq in heart and liver tissues in a panel of rat recombinant inbred and their progenitor strains. We identified extensive variation of histone methylation levels among individuals and mapped hundreds of underlying cis- and trans-acting loci throughout the genome that regulate histone methylation levels in an allele-specific manner. Interestingly, most histone methylation level variation was trans-linked and the most prominent QTL identified influenced H3K4me3 levels at 899 putative promoters throughout the genome in the heart. Cis- acting variation was enriched in binding sites of distinct transcription factors in heart and liver. The integrated analysis of DNA variation together with histone methylation and gene expression levels showed that histoneQTLs are an important predictor of gene expression and that a joint analysis significantly enhanced the prediction of gene expression traits (eQTLs). Our data suggest that genetic variation has a widespread impact on histone trimethylation marks that may help to uncover novel genotype-phenotype relationships.

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway.

The yeast RPL9B gene is regulated by modulation between two modes of transcription termination.

RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.

Futile cycle of transcription initiation and termination modulates the response to nucleotide shortage in S. cerevisiae.

Hidden transcription in eukaryotes carries a large potential of regulatory functions that are only recently beginning to emerge. Cryptic unstable transcripts (CUTs) are generated by RNA polymerase II (Pol II) and rapidly degraded after transcription in wild-type yeast cells. Whether CUTs or the act of transcription without RNA production have a function is presently unclear. We describe here a nonconventional mechanism of transcriptional regulation that relies on the selection of alternative transcription start sites to generate CUTs or mRNAs. Transcription from TATA box proximal start sites generates unstable transcripts and downregulates expression of the URA2 gene under repressing conditions. Uracil deprivation activates selection of distal start sites, leading to the production of stable mRNAs. We describe the elements that govern degradation of the CUT and activation of mRNA production by downstream transcription initiation. Importantly, we show that a similar mechanism applies to other genes in the nucleotides biogenesis pathway.

Widespread bidirectional promoters are the major source of cryptic transcripts in yeast.

Pervasive and hidden transcription is widespread in eukaryotes, but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae. These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1-exosome-TRAMP complexes. Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 3' Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 5' end. Our study of glycolysis genes, as well as recent results from the literature, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

What are the consequences of over and undertreatment of type 2 diabetes mellitus in a frail population? A systematic review.

AIMS: This review aims to identify the evidence base for the consequences of over and undertreatment of type 2 diabetes mellitus in a frail population. METHOD: In this systematic review, we searched MEDLINE, Embase, PubMed, CINAHL and the Cochrane Library for studies from January 2001 to 15th August 2022. We included a variety of study types that assessed and reported frailty including patients ≥18 years old. Studies included those that reported the prevalence of over or undertreatment of diabetes mellitus in a frail population and those examining outcomes related to glucose control in frail older people living with diabetes. Data were extracted using a bespoke extraction table using a narrative synthesis approach. RESULTS: A total of 4114 articles were identified with 112 meeting inclusion criteria. These included 15,130 participants across the 11 studies with sample sizes ranging from 101 to 11,140. Several areas were identified in the included studies where under or overtreatment of diabetes impacted outcomes for patients. These included hospital admissions, readmissions, length of stay, falls, mortality, cognitive impairment and cardiovascular disease outcomes. CONCLUSION: The results showed that there was a high heterogeneity of outcomes between the studies and that many examined small numbers of participants. In this review, both over and undertreatment were shown to increase adverse outcomes in frail older people. Further research around optimal glycaemic control for frail older people living with diabetes is required with the aim to identify ideal target ranges and produce practical clinical guidelines to promote attainment of these.

Stroke Rehabilitation.

Despite contemporary rehabilitation strategies, stroke remains a leading cause of loss of function, limited mobility, psycho-social complications, and decreased quality of life. Stroke rehabilitation is a process that aims to prevent deterioration of function, increase function, and assist the patient in achieving the highest possible level of independence physically, socially, spiritually, psychologically, vocationally, and economically. The process begins with relearning activities of daily living such as grooming, bathing, toileting, eating, and dressing. As the patient progresses, stroke rehabilitation works on instrumental activities of daily living such as housekeeping, cooking, driving, and managing financial responsibilities.

Natural cycles in South Pacific Gyre strength and the Southern Annular Mode.

The South Pacific Gyre (SPG) plays a vital role in regulating Southern Hemisphere climate and ecosystems. The SPG has been intensifying since the twentieth century due to changes in large scale wind forcing. These changes result from variability in the Southern Annular Mode (SAM), causing warming along the eastern SPG which affects local ecosystems. However, our understanding of SPG variability on timescales greater than several decades is poor due to limited observations. Marine sediment cores are traditionally used to determine if recent ocean trends are anomalous, but rarely capture centennial variability in the southwest Pacific and limit our understanding of SPG variability. Here we capture centennial SPG dynamics using a novel high-resolution paleocirculation archive: radiocarbon reservoir ages (R) and local reservoir corrections (∆R) in SPG deep-sea black corals. We find black coral R and ∆R correlates with SAM reconstructions over 0-1000 cal BP and 2000-3000 cal BP. We propose this correlation indicates varying transport of well-ventilated subtropical waters resulting from SPG and SAM interactions. We reconstruct several 'spin up' cycles reminiscent of the recent gyre intensification, which has been attributed to anthropogenic causes. This implies gyre strength and SAM show natural co-variability on anthropogenic timescales which should factor into future climate projections.

Ultra-low Ultraviolet Radiation in Office Lighting Can Moderate Seasonal Vitamin D Cycle: A Pilot Study.

BACKGROUND/AIM: Ultraviolet-B (UV-B) radiation initiates vitamin D synthesis in the skin, making sun exposure a major source of vitamin D. We aimed to determine whether office lighting containing ultra-low levels of UV-B radiation could modify the winter decline in vitamin D status in the UK, while being safe and well tolerated. PATIENTS AND METHODS: Twenty commercial office desk lamps were modified with the addition of UV-B LEDs. Ten hospital office administrative staff received UV-modified lamps with UV-on, and 10 staff received identical placebo lamps with UV switched off, in a double-blind, cross-over pilot study during the winter of 2021/22. Circulating 25-hydroxyvitamin D [25(OH)D] was measured every 4 weeks for 20 weeks: at baseline and during an 8-week trial period, 4-week washout, and a cross-over 8-week trial period. RESULTS: The linear regression combining the complete datasets for phase 1 and 2 of the trial showed that an 8-week UV light intervention significantly increased 25OHD by 7.13 nmol/l with a p-Value=0.02, compared to the placebo group. Similar results were confirmed by cross-over analyses using the datasets of those completing both phases of the trial both with and without using the inverse probability weighing method to handle dropouts. CONCLUSION: The UV-B-modified lighting was well-tolerated and safe with weekly doses of UV-B of 0.5 - 0.9 Standard Erythema Dose [SED=100 Jm-2 erythema weighted UV radiation] measured at chest level. This ultra-low dosing was effective in reducing the winter decline in vitamin D status.

Rapid proteomic analysis for solid tumors reveals LSD1 as a drug target in an end-stage cancer patient.

Recent advances in mass spectrometry (MS)-based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine-specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can be applied broadly to other patients in a similar condition.

RNA helicases DDX5 and DDX17 dynamically orchestrate transcription, miRNA, and splicing programs in cell differentiation.

The RNA helicases DDX5 and DDX17 are members of a large family of highly conserved proteins that are involved in gene-expression regulation; however, their in vivo targets and activities in biological processes such as cell differentiation, which requires reprogramming of gene-expression programs at multiple levels, are not well characterized. Here, we uncovered a mechanism by which DDX5 and DDX17 cooperate with heterogeneous nuclear ribonucleoprotein (hnRNP) H/F splicing factors to define epithelial- and myoblast-specific splicing subprograms. We then observed that downregulation of DDX5 and DDX17 protein expression during myogenesis and epithelial-to-mesenchymal transdifferentiation contributes to the switching of splicing programs during these processes. Remarkably, this downregulation is mediated by the production of miRNAs induced upon differentiation in a DDX5/DDX17-dependent manner. Since DDX5 and DDX17 also function as coregulators of master transcriptional regulators of differentiation, we propose to name these proteins "master orchestrators" of differentiation that dynamically orchestrate several layers of gene expression.

Enrichment of unstable non-coding RNAs and their genome-wide identification.

Cryptic unstable transcripts (CUTs) have been recently described as a major class of non-coding RNAs. These transcripts are, however, extremely unstable in normal cells and their analyzes pose specific technical problems. In this chapter, after a brief introduction discussing general aspects associated with the analysis of non-coding RNAs, we provide details of methods to enrich, map, and quantify this unconventional class of transcripts.

Sck1 activator coordinates glucose transport and glycolysis and is controlled by Rag8 casein kinase I in Kluyveromyces lactis.

Casein kinases I (CKI) are ubiquitous in eukaryotic cells and are crucial factors for nutrient-signalling pathways in yeasts. In Kluyveromyces lactis, the KlRgt1 repressor represses the expression of the glucose transporter RAG1 gene in absence of glucose, but in response to glucose availability, Rag8 CKI cooperates with the Rag4 glucose sensor to inactivate KlRgt1. The SCK1 gene, a rag8 mutation suppressor, encodes a bHLH activator required for maximal expression of the RAG1 and glycolytic genes in the presence of glucose. We investigated further the function of Sck1 and its relationship to Rag8. We demonstrated that Sck1 regulates the RAG1 and glycolytic genes by directly binding to their promoter. We also found that SCK1 gene expression was induced by glucose and repressed by KlRgt1. In addition, we showed that (i) Sck1 was phosphorylated in vivo, (ii) Sck1 was phosphorylated in vitro by Rag8, and (iii) Sck1 was rapidly degraded in a rag8 mutant. We therefore suggest that Sck1 coordinates glucose import and glycolysis in K. lactis and that Rag8 controls this transcription factor by transcriptional and post-translational regulations.

Mutations of the RAG3 gene encoding a regulator of fermentation in Kluyveromyces lactis are suppressed by a mutation of the transcription factor gene KlGCR1.

In Kluyveromyces lactis, Rag3 regulates both fermentative metabolism and thiamine biosynthesis. Regulation of fermentation is exerted at the level of transcription of KlPDC1. We have isolated and identified a mutation of the transcription factor KlGCR1, Klgcr1-1, which suppressed the fermentative-deficient phenotype associated with the RAG3 deletion. In the mutant, the transcription of KlPDC1 was restored. However, we found that the suppression was not specific to the RAG3 mutation, as the Klgcr1-1 mutation could also suppress the fermentative defect associated with mutation of Sck1, another regulator of glycolysis.