RNA Polymerase II during Transcript Elongation: deaiing with DNA damage and staying phosphorylated

This thesis covers two topics related to transcript elongation in Saccharomyces cerevisiae - the regulation of the phosphatase Fcp1 and transcription-coupled DNA damage repair. Formation of RNA polymerase II (RNAPII) complexes throughout the transcription cycle is mediated in part by the phosphorylation state of the C-terminal domain (CTD) of the largest RNAPII subunit. Although a multitude of kinases can phosphorylate the CTD, currently only one CTD-specific phosphatase, Fcp1, has been identified. This work studies the possibility that Fcp1 might be associated with the elongating form of RNAPII. The phosphatase co-fractionates with RNAPII in association with the elongation factor Elongator. Furthermore, genetic studies show that a double mutant that carries a deletion of an Elongator gene as well as a temperature sensitive fcp1 mutation has a synthetic lethal phenotype at the permissive temperature. In vitro assays using crude extracts demonstrate that the CTD of RNAPII becomes dephosphorylated in a Fcp1-dependent manner. In contrast, in a reconstituted DNA-RNA-RNAPII system, the addition of the purified phosphatase does not stimulate such dephosphoryation. These results indicate a close relationship between Fcp1 phosphatase and the elongating form of RNAPII. Transcription-coupled DNA damage repair is a term applied to the preferential repair of DNA damage on the coding strand within active genes. The second half of this thesis describes the characterisation of nucleotide excision repair (NER) of the intrastrand 1,3-(pGpTpG)-cisplatin lesion in Saccharomyces cerevisiae as well as an attempt to reconstitute a transcription-coupled NER reaction (TC-NER) in vitro. Using modified yeast extracts, the excision products of the above lesion by NER were found to be between 23 and 26 nucleotides long, via incisions around the 15th phosphodiester bond 3' and 7th the phosphodiester bond 5' of the damage. The attempt to reconstitute TC-NER in vitro was hindered by difficulties with the transcription substrate and the functional instability of purified NER proteins

Characteristics and Predictive Value of Blood Transcriptome Signature in Males with Autism Spectrum Disorders

Autism Spectrum Disorders (ASD) is a spectrum of highly heritable neurodevelopmental disorders in which known mutations contribute to disease risk in 20% of cases. Here, we report the results of the largest blood transcriptome study to date that aims to identify differences in 170 ASD cases and 115 age/sex-matched controls and to evaluate the utility of gene expression profiling as a tool to aid in the diagnosis of ASD. The differentially expressed genes were enriched for the neurotrophin signaling, long-term potentiation/depression, and notch signaling pathways. We developed a 55-gene prediction model, using a cross-validation strategy, on a sample cohort of 66 male ASD cases and 33 age-matched male controls (P1). Subsequently, 104 ASD cases and 82 controls were recruited and used as a validation set (P2). This 55-gene expression signature achieved 68% classification accuracy with the validation cohort (area under the receiver operating characteristic curve (AUC): 0.70 [95% confidence interval [CI]: 0.62–0.77]). Not surprisingly, our prediction model that was built and trained with male samples performed well for males (AUC 0.73, 95% CI 0.65–0.82), but not for female samples (AUC 0.51, 95% CI 0.36–0.67). The 55-gene signature also performed robustly when the prediction model was trained with P2 male samples to classify P1 samples (AUC 0.69, 95% CI 0.58–0.80). Our result suggests that the use of blood expression profiling for ASD detection may be feasible. Further study is required to determine the age at which such a test should be deployed, and what genetic characteristics of ASD can be identified

Clinical Sequencing Exploratory Research Consortium: Accelerating Evidence-Based Practice of Genomic Medicine

Despite rapid technical progress and demonstrable effectiveness for some types of diagnosis and therapy, much remains to be learned about clinical genome and exome sequencing (CGES) and its role within the practice of medicine. The Clinical Sequencing Exploratory Research (CSER) consortium includes 18 extramural research projects, one National Human Genome Research Institute (NHGRI) intramural project, and a coordinating center funded by the NHGRI and National Cancer Institute. The consortium is exploring analytic and clinical validity and utility, as well as the ethical, legal, and social implications of sequencing via multidisciplinary approaches; it has thus far recruited 5,577 participants across a spectrum of symptomatic and healthy children and adults by utilizing both germline and cancer sequencing. The CSER consortium is analyzing data and creating publically available procedures and tools related to participant preferences and consent, variant classification, disclosure and management of primary and secondary findings, health outcomes, and integration with electronic health records. Future research directions will refine measures of clinical utility of CGES in both germline and somatic testing, evaluate the use of CGES for screening in healthy individuals, explore the penetrance of pathogenic variants through extensive phenotyping, reduce discordances in public databases of genes and variants, examine social and ethnic disparities in the provision of genomics services, explore regulatory issues, and estimate the value and downstream costs of sequencing. The CSER consortium has established a shared community of research sites by using diverse approaches to pursue the evidence-based development of best practices in genomic medicine

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The most abundant transcripts in VCM populations.

<p>A) The Venn diagram shows the distribution of the top 200 most abundant genes in hESC-<b>V</b>CMs, hF-<b>V</b>CMs and hA-<b>V</b>CMs. The number of genes in common is indicated. The labels refer to biological processes that are enriched. B) The tissue-specific distribution of the top 200 genes was examined in public databases including Genenote and bioGPS. The ‘cardiac-specific’ genes among the top 200 most abundant genes are shown. C) 27 cardiac-specific genes within the top 200 most abundant genes in hESC-<b>V</b>CMs, hF-<b>V</b>CMs and/or hA-<b>V</b>CMs. Fold change relative to hESC-VCMs are indicated and fold change >10 is highlighted in bold. Possible function is based on gene ontology association. </p

Developmental classification of hESC-VCMs.

<p>A) A representative FACS plot showing that approximately 55% of hESC-derivatives were positive for cardiac troponin-T (cTnT) prior to selection. B) Selected MLC2V-mCherry+ hESC-<b>V</b>CMs (~95% purified) under brightfield and fluorescence microscopy (100x). C) Purified HESC-<b>V</b>CMs were stained with anti-cTnT and anti-α-actinin antibodies. D) Representative action potential of hESC-<b>V</b>CMs. E) Hierarchical clustering showing that biological replicates cluster together. Red and green indicates up- and down-regulation respectively. F) Principal Component Analysis. HESC-<b>V</b>CMs, hF-<b>V</b>CMs and hA-<b>V</b>CMs lie along a linear developmental axis (dotted black line). Correlation coefficient is shown to indicate the degree of linearity. G) An activation isochronal map of trans-membrane potential with 15ms intervals. H) CX43 staining of hESC-<b>V</b>CMs. Red= CX43 staining, blue=DAPI.</p