Frequent Missense and Insertion/Deletion Polymorphisms in the Ovine Shadoo Gene Parallel Species-Specific Variation in PrP

BACKGROUND: The cellular prion protein PrP(C) is encoded by the Prnp gene. This protein is expressed in the central nervous system (CNS) and serves as a precursor to the misfolded PrP(Sc) isoform in prion diseases. The prototype prion disease is scrapie in sheep, and whereas Prnp exhibits common missense polymorphisms for V136A, R154H and Q171R in ovine populations, genetic variation in mouse Prnp is limited. Recently the CNS glycoprotein Shadoo (Sho) has been shown to resemble PrP(C) both in a central hydrophobic domain and in activity in a toxicity assay performed in cerebellar neurons. Sho protein levels are reduced in prion infections in rodents. Prompted by these properties of the Sho protein we investigated the extent of natural variation in SPRN. PRINCIPAL FINDINGS: Paralleling the case for ovine versus human and murine PRNP, we failed to detect significant coding polymorphisms that alter the mature Sho protein in a sample of neurologically normal humans, or in diverse strains of mice. However, ovine SPRN exhibited 4 missense mutations and expansion/contraction in a series of 5 tandem Ala/Gly-containing repeats R1-R5 encoding Sho's hydrophobic domain. A Val71Ala polymorphism and polymorphic expansion of wt 67(Ala)(3)Gly70 to 67(Ala)(5)Gly72 reached frequencies of 20%, with other alleles including Delta67-70 and a 67(Ala)(6)Gly73 expansion. Sheep V71, A71, Delta67-70 and 67(Ala)(6)Gly73 SPRN alleles encoded proteins with similar stability and posttranslational processing in transfected neuroblastoma cells. SIGNIFICANCE: Frequent coding polymorphisms are a hallmark of the sheep PRNP gene and our data indicate a similar situation applies to ovine SPRN. Whether a common selection pressure balances diversity at both loci remains to be established

Histone H4 Lysine 12 Acetylation Regulates Telomeric Heterochromatin Plasticity in Saccharomyces cerevisiae

Recent studies have established that the highly condensed and transcriptionally silent heterochromatic domains in budding yeast are virtually dynamic structures. The underlying mechanisms for heterochromatin dynamics, however, remain obscure. In this study, we show that histones are dynamically acetylated on H4K12 at telomeric heterochromatin, and this acetylation regulates several of the dynamic telomere properties. Using a de novo heterochromatin formation assay, we surprisingly found that acetylated H4K12 survived the formation of telomeric heterochromatin. Consistently, the histone acetyltransferase complex NuA4 bound to silenced telomeric regions and acetylated H4K12. H4K12 acetylation prevented the over-accumulation of Sir proteins at telomeric heterochromatin and elimination of this acetylation caused defects in multiple telomere-related processes, including transcription, telomere replication, and recombination. Together, these data shed light on a potential histone acetylation mark within telomeric heterochromatin that contributes to telomere plasticity

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type

Semaglutide and cardiovascular outcomes in patients with obesity and prevalent heart failure: a prespecified analysis of the SELECT trial

Background: Semaglutide, a GLP-1 receptor agonist, reduces the risk of major adverse cardiovascular events (MACE) in people with overweight or obesity, but the effects of this drug on outcomes in patients with atherosclerotic cardiovascular disease and heart failure are unknown. We report a prespecified analysis of the effect of once-weekly subcutaneous semaglutide 2·4 mg on ischaemic and heart failure cardiovascular outcomes. We aimed to investigate if semaglutide was beneficial in patients with atherosclerotic cardiovascular disease with a history of heart failure compared with placebo; if there was a difference in outcome in patients designated as having heart failure with preserved ejection fraction compared with heart failure with reduced ejection fraction; and if the efficacy and safety of semaglutide in patients with heart failure was related to baseline characteristics or subtype of heart failure. Methods: The SELECT trial was a randomised, double-blind, multicentre, placebo-controlled, event-driven phase 3 trial in 41 countries. Adults aged 45 years and older, with a BMI of 27 kg/m2 or greater and established cardiovascular disease were eligible for the study. Patients were randomly assigned (1:1) with a block size of four using an interactive web response system in a double-blind manner to escalating doses of once-weekly subcutaneous semaglutide over 16 weeks to a target dose of 2·4 mg, or placebo. In a prespecified analysis, we examined the effect of semaglutide compared with placebo in patients with and without a history of heart failure at enrolment, subclassified as heart failure with preserved ejection fraction, heart failure with reduced ejection fraction, or unclassified heart failure. Endpoints comprised MACE (a composite of non-fatal myocardial infarction, non-fatal stroke, and cardiovascular death); a composite heart failure outcome (cardiovascular death or hospitalisation or urgent hospital visit for heart failure); cardiovascular death; and all-cause death. The study is registered with ClinicalTrials.gov, NCT03574597. Findings: Between Oct 31, 2018, and March 31, 2021, 17 604 patients with a mean age of 61·6 years (SD 8·9) and a mean BMI of 33·4 kg/m2 (5·0) were randomly assigned to receive semaglutide (8803 [50·0%] patients) or placebo (8801 [50·0%] patients). 4286 (24·3%) of 17 604 patients had a history of investigator-defined heart failure at enrolment: 2273 (53·0%) of 4286 patients had heart failure with preserved ejection fraction, 1347 (31·4%) had heart failure with reduced ejection fraction, and 666 (15·5%) had unclassified heart failure. Baseline characteristics were similar between patients with and without heart failure. Patients with heart failure had a higher incidence of clinical events. Semaglutide improved all outcome measures in patients with heart failure at random assignment compared with those without heart failure (hazard ratio [HR] 0·72, 95% CI 0·60-0·87 for MACE; 0·79, 0·64-0·98 for the heart failure composite endpoint; 0·76, 0·59-0·97 for cardiovascular death; and 0·81, 0·66-1·00 for all-cause death; all pinteraction>0·19). Treatment with semaglutide resulted in improved outcomes in both the heart failure with reduced ejection fraction (HR 0·65, 95% CI 0·49-0·87 for MACE; 0·79, 0·58-1·08 for the composite heart failure endpoint) and heart failure with preserved ejection fraction groups (0·69, 0·51-0·91 for MACE; 0·75, 0·52-1·07 for the composite heart failure endpoint), although patients with heart failure with reduced ejection fraction had higher absolute event rates than those with heart failure with preserved ejection fraction. For MACE and the heart failure composite, there were no significant differences in benefits across baseline age, sex, BMI, New York Heart Association status, and diuretic use. Serious adverse events were less frequent with semaglutide versus placebo, regardless of heart failure subtype. Interpretation: In patients with atherosclerotic cardiovascular diease and overweight or obesity, treatment with semaglutide 2·4 mg reduced MACE and composite heart failure endpoints compared with placebo in those with and without clinical heart failure, regardless of heart failure subtype. Our findings could facilitate prescribing and result in improved clinical outcomes for this patient group. Funding: Novo Nordisk

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

The tax protein-DNA interaction is essential for HTLV-I transactivation in vitro

The human T-cell leukemia virus type-1 (HTLV-I)-encoded Tax protein enhances viral gene transcription through interaction with three repeated DNA elements located in the viral promoter. These elements, called viral CREs, are composed of an off-consensus eight base-pair cyclic AMP response element (CRE), immediately flanked by sequences that are rich in guanine and cytosine residues. Recent biochemical experiments have demonstrated that in the presence of the cellular protein CREB, Tax directly binds the viral CRE G+C-rich sequences via interaction with the minor groove. To determine the functional significance of the Tax-DNA interaction, we synthesized minor groove-binding pyrrole-imidazole polyamides which bind specifically to the G+C-rich sequences in the viral CREs. At concentrations where the polyamides specifically protect the G+C-rich sequences from MPE:Fe cleavage, the polyamides block the Tax-DNA interaction. At precisely these same concentrations, the polyamides specifically inhibit Tax transactivation in vitro, without altering CREB-activated transcription or basal transcription from the same promoter. Together, these data provide strong evidence that Tax-viral CRE interaction is essential for Tax function in vitro, and suggest that targeted disruption of the Tax-DNA minor groove interaction with polyamides may provide a novel approach for inhibiting viral replication in vivo

Human T-Lymphotropic Virus Type 1 p30(II) Regulates Gene Transcription by Binding CREB Binding Protein/p300

The highly conserved coadapters CREB binding protein (CBP) and p300 form complexes with CREB as well as other DNA binding transcription factors to modulate chromatin remodeling and thus transcription. Human T-lymphotropic virus type 1 (HTLV-1) transcription is controlled, in part, by the CREB/ATF family of transcription factors which bind promoter sequences and function as complexes with the viral oncogenic protein Tax. We have reported that the nuclear localizing protein p30(II) of HTLV-1 functions as a transcription factor, differentially modulates CREB-responsive promoters, and is critical for maintenance of proviral loads in rabbits. In this study, we tested whether p30(II) directly interacts with CBP/p300 to modulate gene transcription. Gal4(BD)-p30(II)-mediated transactivation was enhanced following exogenous expression of p300 and was competitively repressed by the p300 binding protein, adenovirus E1A, and E1ACR2 (mutated for retinoblastoma binding but retaining p300 binding). In contrast, E1ACR1 (mutated for p300 binding) failed to alter Gal4(BD)-p30(II)-mediated transactivation. In addition, Gal4(BD)-p30(II)-mediated transactivation was competitively inhibited by the cotransfection of CMV-p30(II)-HA and CMV-Tax but could be rescued by exogenous p300. Importantly, we demonstrate that p30(II) colocalizes with p300 in cell nuclei and directly binds to CBP/p300 in cells. Deletion mutants of CBP/p300 were used to localize the site critical for binding p30(II) to a highly conserved KIX region. DNA binding assays confirmed the interference of p30(II) with the assembly of CREB-Tax-p300/CBP multiprotein complexes on 21-bp repeat oligonucleotides in vitro. Collectively, our results demonstrate that CBP/p300 is a cellular protein target for HTLV-1 p30(II) and mediates its transcriptional effects in vivo

Topoisomerase 1 and Single-Strand Break Repair Modulate Transcription-Induced CAG Repeat Contraction in Human Cells ▿ †

Expanded trinucleotide repeats are responsible for a number of neurodegenerative diseases, such as Huntington disease and myotonic dystrophy type 1. The mechanisms that underlie repeat instability in the germ line and in the somatic tissues of human patients are undefined. Using a selection assay based on contraction of CAG repeat tracts in human cells, we screened the Prestwick chemical library in a moderately high-throughput assay and identified 18 novel inducers of repeat contraction. A subset of these compounds targeted pathways involved in the management of DNA supercoiling associated with transcription. Further analyses using both small molecule inhibitors and small interfering RNA (siRNA)-mediated knockdowns demonstrated the involvement of topoisomerase 1 (TOP1), tyrosyl-DNA phosphodiesterase 1 (TDP1), and single-strand break repair (SSBR) in modulating transcription-dependent CAG repeat contractions. The TOP1-TDP1-SSBR pathway normally functions to suppress repeat instability, since interfering with it stimulated repeat contractions. We further showed that the increase in repeat contractions when the TOP1-TDP1-SSBR pathway is compromised arises via transcription-coupled nucleotide excision repair, a previously identified contributor to transcription-induced repeat instability. These studies broaden the scope of pathways involved in transcription-induced CAG repeat instability and begin to define their interrelationships