Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network.

Non-coding RNAs (ncRNAs), including the more recently identified Stable Unannotated Transcripts (SUTs) and Cryptic Unstable Transcripts (CUTs), are increasingly being shown to play pivotal roles in the transcriptional and post-transcriptional regulation of genes in eukaryotes. Here, we carried out a large-scale screening of ncRNAs in Saccharomyces cerevisiae, and provide evidence for SUT and CUT function. Phenotypic data on 372 ncRNA deletion strains in 23 different growth conditions were collected, identifying ncRNAs responsible for significant cellular fitness changes. Transcriptome profiles were assembled for 18 haploid ncRNA deletion mutants and 2 essential ncRNA heterozygous deletants. Guided by the resulting RNA-seq data we analysed the genome-wide dysregulation of protein coding genes and non-coding transcripts. Novel functional ncRNAs, SUT125, SUT126, SUT035 and SUT532 that act in trans by modulating transcription factors were identified. Furthermore, we described the impact of SUTs and CUTs in modulating coding gene expression in response to different environmental conditions, regulating important biological process such as respiration (SUT125, SUT126, SUT035, SUT432), steroid biosynthesis (CUT494, SUT053, SUT468) or rRNA processing (SUT075 and snR30). Overall, these data capture and integrate the regulatory and phenotypic network of ncRNAs and protein-coding genes, providing genome-wide evidence of the impact of ncRNAs on cellular homeostasis.

Stress-Activated Kinase Mitogen-Activated Kinase Kinase-7 Governs Epigenetics of Cardiac Repolarization for Arrhythmia Prevention.

BACKGROUND: Ventricular arrhythmia is a leading cause of cardiac mortality. Most antiarrhythmics present paradoxical proarrhythmic side effects, culminating in a greater risk of sudden death. METHODS: We describe a new regulatory mechanism linking mitogen-activated kinase kinase-7 deficiency with increased arrhythmia vulnerability in hypertrophied and failing hearts using mouse models harboring mitogen-activated kinase kinase-7 knockout or overexpression. The human relevance of this arrhythmogenic mechanism is evaluated in human-induced pluripotent stem cell-derived cardiomyocytes. Therapeutic potentials by targeting this mechanism are explored in the mouse models and human-induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Mechanistically, hypertrophic stress dampens expression and phosphorylation of mitogen-activated kinase kinase-7. Such mitogen-activated kinase kinase-7 deficiency leaves histone deacetylase-2 unphosphorylated and filamin-A accumulated in the nucleus to form a complex with Krüppel-like factor-4. This complex leads to Krüppel-like factor-4 disassociation from the promoter regions of multiple key potassium channel genes (Kv4.2, KChIP2, Kv1.5, ERG1, and Kir6.2) and reduction of their transcript levels. Consequent repolarization delays result in ventricular arrhythmias. Therapeutically, targeting the repressive function of the Krüppel-like factor-4/histone deacetylase-2/filamin-A complex with the histone deacetylase-2 inhibitor valproic acid restores K+ channel expression and alleviates ventricular arrhythmias in pathologically remodeled hearts. CONCLUSIONS: Our findings unveil this new gene regulatory avenue as a new antiarrhythmic target where repurposing of the antiepileptic drug valproic acid as an antiarrhythmic is supported.

H2A.Z marks antisense promoters and has positive effects on antisense transcript levels in budding yeast.

BACKGROUND: The histone variant H2A.Z, which has been reported to have both activating and repressive effects on gene expression, is known to occupy nucleosomes at the 5' ends of protein-coding genes. RESULTS: We now find that H2A.Z is also significantly enriched in gene coding regions and at the 3' ends of genes in budding yeast, where it co-localises with histone marks associated with active promoters. By comparing H2A.Z binding to global gene expression in budding yeast strains engineered so that normally unstable transcripts are abundant, we show that H2A.Z is required for normal levels of antisense transcripts as well as sense ones. High levels of H2A.Z at antisense promoters are associated with decreased antisense transcript levels when H2A.Z is deleted, indicating that H2A.Z has an activating effect on antisense transcripts. Decreases in antisense transcripts affected by H2A.Z are accompanied by increased levels of paired sense transcripts. CONCLUSIONS: The effect of H2A.Z on protein coding gene expression is a reflection of its importance for normal levels of both sense and antisense transcripts.

Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes.

Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.

Organizing the genome with H2A histone variants.

Chromatin acts as an organizer and indexer of genomic DNA and is a highly dynamic and regulated structure with properties directly related to its constituent parts. Histone variants are abundant components of chromatin that replace canonical histones in a subset of nucleosomes, thereby altering nucleosomal characteristics. The present review focuses on the H2A variant histones, summarizing current knowledge of how H2A variants can introduce chemical and functional heterogeneity into chromatin, the positions that nucleosomes containing H2A variants occupy in eukaryotic genomes, and the regulation of these localization patterns.

Wastewater-based surveillance of SARS-CoV-2: Short-term projection (forecasting), smoothing and outlier identification using Bayesian smoothing.

BACKGROUND: Day-to-day variation in the measurement of SARS-CoV-2 in wastewater can challenge public health interpretation. We assessed a Bayesian smoothing and forecasting method previously used for surveillance and short-term projection of COVID-19 cases, hospitalizations, and deaths. METHODS: SARS-CoV-2 viral measurement from the sewershed in Ottawa, Canada, sampled at the municipal wastewater treatment plant from July 1, 2020, to February 15, 2022, was used to assess and internally validate measurement averaging and prediction. External validation was performed using viral measurement data from influent wastewater samples from 15 wastewater treatment plants and municipalities across Ontario. RESULTS: Plots of SARS-CoV-2 viral measurement over time using Bayesian smoothing visually represented distinct COVID-19 "waves" described by case and hospitalization data in both initial (Ottawa) and external validation in 15 Ontario communities. The time-varying growth rate of viral measurement in wastewater samples approximated the growth rate observed for cases and hospitalization. One-week predicted viral measurement approximated the observed viral measurement throughout the assessment period from December 23, 2020, to August 8, 2022. An uncalibrated model showed underprediction during rapid increases in viral measurement (positive growth) and overprediction during rapid decreases. After recalibration, the model showed a close approximation between observed and predicted estimates. CONCLUSION: Bayesian smoothing of wastewater surveillance data of SARS-CoV-2 allows for accurate estimates of COVID-19 growth rates and one- and two-week forecasting of SARS-CoV-2 in wastewater for 16 municipalities in Ontario, Canada. Further assessment is warranted in other communities representing different sewersheds and environmental conditions.

A conserved function for the H2A.Z C terminus.

Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.

Eisosome disruption by noncoding RNA deletion increases protein secretion in yeast.

Noncoding RNAs (ncRNAs) regulate many aspects of gene expression. We investigated how ncRNAs affected protein secretion in yeast by large-scale screening for improved endogenous invertase secretion in ncRNA deletion strains with deletion of stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs), tRNAs, or snRNAs. We identified three candidate ncRNAs, SUT418, SUT390, and SUT125, that improved endogenous invertase secretion when deleted. As SUTs can affect expression of nearby genes, we quantified adjacent gene transcription and found that the PIL1 gene was down-regulated in the SUT125 deletion strain. Pil1 is a core component of eisosomes, nonmobile invaginations found throughout the plasma membrane. PIL1 knockout alone, or in combination with eisosome components LSP1 or SUR7, resulted in further increased secretion of invertase. Secretion of heterologous GFP was also increased upon PIL1 deletion, but this increase was signal sequence dependent. To reveal the potential for increased biopharmaceutical production, secretion of monoclonal antibody Pexelizumab scFv peptide was increased by PIL1 deletion. Global analysis of secreted proteins revealed that approximately 20% of secreted proteins, especially serine-enriched secreted proteins, including invertase, were increased upon eisosome disruption. Eisosomes are enriched with APC transporters and sphingolipids, which are essential components for secretory vesicle formation and protein sorting. Sphingolipid and serine biosynthesis pathways were up-regulated upon PIL1 deletion. We propose that increased secretion of endogenous and heterologous proteins upon PIL1 deletion resulted from sphingolipid redistribution in the plasma membrane and up-regulated sphingolipid biosynthesis. Overall, a new pathway to improve protein secretion in yeast via eisosome disruption has been identified.

Fate and developmental effects of dietary uptake of methylmercury in Silurana tropicalis tadpoles.

Adverse effects of methylmercury (MeHg) exposure during amphibian metamorphosis remain to be fully characterized. Most previous investigations determined effects of short-term exposure to elevated dose rates, without information on mercury (Hg) depuration and degradation pathways. Since metamorphosis is primarily controlled by thyroid hormones (TH), alterations in this process suggest a disruption of the TH endocrine axis. The aim of this research was to (1) characterize patterns of MeHg accumulation and depuration in tadpoles and (2) examine effects of MeHg accumulation on metamorphosis and the TH axis. Silurana tropicalis tadpoles were exposed to environmental levels of dietary MeHg until metamorphic climax. Whole-body MeHg and total Hg (THg) levels were measured, as well as the number of metamorphs, rate of metamorphosis, body size, and whole-body triiodothyronine (T3) levels at metamorphosis. Tadpoles exposed to a higher level of MeHg exhibited increased mortality and size, and reduced metamorphosis. At lower levels of MeHg, body burdens increased rapidly and eventually reached a plateau, whereas no plateau was reached at a higher level of MeHg exposure. T3 levels were not affected. Data indicate that at low and medium levels of exposure, depuration of MeHg may prevent toxicity in tadpoles. However, depuration mechanisms may be insufficient at high doses, producing disruption of metamorphosis and death. Although there were no marked effects of MeHg on whole-body T3 levels, further investigation of other components of the TH axis is warranted.

Complex care core curriculum for pediatric post-graduate trainees: Results of a North American needs assessment.

PURPOSE: There are increasing numbers of children with chronic, complex conditions requiring comprehensive care, however post-graduate training in this field is limited. Lack of training may contribute to reticence in engaging with their care. Although children with medical complexity (CMC) are heterogeneous, their care needs are similar. We aimed to address a lack of explicit training via a standardized curriculum. As an initial step, a collaborative needs assessment of key content was developed. METHODS: An on-line survey with drafted learning objectives was sent to professionals skilled in complex care. Participants indicated if an objective should be obtained by the end of Junior residency (PGY2), Senior residency (PGY4), or not at all. RESULTS: Eighty-two Canadian and US professionals participated; 60.3% practiced in a University Health Centre and 60% cared for CMC and participated in pediatric postgraduate education. Over 80% felt that the medical home concept should be understood by the end of PGY2, and that trainees should develop a care plan by the end of PGY4. CONCLUSION: This needs assessment supported the learning objectives and provided information on the expected competency time line for knowledge and skill acquisition. Additional comments will be used to revise the objectives.

Mutations in non-acid patch residues disrupt H2A.Z's association with chromatin through multiple mechanisms.

The incorporation of histone variants into nucleosomes is a critical mechanism for regulating essential DNA-templated processes and for establishing distinct chromatin architectures with specialised functions. H2A.Z is an evolutionarily conserved H2A variant that has diverse roles in transcriptional regulation, heterochromatin boundary definition, chromosome stability and DNA repair. The H2A.Z C-terminus diverges in sequence from canonical H2A and imparts unique functions to H2A.Z in the yeast S. cerevisiae. Although mediated in part through the acid patch-containing M6 region, many molecular determinants of this divergent structure-function relationship remain unclear. Here, by using an unbiased random mutagenesis screen of H2A.Z alleles, we identify point mutations in the C-terminus outside of the M6 region that disrupt the normal function of H2A.Z in response to cytotoxic stress. These functional defects correlate with reduced chromatin association, which we attribute to reduced physical stability within chromatin, but also to altered interactions with the SWR and INO80 chromatin remodeling complexes. Together with experimental data, computational modelling of these residue changes in the context of protein structure suggests the importance of C-terminal domain integrity and configuration for maintaining the level of H2A.Z in nucleosomes.

Genome-wide patterns of histone modifications in yeast.

Post-translational histone modifications and histone variants generate complexity in chromatin to enable the many functions of the chromosome. Recent studies have mapped histone modifications across the Saccharomyces cerevisiae genome. These experiments describe how combinations of modified and unmodified states relate to each other and particularly to chromosomal landmarks that include heterochromatin, subtelomeric chromatin, centromeres, origins of replication, promoters and coding regions. Such patterns might be important for the regulation of heterochromatin-mediated silencing, chromosome segregation, DNA replication and gene expression.

Acetylation of H2AZ Lys 14 is associated with genome-wide gene activity in yeast.

Histone variants and their post-translational modifications help regulate chromosomal functions. Htz1 is an evolutionarily conserved H2A variant found at several promoters in the yeast Saccharomyces cerevisiae. In this study, we undertook a genome-wide analysis of Htz1 and its modifications in yeast. Using mass spectrometric analysis, we determined that Htz1 is acetylated at Lys 3, Lys 8, Lys 10, and Lys 14 within its N-terminal tail, with K14 being the most abundant acetylated site. ChIP and microarray analysis were then used to compare the location of Htz1-K14 acetylation to that of Htz1 genome-wide. The data presented here demonstrate that while Htz1 is associated preferentially with the promoters of repressed genes, K14 acetylation is enriched at the promoters of active genes, and requires two known histone acetyltransferases, Gcn5 and Esa1. In support of our genome-wide analysis, we found that the acetylatable lysines of Htz1 are required for its full deposition during nucleosome reassembly upon repression of PHO5. Since the majority of Htz1 acetylation is seen at active promoters, where nucleosomes are known to be disassembled, our data argue for a dynamic process in which reassembly of Htz1 is regulated by its acetylation at promoters during transcription.

Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice.

The mammalian protein MBD4 contains a methyl-CpG binding domain and can enzymatically remove thymine (T) or uracil (U) from a mismatched CpG site in vitro. These properties suggest that MBD4 might function in vivo to minimize the mutability of 5-methylcytosine by removing its deamination product from DNA. We tested this hypothesis by analyzing Mbd4-/- mice and found that the frequency of of C --> T transitions at CpG sites was increased by a factor of three. On a cancer-susceptible Apc(Min/+) background, Mbd4-/- mice showed accelerated tumor formation with CpG --> TpG mutations in the Apc gene. Thus MBD4 suppresses CpG mutability and tumorigenesis in vivo.

Fas-associated death domain protein interacts with methyl-CpG binding domain protein 4: a potential link between genome surveillance and apoptosis.

Fas-associated death domain protein (FADD) is an adaptor protein bridging death receptors with initiator caspases. Thus, its function and localization are assumed to be cytoplasmic, although the localization of endogenous FADD has not been reported. Surprisingly, the data presented here demonstrate that FADD is mainly nuclear in several adherent cell lines. Its accumulation in the nucleus and export to the cytoplasm required the phosphorylation site Ser-194, which was also required for its interaction with the nucleocytoplasmic shuttling protein exportin-5. Within the nucleus, FADD interacted with the methyl-CpG binding domain protein 4 (MBD4), which excises thymine from GT mismatches in methylated regions of chromatin. The MBD4-interacting mismatch repair factor MLH1 was also found in a complex with FADD. The FADD-MBD4 interaction involved the death effector domain of FADD and a region of MBD4 adjacent to the glycosylase domain. The FADD-binding region of MBD4 was downstream of a frameshift mutation that occurs in a significant fraction of human colorectal carcinomas. Consistent with the idea that MBD4 can signal to an apoptotic effector, MBD4 regulated DNA damage-, Fas ligand-, and cell detachment-induced apoptosis. The nuclear localization of FADD and its interaction with a genome surveillance/DNA repair protein that can regulate apoptosis suggests a novel function of FADD distinct from direct participation in death receptor signaling complexes.