Comprehensive evaluation of smoking exposures and their interactions on DNA methylation.

BACKGROUND: Smoking impacts DNA methylation, but data are lacking on smoking-related differential methylation by sex or dietary intake, recent smoking cessation (<1 year), persistence of differential methylation from in utero smoking exposure, and effects of environmental tobacco smoke (ETS). METHODS: We meta-analysed data from up to 15,014 adults across 5 cohorts with DNA methylation measured in blood using Illumina's EPIC array for current smoking (2560 exposed), quit < 1 year (500 exposed), in utero (286 exposed), and ETS exposure (676 exposed). We also evaluated the interaction of current smoking with sex or diet (fibre, folate, and vitamin C). FINDINGS: Using false discovery rate (FDR < 0.05), 65,857 CpGs were differentially methylated in relation to current smoking, 4025 with recent quitting, 594 with in utero exposure, and 6 with ETS. Most current smoking CpGs attenuated within a year of quitting. CpGs related to in utero exposure in adults were enriched for those previously observed in newborns. Differential methylation by current smoking at 4-71 CpGs may be modified by sex or dietary intake. Nearly half (35-50%) of differentially methylated CpGs on the 450 K array were associated with blood gene expression. Current smoking and in utero smoking CpGs implicated 3049 and 1067 druggable targets, including chemotherapy drugs. INTERPRETATION: Many smoking-related methylation sites were identified with Illumina's EPIC array. Most signals revert to levels observed in never smokers within a year of cessation. Many in utero smoking CpGs persist into adulthood. Smoking-related druggable targets may provide insights into cancer treatment response and shared mechanisms across smoking-related diseases. FUNDING: Intramural Research Program of the National Institutes of Health, Norwegian Ministry of Health and Care Services and the Ministry of Education and Research, Chief Scientist Office of the Scottish Government Health Directorates and the Scottish Funding Council, Medical Research Council UK and the Wellcome Trust.

Synergistic roles of tristetraprolin family members in myeloid cells in the control of inflammation.

Members of the tristetraprolin (TTP) family of RNA-binding proteins can bind to and promote the decay of specific transcripts containing AU-rich motifs. ZFP36 (TTP) is best known for regulating pro-inflammatory cytokine expression in myeloid cells; however, its mammalian paralogues ZFP36L1 and ZFP36L2 have not been viewed as important in controlling inflammation. We knocked out these genes in myeloid cells in mice, singly and together. Single-gene myeloid-specific knockouts resulted in almost no spontaneous phenotypes. In contrast, mice with myeloid cell deficiency of all three genes developed severe inflammation, with a median survival of 8 wk. Macrophages from these mice expressed many more stabilized transcripts than cells from myeloid-specific TTP knockout mice; many of these encoded pro-inflammatory cytokines and chemokines. The failure of weight gain, arthritis, and early death could be prevented completely by two normal alleles of any of the three paralogues, and even one normal allele of Zfp36 or Zfp36l2 was enough to prevent the inflammatory phenotype. Our findings emphasize the importance of all three family members, acting in concert, in myeloid cell function.

Vanadium Pentoxide Exposure Causes Strain-Dependent Changes in Mitochondrial DNA Heteroplasmy, Copy Number, and Lesions, but Not Nuclear DNA Lesions.

Interstitial lung diseases (ILDs) are lethal lung diseases characterized by pulmonary inflammation and progressive lung interstitial scarring. We previously developed a mouse model of ILD using vanadium pentoxide (V2O5) and identified several gene candidates on chromosome 4 associated with pulmonary fibrosis. While these data indicated a significant genetic contribution to ILD susceptibility, they did not include any potential associations and interactions with the mitochondrial genome that might influence disease risk. To conduct this pilot work, we selected the two divergent strains we previously categorized as V2O5-resistant C57BL6J (B6) and -responsive DBA/2J (D2) and compared their mitochondrial genome characteristics, including DNA variants, heteroplasmy, lesions, and copy numbers at 14- and 112-days post-exposure. While we did not find changes in the mitochondrial genome at 14 days post-exposure, at 112 days, we found that the responsive D2 strain exhibited significantly fewer mtDNA copies and more lesions than control animals. Alongside these findings, mtDNA heteroplasmy frequency decreased. These data suggest that mice previously shown to exhibit increased susceptibility to pulmonary fibrosis and inflammation sustain damage to the mitochondrial genome that is evident at 112 days post-V2O5 exposure.

Glycidamide-induced hypermutation in yeast single-stranded DNA reveals a ubiquitous clock-like mutational motif in humans.

Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents.

Race/ethnicity-stratified fine-mapping of the MHC locus reveals genetic variants associated with late-onset asthma.

Introduction: Asthma is a chronic disease of the airways that impairs normal breathing. The etiology of asthma is complex and involves multiple factors, including the environment and genetics, especially the distinct genetic architecture associated with ancestry. Compared to early-onset asthma, little is known about genetic predisposition to late-onset asthma. We investigated the race/ethnicity-specific relationship among genetic variants within the major histocompatibility complex (MHC) region and late-onset asthma in a North Carolina-based multiracial cohort of adults. Methods: We stratified all analyses by self-reported race (i.e., White and Black) and adjusted all regression models for age, sex, and ancestry. We conducted association tests within the MHC region and performed fine-mapping analyses conditioned on the race/ethnicity-specific lead variant using whole-genome sequencing (WGS) data. We applied computational methods to infer human leukocyte antigen (HLA) alleles and residues at amino acid positions. We replicated findings in the UK Biobank. Results: The lead signals, rs9265901 on the 5' end of HLA-B, rs55888430 on HLA-DOB, and rs117953947 on HCG17, were significantly associated with late-onset asthma in all, White, and Black participants, respectively (OR = 1.73, 95%CI: 1.31 to 2.14, p = 3.62 × 10-5; OR = 3.05, 95%CI: 1.86 to 4.98, p = 8.85 × 10-6; OR = 19.5, 95%CI: 4.37 to 87.2, p = 9.97 × 10-5, respectively). For the HLA analysis, HLA-B*40:02 and HLA-DRB1*04:05, HLA-B*40:02, HLA-C*04:01, and HLA-DRB1*04:05, and HLA-DRB1*03:01 and HLA-DQB1 were significantly associated with late-onset asthma in all, White, and Black participants. Conclusion: Multiple genetic variants within the MHC region were significantly associated with late-onset asthma, and the associations were significantly different by race/ethnicity group.

Detection of DNA replication errors and 8-oxo-dGTP-mediated mutations in E. coli by Duplex DNA Sequencing.

Mutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107 base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations in E. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using a mutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10-7 to 10-8 depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurred in vivo. In contrast, bona-fide in vivo mutations were identified for DNA from both the mutL and mutT strains, as indicated by specific increases in base substitutions that are fully consistent with their established in vivo roles. Notably, the data reproduce the specific context effects of in vivo mutations as well as the leading vs. lagging strand bias among DNA replication errors.

DUX4 double whammy: The transcription factor that causes a rare muscular dystrophy also kills the precursors of the human nose.

SMCHD1 mutations cause congenital arhinia (absent nose) and a muscular dystrophy called FSHD2. In FSHD2, loss of SMCHD1 repressive activity causes expression of double homeobox 4 (DUX4) in muscle tissue, where it is toxic. Studies of arhinia patients suggest a primary defect in nasal placode cells (human nose progenitors). Here, we show that upon SMCHD1 ablation, DUX4 becomes derepressed in H9 human embryonic stem cells (hESCs) as they differentiate toward a placode cell fate, triggering cell death. Arhinia and FSHD2 patient-derived induced pluripotent stem cells (iPSCs) express DUX4 when converted to placode cells and demonstrate variable degrees of cell death, suggesting an environmental disease modifier. HSV-1 may be one such modifier as herpesvirus infection amplifies DUX4 expression in SMCHD1 KO hESC and patient iPSC. These studies suggest that arhinia, like FSHD2, is due to compromised SMCHD1 repressive activity in a cell-specific context and provide evidence for an environmental modifier.

Questionnaire-Based Polyexposure Assessment Outperforms Polygenic Scores for Classification of Type 2 Diabetes in a Multiancestry Cohort.

OBJECTIVE: Environmental exposures may have greater predictive power for type 2 diabetes than polygenic scores (PGS). Studies examining environmental risk factors, however, have included only individuals with European ancestry, limiting the applicability of results. We conducted an exposome-wide association study in the multiancestry Personalized Environment and Genes Study to assess the effects of environmental factors on type 2 diabetes. RESEARCH DESIGN AND METHODS: Using logistic regression for single-exposure analysis, we identified exposures associated with type 2 diabetes, adjusting for age, BMI, household income, and self-reported sex and race. To compare cumulative genetic and environmental effects, we computed an overall clinical score (OCS) as a weighted sum of BMI and prediabetes, hypertension, and high cholesterol status and a polyexposure score (PXS) as a weighted sum of 13 environmental variables. Using UK Biobank data, we developed a multiancestry PGS and calculated it for participants. RESULTS: We found 76 significant associations with type 2 diabetes, including novel associations of asbestos and coal dust exposure. OCS, PXS, and PGS were significantly associated with type 2 diabetes. PXS had moderate power to determine associations, with larger effect size and greater power and reclassification improvement than PGS. For all scores, the results differed by race. CONCLUSIONS: Our findings in a multiancestry cohort elucidate how type 2 diabetes odds can be attributed to clinical, genetic, and environmental factors and emphasize the need for exposome data in disease-risk association studies. Race-based differences in predictive scores highlight the need for genetic and exposome-wide studies in diverse populations.

Pif1 family helicases promote mutation avoidance during DNA replication.

Pif1 family 5' → 3' DNA helicases are important for replication fork progression and genome stability. The budding yeast Saccharomyces cerevisiae encodes two Pif1 family helicases, Rrm3 and Pif1, both of which are multi-functional. Here we describe novel functions for Rrm3 in promoting mutation avoidance during DNA replication. We show that loss of RRM3 results in elevated spontaneous mutations made by DNA polymerases Pols ϵ and δ, which are subject to DNA mismatch repair. The absence of RRM3 also causes higher mutagenesis by the fourth B-family DNA polymerase Pol ζ. By genome-wide analysis, we show that the mutational consequences due to loss of RRM3 vary depending on the genomic locus. Rrm3 promotes the accuracy of DNA replication by Pols ϵ and δ across the genome, and it is particularly important for preventing Pol ζ-dependent mutagenesis at tRNA genes. In addition, mutation avoidance by Rrm3 depends on its helicase activity, and Pif1 serves as a backup for Rrm3 in suppressing mutagenesis. We present evidence that the sole human Pif1 family helicase in human cells likely also promotes replication fidelity, suggesting that a role for Pif1 family helicases in mutation avoidance may be evolutionarily conserved, a possible underlying mechanism for its potential tumor-suppressor function.

Mitochondrial-nuclear epistasis underlying phenotypic variation in breast cancer pathology.

The interplay between genes harboring single nucleotide polymorphisms (SNPs) is vital to better understand underlying contributions to the etiology of breast cancer. Much attention has been paid to epistasis between nuclear genes or mutations in the mitochondrial genome. However, there is limited understanding about the epistatic effects of genetic variants in the nuclear and mitochondrial genomes jointly on breast cancer. We tested the interaction of germline SNPs in the mitochondrial (mtSNPs) and nuclear (nuSNPs) genomes of female breast cancer patients in The Cancer Genome Atlas (TCGA) for association with morphological features extracted from hematoxylin and eosin (H&E)-stained pathology images. We identified 115 significant (q-value < 0.05) mito-nuclear interactions that increased nuclei size by as much as 12%. One interaction between nuSNP rs17320521 in an intron of the WSC Domain Containing 2 (WSCD2) gene and mtSNP rs869096886, a synonymous variant mapped to the mitochondrially-encoded NADH dehydrogenase 4 (MT-ND4) gene, was confirmed in an independent breast cancer data set from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). None of the 10 mito-nuclear interactions identified from non-diseased female breast tissues from the Genotype-Expression (GTEx) project resulted in an increase in nuclei size. Comparisons of gene expression data from the TCGA breast cancer patients with the genotype homozygous for the minor alleles of the SNPs in WSCD2 and MT-ND4 versus the other genotypes revealed core transcriptional regulator interactions and an association with insulin. Finally, a Cox proportional hazards ratio = 1.7 (C.I. 0.98-2.9, p-value = 0.042) and Kaplan-Meier plot suggest that the TCGA female breast cancer patients with low gene expression of WSCD2 coupled with large nuclei have an increased risk of mortality. The intergenomic dependency between the two variants may constitute an inherent susceptibility of a more severe form of breast cancer and points to genetic targets for further investigation of additional determinants of the disease.

Opioid medication use and blood DNA methylation: epigenome-wide association meta-analysis.

Aim: To identify differential methylation related to prescribed opioid use. Methods: This study examined whether blood DNA methylation, measured using Illumina arrays, differs by recent opioid medication use in four population-based cohorts. We meta-analyzed results (282 users; 10,560 nonusers) using inverse-variance weighting. Results: Differential methylation (false discovery rate <0.05) was observed at six CpGs annotated to the following genes: KIAA0226, CPLX2, TDRP, RNF38, TTC23 and GPR179. Integrative epigenomic analyses linked implicated loci to regulatory elements in blood and/or brain. Additionally, 74 CpGs were differentially methylated in males or females. Methylation at significant CpGs correlated with gene expression in blood and/or brain. Conclusion: This study identified DNA methylation related to opioid medication use in general populations. The results could inform the development of blood methylation biomarkers of opioid use.

How asymmetric DNA replication achieves symmetrical fidelity.

Accurate DNA replication of an undamaged template depends on polymerase selectivity for matched nucleotides, exonucleolytic proofreading of mismatches, and removal of remaining mismatches via DNA mismatch repair (MMR). DNA polymerases (Pols) δ and ε have 3'-5' exonucleases into which mismatches are partitioned for excision in cis (intrinsic proofreading). Here we provide strong evidence that Pol δ can extrinsically proofread mismatches made by itself and those made by Pol ε, independently of both Pol δ's polymerization activity and MMR. Extrinsic proofreading across the genome is remarkably efficient. We report, with unprecedented accuracy, in vivo contributions of nucleotide selectivity, proofreading, and MMR to the fidelity of DNA replication in Saccharomyces cerevisiae. We show that extrinsic proofreading by Pol δ improves and balances the fidelity of the two DNA strands. Together, we depict a comprehensive picture of how nucleotide selectivity, proofreading, and MMR cooperate to achieve high and symmetrical fidelity on the two strands.

A post-transcriptional regulon controlled by TtpA, the single tristetraprolin family member expressed in Dictyostelium discoideum.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4-NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3'-untranslated regions (3'-UTRs) of all six 'TtpA-target' mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3'-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an 'RNA regulon', where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.

The fidelity of DNA replication, particularly on GC-rich templates, is reduced by defects of the Fe-S cluster in DNA polymerase δ.

Iron-sulfur clusters (4Fe-4S) exist in many enzymes concerned with DNA replication and repair. The contribution of these clusters to enzymatic activity is not fully understood. We identified the MET18 (MMS19) gene of Saccharomyces cerevisiae as a strong mutator on GC-rich genes. Met18p is required for the efficient insertion of iron-sulfur clusters into various proteins. met18 mutants have an elevated rate of deletions between short flanking repeats, consistent with increased DNA polymerase slippage. This phenotype is very similar to that observed in mutants of POL3 (encoding the catalytic subunit of Pol Î´) that weaken binding of the iron-sulfur cluster. Comparable mutants of POL2 (Pol ϵ) do not elevate deletions. Further support for the conclusion that met18 strains result in impaired DNA synthesis by Pol Î´ are the observations that Pol Î´ isolated from met18 strains has less bound iron and is less processive in vitro than the wild-type holoenzyme.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Increased Burden of Rare Sequence Variants in GnRH-Associated Genes in Women With Hypothalamic Amenorrhea.

CONTEXT: Functional hypothalamic amenorrhea (HA) is a common, acquired form of hypogonadotropic hypogonadism that occurs in the setting of energy deficits and/or stress. Variability in individual susceptibility to these stressors, HA heritability, and previous identification of several rare sequence variants (RSVs) in genes associated with the rare disorder, isolated hypogonadotropic hypogonadism (IHH), in individuals with HA suggest a possible genetic contribution to HA susceptibility. OBJECTIVE: We sought to determine whether the burden of RSVs in IHH-related genes is greater in women with HA than controls. DESIGN: We compared patients with HA to control women. SETTING: The study was conducted at secondary referral centers. PATIENTS AND OTHER PARTICIPANTS: Women with HA (n = 106) and control women (ClinSeq study; n = 468). INTERVENTIONS: We performed exome sequencing in all patients and controls. MAIN OUTCOME MEASURE(S): The frequency of RSVs in 53 IHH-associated genes was determined using rare variant burden and association tests. RESULTS: RSVs were overrepresented in women with HA compared with controls (P = .007). Seventy-eight heterozygous RSVs in 33 genes were identified in 58 women with HA (36.8% of alleles) compared to 255 RSVs in 41 genes among 200 control women (27.2%). CONCLUSIONS: Women with HA are enriched for RSVs in genes that cause IHH, suggesting that variation in genes associated with gonadotropin-releasing hormone neuronal ontogeny and function may be a major determinant of individual susceptibility to developing HA in the face of diet, exercise, and/or stress.

Association between Mitochondrial DNA Sequence Variants and V˙O2 max Trainability.

PURPOSE: We designed the study to determine whether mitochondrial DNA (mtDNA) haplogroup, sequence, and heteroplasmy differed between individuals previously characterized as low (LR) or high responders (HR) as defined by their maximal oxygen uptake response to a standardized aerobic exercise training program. METHODS: DNA was isolated from whole blood in subjects from the HERITAGE Family Study that were determined to be either HR (n = 15) or LR (n = 15). mtDNA was amplified by long-range polymerase chain reaction, then tagged with Nextera libraries and sequenced on a MiSeq instrument. RESULTS: Different mtDNA haplogroup subtypes were found in HR and LR individuals. Compared with HR subjects, significantly more LR subjects had variants in 13 sites, including 7 in hypervariable (HV) regions: HV2 (G185A: 0 vs 6, P = 0.02; G228A: 0 vs 5, P = 0.04; C295T: 0 vs 6; P = 0.04), HV3 (C462T: 0 vs 5, P = 0.04; T489C: 0 vs 5; P = 0.04), and HV1 (C16068T: 0 vs 6, P = 0.02; T16125C: 0 vs 6, P = 0.02). Remaining variants were in protein coding genes, mtND1 (1 vs 8, P = 0.02), mtND3 (A10397G: 0 vs 5, P = 0.04), mtND4 (A11250G: 1 vs 8, P = 0.02), mtND5 (G13707A: 0 vs 5, P = 0.04), and mtCYTB (T14797C: 0 vs 5, P = 0.04; C15451A: 1 vs 8, P = 0.02). Average total numbers of heteroplasmies (P = 0.83) and frequency of heteroplasmies (P = 0.05) were similar between the groups. CONCLUSIONS: Our findings provide specific sites across the mitochondrial genome that may be related to maximal oxygen uptake trainability.

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging.

BACKGROUND: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease. RESULTS: To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns. CONCLUSIONS: These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.

H/ACA snoRNA levels are regulated during stem cell differentiation.

H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA levels in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA levels are dynamic during differentiation, we comprehensively profiled H/ACA snoRNA abundance in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that the profiles of H/ACA snoRNA abundance are cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 abundance upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Together, these data point toward a potential model in which H/ACA snoRNAs are specifically regulated during differentiation to alter pseudouridylation and fine tune ribosome function.

Investigation of the adolescent female breast transcriptome and the impact of obesity.

BACKGROUND: Early life environmental exposures affect breast development and breast cancer risk in adulthood. The breast is particularly vulnerable during puberty when mammary epithelial cells proliferate exponentially. In overweight/obese (OB) women, inflammation increases breast aromatase expression and estrogen synthesis and promotes estrogen-receptor (ER)-positive breast cancer. In contrast, recent epidemiological studies suggest that obesity during childhood decreases future breast cancer risk. Studies on environmental exposures and breast cancer risk have thus far been limited to animal models. Here, we present the first interrogation of the human adolescent breast at the molecular level and investigate how obesity affects the immature breast. METHODS: We performed RNA-seq in 62 breast tissue samples from adolescent girls/young women (ADOL; mean age 17.8 years) who underwent reduction mammoplasty. Thirty-one subjects were non-overweight/obese (NOB; mean BMI 23.4 kg/m2) and 31 were overweight/obese (OB; BMI 32.1 kg/m2). We also compared our data to published mammary transcriptome datasets from women (mean age 39 years) and young adult mice, rats, and macaques. RESULTS: The ADOL breast transcriptome showed limited (30%) overlap with other species, but 88% overlap with adult women for the 500 most highly expressed genes in each dataset; only 43 genes were shared by all groups. In ADOL, there were 120 differentially expressed genes (DEG) in OB compared with NOB samples (padj < 0.05). Based on these DEG, Ingenuity Pathway Analysis (IPA) identified the cytokines CSF1 and IL-10 and the chemokine receptor CCR2 as among the most highly activated upstream regulators, suggesting increased inflammation in the OB breast. Classical ER targets (e.g., PR, AREG) were not differentially expressed, yet IPA identified the ER and PR and growth factors/receptors (VEGF, HGF, HER3) and kinases (AKT1) involved in hormone-independent ER activation as activated upstream regulators in OB breast tissue. CONCLUSIONS: These studies represent the first investigation of the human breast transcriptome during late puberty/young adulthood and demonstrate that obesity is associated with a transcriptional signature of inflammation which may augment estrogen action in the immature breast microenvironment. We anticipate that these studies will prompt more comprehensive cellular and molecular investigations of obesity and its effect on the breast during this critical developmental window.