DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA ...(mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization.
Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA ...adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.
HDACs (histone deacetylases) 1 and 2 are ubiquitous long-lived proteins, which are often found together in three major multiprotein co-repressor complexes: Sin3, NuRD (nucleosome remodelling and ...deacetylation) and CoREST (co-repressor for element-1-silencing transcription factor). Although there is a burgeoning number of non-histone proteins within the acetylome, these complexes contain multiple DNA/chromatin-recognition motifs, which, in combination with transcription factors, target HDAC1/2 to chromatin. Their physiological roles should therefore be viewed within the framework of chromatin manipulation. Classically, HDACs were thought to be recruited predominantly by transcriptional repressors to facilitate local histone deacetylation and transcriptional repression. More recently, genome-wide assays have mapped HDAC1/2 and their associated proteins to transcriptionally active loci and have provided alternative context-specific functions, whereby their repressive functions are subtly exerted to balance transcriptional activation and repression. With a few significant exceptions (early embryogenesis, brain development), HDAC1 and HDAC2 are functionally redundant. In most mouse knockout studies, deletion of both enzymes is required in order to produce a substantial phenotype. HDAC1/2 activity has been implicated in the development of numerous tissue and cell types, including heart, skin, brain, B-cells and T-cells. A common feature in all HDAC1/2-knockout, -knockdown and small-molecule inhibitor studies is a reduction in cell proliferation. A generic role in cell cycle progression could be exploited in cancer cells, by blocking HDAC1/2 activity with small-molecule inhibitors, making them potentially useful drug targets.
Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in ...chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of
and
in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors
,
, and
(PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 cause a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.
Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to ...generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2 -deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP ₄) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP ₄ regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP ₄ binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.
Replication stress results from obstacles to replication fork progression, including ongoing transcription, which can cause transcription–replication conflicts. Oncogenic signaling can promote global ...increases in transcription activity, also termed hypertranscription. Despite the widely accepted importance of oncogene-induced hypertranscription, its study remains neglected compared with other causes of replication stress and genomic instability in cancer. A growing number of recent studies are reporting that oncogenes, such as RAS, and targeted cancer treatments, such as bromodomain and extraterminal motif (BET) bromodomain inhibitors, increase global transcription, leading to R-loop accumulation, transcription–replication conflicts, and the activation of replication stress responses. Here we discuss our mechanistic understanding of hypertranscription-induced replication stress and the resulting cellular responses, in the context of oncogenes and targeted cancer therapies.
Hypertranscription is defined as a relative increase in global transcription to support proliferation and cell growth.Oncogenes and oncogenic signaling pathways stimulate the activity of general transcription factors for all three RNA polymerases to induce hypertranscription.Oncogene-induced hypertranscription correlates with increased R-loop formation and transcription–replication conflicts, resulting in replication stress and genomic instability.Targeted cancer therapies such as bromodomain and extraterminal motif (BET) and histone deacetylase (HDAC) inhibitors cause widespread hypertranscription and elevated R-loop levels, which are accompanied by replication stress.Hypertranscription-induced replication stress does not always activate canonical DNA damage response pathways, and recent work suggests that downregulation of homologous recombination could be a frequent event in response to elevated transcription.
Undifferentiated mouse embryonic stem cells (ESCs) possess low numbers of mitochondrial DNA (mtDNA), which encodes key subunits associated with the generation of ATP through oxidative phosphorylation ...(OXPHOS). As ESCs differentiate, mtDNA copy number is regulated by the nuclear-encoded mtDNA replication factors, which initiate a major replication event on Day 6 of differentiation. Here, we examined mtDNA replication events in somatic cells reprogrammed to pluripotency, namely somatic cell-ES (SC-ES), somatic cell nuclear transfer ES (NT-ES) and induced pluripotent stem (iPS) cells, all at low-passage. MtDNA copy number in undifferentiated iPS cells was similar to ESCs whilst SC-ES and NT-ES cells had significantly increased levels, which correlated positively and negatively with Nanog and Sox2 expression, respectively. During pluripotency and differentiation, the expression of the mtDNA-specific replication factors, PolgA and Peo1, were differentially expressed in iPS and SC-ES cells when compared to ESCs. Throughout differentiation, reprogrammed somatic cells were unable to accumulate mtDNA copy number, characteristic of ESCs, especially on Day 6. In addition, iPS and SC-ES cells were also unable to regulate ATP content in a manner similar to differentiating ESCs prior to Day 14. The treatment of reprogrammed somatic cells with an inhibitor of de novo DNA methylation, 5-Azacytidine, prior to differentiation enabled iPS cells, but not SC-ES and NT-ES cells, to accumulate mtDNA copies per cell in a manner similar to ESCs. These data demonstrate that the reprogramming process disrupts the regulation of mtDNA replication during pluripotency but this can be re-established through the use of epigenetic modifiers.
Mitochondrial DNA haplotypes are associated with various phenotypes, such as altered susceptibility to disease, environmental adaptations, and aging. Accumulating evidence suggests that mitochondrial ...DNA is essential for cell differentiation and the cell phenotype. However, the effects of different mitochondrial DNA haplotypes on differentiation and development remain to be determined. Using embryonic stem cell lines possessing the same Mus musculus chromosomes but harboring one of Mus musculus, Mus spretus, or Mus terricolor mitochondrial DNA haplotypes, we have determined the effects of different mitochondrial DNA haplotypes on chromosomal gene expression, differentiation, and mitochondrial metabolism. In undifferentiated and differentiating embryonic stem cells, we observed mitochondrial DNA haplotype-specific expression of genes involved in pluripotency, differentiation, mitochondrial energy metabolism, and DNA methylation. These mitochondrial DNA haplotypes also influenced the potential of embryonic stem cells to produce spontaneously beating cardiomyocytes. The differences in gene expression patterns and cardiomyocyte production were independent of ATP content, oxygen consumption, and respiratory capacity, which until now have been considered to be the primary roles of mitochondrial DNA. Differentiation of embryonic stem cells harboring the different mitochondrial DNA haplotypes in a 3D environment significantly increased chromosomal gene expression for all haplotypes during differentiation. However, haplotype-specific differences in gene expression patterns were maintained in this environment. Taken together, these results provide significant insight into the phenotypic consequences of mitochondrial DNA haplotypes and demonstrate their influence on differentiation and development. We propose that mitochondrial DNA haplotypes play a pivotal role in the process of differentiation and mediate the fate of the cell.
Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian ...grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.
Table of Contents Preamble2647 Introduction2649 Methodology and Evidence Review2649 Organization of the GWC2649 Document Review and Approval2649 Scope of the CPG2650 Overview of ACS2650 Initial ...Evaluation and Management: Recommendations2650 Clinical Assessment and Initial Evaluation2650 Emergency Department or Outpatient Facility Presentation2650 Prognosis--Early Risk Stratification2650 Cardiac Biomarkers and the Universal Definition of Myocardial Infarction2654 Biomarkers: Diagnosis2654 Biomarkers: Prognosis2654 Discharge From the ED or Chest Pain Unit2655 Early Hospital Care: Recommendations2655 Standard Medical Therapies2655 Oxygen2655 Nitrates2655 Analgesic Therapy2655 Beta-Adrenergic Blockers2656 Calcium Channel Blockers2657 Cholesterol Management2657 Inhibitors of the Renin-Angiotensin-Aldosterone System2657 Initial Antiplatelet/Anticoagulant Therapy in Patients With Definite or Likely NSTE-ACS2657 Initial Oral and Intravenous Antiplatelet Therapy in Patients With Definite or Likely NSTE-ACS Treated With an Initial Invasive or Ischemia-Guided Strategy2657 Initial Parenteral Anticoagulant Therapy in Patients With Definite NSTE-ACS2659 Ischemia-Guided Strategy Versus Early Invasive Strategies2659 Early Invasive and Ischemia-Guided Strategies2659 Risk Stratification Before Discharge for Patients With an Ischemia-Guided Strategy of NSTE-ACS2661 Myocardial Revascularization: Recommendations2661 PCI--General Considerations2661 PCI--Oral and Intravenous Antiplatelet Agents2661 PCI--GP IIb/IIIa Inhibitors2662 Anticoagulant Therapy in Patients Undergoing PCI2663 Timing of Urgent Coronary Artery Bypass Graft in Patients With NSTE-ACS in Relation to Use of Antiplatelet Agents2663 Late Hospital Care, Hospital Discharge, and Posthospital Discharge Care: Recommendations2663 Medical Regimen and Use of Medications at Discharge2663 Late Hospital and Posthospital Oral Antiplatelet Therapy2664 Combined Oral Anticoagulant Therapy and Antiplatelet Therapy in Patients With NSTE-ACS2664 Risk Reduction Strategies for Secondary Prevention2664 Plan of Care for Patients With NSTE-ACS2665 Special Patient Groups: Recommendations2665 NSTE-ACS in Older Patients2665 Heart Failure and Cardiogenic Shock2665 Diabetes Mellitus2667 Post-CABG2668 Perioperative NSTE-ACS Related to Noncardiac Surgery2668 Chronic Kidney Disease2668 Women2668 Anemia, Bleeding, and Transfusion2668 Cocaine and Methamphetamine Users2668 Vasospastic (Prinzmetal) Angina2668 ACS With Angiographically Normal Coronary Arteries2669 Stress (Takotsubo) Cardiomyopathy2669 Quality of Care and Outcomes for ACS--Use of Performance Measures and Registries: Recommendation2669 Summary and Evidence Gaps2669 References2670 Appendix 1 Author Relationships With Industry and Other Entities (Relevant)2680 Appendix 2 Reviewer Relationships With Industry and Other Entities (Relevant)2683 Preamble The American College of Cardiology (ACC) and the American Heart Association (AHA) are committed to the prevention and management of cardiovascular diseases through professional education and research for clinicians, providers, and patients. Since 1980, the ACC and AHA have shared a responsibility to translate scientific evidence into clinical practice guidelines (CPGs) with recommendations to standardize and improve cardiovascular health.
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