ARMC5 controls the degradation of most Pol II subunits, and ARMC5 mutation increases neural tube defect risks in mice and humans.

BACKGROUND: Neural tube defects (NTDs) are caused by genetic and environmental factors. ARMC5 is part of a novel ubiquitin ligase specific for POLR2A, the largest subunit of RNA polymerase II (Pol II). RESULTS: We find that ARMC5 knockout mice have increased incidence of NTDs, such as spina bifida and exencephaly. Surprisingly, the absence of ARMC5 causes the accumulation of not only POLR2A but also most of the other 11 Pol II subunits, indicating that the degradation of the whole Pol II complex is compromised. The enlarged Pol II pool does not lead to generalized Pol II stalling or a generalized decrease in mRNA transcription. In neural progenitor cells, ARMC5 knockout only dysregulates 106 genes, some of which are known to be involved in neural tube development. FOLH1, critical in folate uptake and hence neural tube development, is downregulated in the knockout intestine. We also identify nine deleterious mutations in the ARMC5 gene in 511 patients with myelomeningocele, a severe form of spina bifida. These mutations impair the interaction between ARMC5 and Pol II and reduce Pol II ubiquitination. CONCLUSIONS: Mutations in ARMC5 increase the risk of NTDs in mice and humans. ARMC5 is part of an E3 controlling the degradation of all 12 subunits of Pol II under physiological conditions. The Pol II pool size might have effects on NTD pathogenesis, and some of the effects might be via the downregulation of FOLH1. Additional mechanistic work is needed to establish the causal effect of the findings on NTD pathogenesis.

Genome-scale mapping of DNA damage suppressors through phenotypic CRISPR-Cas9 screens.

To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.

ARMC5 is part of an RPB1-specific ubiquitin ligase implicated in adrenal hyperplasia.

ARMC5 is implicated in several pathological conditions, but its function remains unknown. We have previously identified CUL3 and RPB1 (the largest subunit of RNA polymerase II (Pol II) as potential ARMC5-interacting proteins. Here, we show that ARMC5, CUL3 and RBX1 form an active E3 ligase complex specific for RPB1. ARMC5, CUL3, and RBX1 formed an active E3 specific for RPB1. Armc5 deletion caused a significant reduction in RPB1 ubiquitination and an increase in an accumulation of RPB1, and hence an enlarged Pol II pool in normal tissues and organs. The compromised RPB1 degradation did not cause generalized Pol II stalling nor depressed transcription in the adrenal glands but did result in dysregulation of a subset of genes, with most upregulated. We found RPB1 to be highly expressed in the adrenal nodules from patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) harboring germline ARMC5 mutations. Mutant ARMC5 had altered binding with RPB1. In summary, we discovered that wildtype ARMC5 was part of a novel RPB1-specific E3. ARMC5 mutations resulted in an enlarged Pol II pool, which dysregulated a subset of effector genes. Such an enlarged Pol II pool and gene dysregulation was correlated to adrenal hyperplasia in humans and KO mice.

Armc5 deletion causes developmental defects and compromises T-cell immune responses.

Armadillo repeat containing 5 (ARMC5) is a cytosolic protein with no enzymatic activities. Little is known about its function and mechanisms of action, except that gene mutations are associated with risks of primary macronodular adrenal gland hyperplasia. Here we map Armc5 expression by in situ hybridization, and generate Armc5 knockout mice, which are small in body size. Armc5 knockout mice have compromised T-cell proliferation and differentiation into Th1 and Th17 cells, increased T-cell apoptosis, reduced severity of experimental autoimmune encephalitis, and defective immune responses to lymphocytic choriomeningitis virus infection. These mice also develop adrenal gland hyperplasia in old age. Yeast 2-hybrid assays identify 16 ARMC5-binding partners. Together these data indicate that ARMC5 is crucial in fetal development, T-cell function and adrenal gland growth homeostasis, and that the functions of ARMC5 probably depend on interaction with multiple signalling pathways.

[Malnutrition in early life and cardiovascular disease in adulthood].

Epidemiological and experimental studies have demonstrated that the risk of cardiovascular disease (CVD) in adulthood is influenced by the environment in early life. Intrauterine and early postnatal malnutrition and the following catch-up growth have a long-term effect on blood pressure and endothelial function in adulthood. Well-established prenatal or/and postnatal animal models are used to study the impact of different nutritional intervention on CVD in adulthood. This article reviews the early original cause of chronic CVD in adulthood with the hypothesis of DOHaD (the developmental origins of health and disease), and proposes possible preventions in early life on the basis of this theory.

Epigenetics of Notch1 regulation in pulmonary microvascular rarefaction following extrauterine growth restriction.

BACKGROUND: Extrauterine growth restriction (EUGR) plays an important role in the developmental origin of adult cardiovascular diseases. In an EUGR rat model, we reported an elevated pulmonary arterial pressure in adults and genome-wide epigenetic modifications in pulmonary vascular endothelial cells (PVECs). However, the underlying mechanism of the early nutritional insult that results in pulmonary vascular consequences later in life remains unclear. METHODS: A rat model was used to investigate the physiological and structural effect of EUGR on early pulmonary vasculature by evaluating right ventricular systolic pressure and pulmonary vascular density in male rats. Epigenetic modifications of the Notch1 gene in PVECs were evaluated. RESULTS: EUGR decreased pulmonary vascular density with no significant impact on right ventricular systolic pressure at 3 weeks. Decreased transcription of Notch1 was observed both at 3 and 9 weeks, in association with decreased downstream target gene, Hes-1. Chromatin immunoprecipitation and bisulfite sequencing were performed to analyze the epigenetic modifications of the Notch1 gene promoter in PVECs. EUGR caused a significantly increased H3K27me3 in the proximal Notch1 gene promoter, and increased methylation of single CpG sites in the distal Notch1 gene promoter, both at 3 and 9 weeks. CONCLUSIONS: We conclude that EUGR results in decreased pulmonary vascular growth in association with decreased Notch1 in PVECs. This may be mediated by increased CpG methylation and H3K27me3 in the Notch1 gene promoter region.

Extrauterine growth restriction on pulmonary vascular endothelial dysfunction in adult male rats: the role of epigenetic mechanisms.

OBJECTIVE: Early postnatal life is considered as a critical time window for the determination of long-term metabolic states and organ functions. Extrauterine growth restriction (EUGR) causes the development of adult-onset chronic diseases, including pulmonary hypertension. However, the effects of nutritional disadvantages during the early postnatal period on pulmonary vascular consequences in later life are not fully understood. Our study was designed to test whether epigenetics dysregulation mediates the cellular memory of this early postnatal event. METHODS AND RESULTS: To test this hypothesis, we isolated pulmonary vascular endothelial cells by magnetic-activated cell sorting from EUGR and control rats. A postnatal insult, nutritional restriction-induced EUGR caused development of an increased pulmonary artery pressure at 9 weeks of age in male Sprague-Dawley rats. Methyl-DNA immune precipitation chip, genome-scale mapping studies to search for differentially methylated loci between control and EUGR rats, revealed significant difference in cytosine methylation between EUGR and control rats. EUGR changes the cytosine methylation at approximately 500 loci in male rats at 9 weeks of age, preceding the development of pulmonary hypertension and these represent the candidate loci for mediating the pathogenesis of pulmonary vascular disease that occurs later in life. Gene ontology analysis on differentially methylated genes showed that hypermethylated genes in EUGR are vascular development-associated genes and hypomethylated genes in EUGR are late-differentiation-associated and signal transduction genes. We validated candidate dysregulated loci with the quantitative assays of cytosine methylation and gene expressions. CONCLUSION: These results demonstrate that epigenetics dysregulation is a strong mechanism for propagating the cellular memory of early postnatal events, causing changes in the expression of genes and long-term susceptibility to pulmonary hypertension, and further providing a new insight into the prevention and treatment of EUGR-related pulmonary hypertension.