Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients

Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1

Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis

Infectious Bronchitis Virus (IBV) is a highly contagious virus of chicken, causing huge economic losses in the poultry industry. Many genotypes circulate in a given area, and optimal protection relies on vaccination with live attenuated vaccines of the same genotype. As these live vaccines are derived from field viruses and circulate, understanding the prevalence of different IBV genotypes in any area is complex. In a recent study, the genome comparison of an IBV QX vaccine and its progenitor field strain led to the identification of vaccine markers. Here we developed a simplex SYBRgreen RT-qPCR assay for differentiation between QX-like field and vaccine strains and a multiplex SYBRgreen RT-qPCR assay for IBV genotyping with melting curve analysis, as each virus produced distinct and reliable melting peaks. Both the simplex and the multiplex assays showed excellent efficiency, sensitivity and specificity representing a low cost diagnostic tool for IBV genotyping and vaccine differentiation

Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis

Infectious Bronchitis Virus (IBV) is a highly contagious virus of chicken, causing huge economic losses in the poultry industry. Many genotypes circulate in a given area, and optimal protection relies on vaccination with live attenuated vaccines of the same genotype. As these live vaccines are derived from field viruses and circulate, understanding the prevalence of different IBV genotypes in any area is complex. In a recent study, the genome comparison of an IBV QX vaccine and its progenitor field strain led to the identification of vaccine markers. Here we developed a simplex SYBRgreen RT-qPCR assay for differentiation between QX-like field and vaccine strains and a multiplex SYBRgreen RT-qPCR assay for IBV genotyping with melting curve analysis, as each virus produced distinct and reliable melting peaks. Both the simplex and the multiplex assays showed excellent efficiency, sensitivity and specificity representing a low cost diagnostic tool for IBV genotyping and vaccine differentiation

Infectious bronchitis virus Mass-type (GI-1) and QX-like (GI-19) genotyping and vaccine differentiation using SYBR green RT-qPCR paired with melting curve analysis

Infectious Bronchitis Virus (IBV) is a highly contagious virus of chicken, causing huge economic losses in the poultry industry. Many genotypes circulate in a given area, and optimal protection relies on vaccination with live attenuated vaccines of the same genotype. As these live vaccines are derived from field viruses and circulate, understanding the prevalence of different IBV genotypes in any area is complex. In a recent study, the genome comparison of an IBV QX vaccine and its progenitor field strain led to the identification of vaccine markers. Here we developed a simplex SYBRgreen RT-qPCR assay for differentiation between QX-like field and vaccine strains and a multiplex SYBRgreen RT-qPCR assay for IBV genotyping with melting curve analysis, as each virus produced distinct and reliable melting peaks. Both the simplex and the multiplex assays showed excellent efficiency, sensitivity and specificity representing a low cost diagnostic tool for IBV genotyping and vaccine differentiation

Genetic Dissection of a Super Enhancer Controlling the Nppa-Nppb Cluster in the Heart

RATIONALE: ANP (atrial natriuretic peptide) and BNP (B-type natriuretic peptide), encoded by the clustered genes Nppa and Nppb, are important prognostic, diagnostic, and therapeutic proteins in cardiac disease. The spatiotemporal expression pattern and stress-induction of the Nppa and Nppb are tightly regulated, possibly involving their coregulation by an evolutionary conserved enhancer cluster. OBJECTIVE: To explore the physiological functions of the enhancer cluster and elucidate the genomic mechanism underlying Nppa-Nppb coregulation in vivo. METHODS AND RESULTS: By analyzing epigenetic data we uncovered an enhancer cluster with super enhancer characteristics upstream of Nppb. Using CRISPR/Cas9 genome editing, the enhancer cluster or parts thereof, Nppb and flanking regions or the entire genomic block spanning Nppa-Nppb, respectively, were deleted from the mouse genome. The impact on gene regulation and phenotype of the respective mouse lines was investigated by transcriptomic, epigenomic, and phenotypic analyses. The enhancer cluster was essential for prenatal and postnatal ventricular expression of Nppa and Nppb but not of any other gene. Enhancer cluster-deficient mice showed enlarged hearts before and after birth, similar to Nppa-Nppb compound knockout mice we generated. Analysis of the other deletion alleles indicated the enhancer cluster engages the promoters of Nppa and Nppb in a competitive rather than a cooperative mode, resulting in increased Nppa expression when Nppb and flanking sequences were deleted. The enhancer cluster maintained its active epigenetic state and selectivity when its target genes are absent. In enhancer cluster-deficient animals, Nppa was induced but remained low in the postmyocardial infarction border zone and in the hypertrophic ventricle, involving regulatory sequences proximal to Nppa. CONCLUSIONS: Coordinated ventricular expression of Nppa and Nppb is controlled in a competitive manner by a shared super enhancer, which is also required to augment stress-induced expression and to prevent premature hypertrophy