TFIIH: at the crossroads of cancer and ageing

It is widely although not uniformly accepted that ageing is caused by time-dependent accumulation of damage. A side effect of ageing is an increased risk of developing a disease, such as cancer. Yet, the primary molecular target of damage accumulation has remained obscure. A clue has emerged from the notion that human inborn premature ageing syndromes are often associated with mutations in genes involved in DNA metabolism such as nucleotide excision repair (NER). In this thesis, mechanisms of pathological and normal ageing are addressed by genocopying naturally occurring human NER mutants with accelerated ageing and/or cancer phenotype in mouse model systems. Pathological conditions associated with mutations in the XPD and XPB genes, encoding the helicase components of the multifunctional TFIIH complex, range from a dramatic 1000 times elevated cancer predisposition (Xeroderma pigmentosum) to the severe neurodevelopmental premature ageing disorders trichothiodystrophy (TTD) and XP combined with Cockayne syndrome (XPCS). The aim of this thesis is to understand the mechanisms of accelerated ageing and to uncover variables determining the immense clinical heterogeneity ofNER disorders. Chapter 1 introduces the theory of ageing, summarizes clinical consequences ofNER mutations and describes the basic DNA repair mechanims involved. Chapter 2 presents experiments revealing TTD as a progeroid syndrome and defines the crucial role of DNA damage and repair in the rate ofTTD-related ageing. Chapter 3 reveals interallelic complementation between differentially compromised XPD molecules in mice and suggests a new variable in genotype-phenotype relationships within human recessive disease. Chapter 4 shows that CS and TTD share a common root cause in defective DNA repair. Chapter 5 uncovers the widely pleiotropic effects of combining different NER defects with the latent Xp!JXPCS mutation, ranging from moderately enhanced ageing to immediate postnatal lethality. Chapter 6 describes how interallelic complementation can be used for the reconstitution of CS pathology in mice and outlines some potentials for therapy. Chapter 7 reviews all major features of the NER mouse models generated in this thesis and elsewhere and sets forward a model for NER- associated diseas

Improving CRISPR/Cas9 mutagenesis efficiency by delaying the early development of zebrafish embryos

CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (H2S) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 degrees C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.Peer reviewe

Deficiency in Nucleotide Excision Repair Family Gene Activity, Especially ERCC3, Is Associated with Non-Pigmented Hair Fiber Growth

We conducted a microarray study to discover gene expression patterns associated with a lack of melanogenesis in non-pigmented hair follicles (HF) by microarray. Pigmented and non-pigmented HFs were collected and micro-dissected into the hair bulb (HB) and the upper hair sheaths (HS) including the bulge region. In comparison to pigmented HS and HBs, nucleotide excision repair (NER) family genes ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH exhibited statistically significantly lower expression in non- pigmented HS and HBs. Quantitative PCR verified microarray data and identified ERCC3 as highly differentially expressed. Immunohistochemistry confirmed ERCC3 expression in HF melanocytes. A reduction in ERCC3 by siRNA interference in human melanocytes in vitro reduced their tyrosinase production ability. Our results suggest that loss of NER gene function is associated with a loss of melanin production capacity. This may be due to reduced gene transcription and/or reduced DNA repair in melanocytes which may eventually lead to cell death. These results provide novel information with regard to melanogenesis and its regulation

Pro-Survival Role for Parkinson's Associated Gene DJ-1 Revealed in Trophically Impaired Dopaminergic Neurons

A mouse genetic study reveals a novel cell-survival role for the Parkinson's disease-associated gene DJ-1 in dopaminergic neurons that have reduced support from endogenous survival factors

Extended longevity mechanisms in short-lived progeroid mice: Identification of a preservative stress response associated with successful aging

Semantic distinctions between "normal" aging, "pathological" aging (or age-related disease) and "premature" aging (otherwise known as segmental progeria) potentially confound important insights into the nature of each of the complex processes. Here we review a recent, unexpected discovery: the presence of longevity-associated characteristics typical of long-lived endocrine-mutant and dietary-restricted animals in short-lived progeroid mice. These data suggest that a subset of symptoms observed in premature aging, and possibly normal aging as well, may be indirect manifestations of a beneficial adaptive stress response to endogenous oxidative damage, rather than a detrimental result of the damage itself