Björn Schumacher

The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis

In mammals, one of the key regulators necessary for responding to genotoxic stress is the p53 transcription factor. p53 is the single most commonly mutated tumor suppressor gene in human cancers. Here we report the identification of a C. elegans homolog of mammalian p53. Using RNAi and DNA cosuppression technology, we show that C. elegans p53 (cep-1) is required for DNA damage-induced apoptosis in the C. elegans germline. However,cep-1 RNAi does not affect programmed cell death occurring during worm development and physiological (radiation-independent) germ cell death. The DNA binding domain of CEP-1 is related to vertebrate p53 members and possesses the conserved residues most frequently mutated in human tumors. Consistent with this, CEP-1 acts as a transcription factor and is able to activate a transcriptional reporter containing consensus human p53 binding sites. Our data support the notion that p53-mediated transcriptional regulation is part of an ancestral pathway mediating DNA damage-induced apoptosis and reveals C. elegans as a genetically tractable model organism for studying the p53 apoptotic pathway.

The p53 network: cellular and systemic DNA damage responses in aging and cancer.

Genome instability contributes to cancer development and accelerates age-related pathologies as evidenced by a variety of congenital cancer susceptibility and progeroid syndromes that are caused by defects in genome maintenance mechanisms. DNA damage response (DDR) pathways that are mediated through the tumor suppressor p53 play an important role in the cell-intrinsic responses to genome instability, including a transient cell cycle arrest, senescence and apoptosis. Both senescence and apoptosis are powerful tumor-suppressive pathways preventing the uncontrolled proliferation of transformed cells. However, both pathways can potentially deplete stem and progenitor cell pools, thus promoting tissue degeneration and organ failure, which are both hallmarks of aging. p53 signaling is also involved in mediating non-cell-autonomous interactions with the innate immune system and in the systemic adjustments during the aging process. The network of p53 target genes thus functions as an important regulator of cancer prevention and aging.

Delayed and accelerated aging share common longevity assurance mechanisms

Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of “survival” responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension.

C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage

The p53 tumor suppressor promotes apoptosis in response to DNA damage. Here we describe the Caenorhabditis elegans gene ced-13, which encodes a conserved BH3-only protein. We show that ced-13 mRNA accumulates following DNA damage, and that this accumulation is dependent on an intact C. elegans cep-1/p53 gene. We demonstrate that CED-13 protein physically interacts with the antiapoptotic Bcl-2-related protein CED-9. Furthermore, overexpression of ced-13 in somatic cells leads to the death of cells that normally survive, and this death requires the core apoptotic pathway of C. elegans. Recent studies have implicated two BH3-only proteins, Noxa and PUMA, in p53-induced apoptosis in mammals. Our studies suggest that in addition to the BH3-only protein EGL-1, CED-13 might also promote apoptosis in the C. elegans germ line in response to p53 activation. We propose that an evolutionarily conserved pathway exists in which p53 promotes cell death by inducing expression of two BH3-only genes.

DNA damage in germ cells induces an innate immune response that triggers systemic stress resistance

DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin–proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.

Persistent transcription-blocking DNA lesions trigger somatic growth attenuation associated with longevity

The accumulation of stochastic DNA damage throughout an organisms lifespan is thought to contribute to ageing. Conversely, ageing seems to be phenotypically reproducible and regulated through genetic pathways such as the insulin-like growth factor-1 (IGF-1) and growth hormone (GH) receptors, which are central mediators of the somatic growth axis. Here we report that persistent DNA damage in primary cells from mice elicits changes in global gene expression similar to those occurring in various organs of naturally aged animals. We show that, as in ageing animals, the expression of IGF-1 receptor and GH receptor is attenuated, resulting in cellular resistance to IGF-1. This cell-autonomous attenuation is specifically induced by persistent lesions leading to stalling of RNA polymerase II in proliferating, quiescent and terminally differentiated cells; it is exacerbated and prolonged in cells from progeroid mice and confers resistance to oxidative stress. Our findings suggest that the accumulation of DNA damage in transcribed genes in most if not all tissues contributes to the ageing-associated shift from growth to somatic maintenance that triggers stress resistance and is thought to promote longevity.

Involvement of global genome repair, transcription coupled repair, and chromatin remodeling in UV DNA damage response changes during development.

Nucleotide Excision Repair (NER), which removes a variety of helix-distorting lesions from DNA, is initiated by two distinct DNA damage-sensing mechanisms. Transcription Coupled Repair (TCR) removes damage from the active strand of transcribed genes and depends on the SWI/SNF family protein CSB. Global Genome Repair (GGR) removes damage present elsewhere in the genome and depends on damage recognition by the XPC/RAD23/Centrin2 complex. Currently, it is not well understood to what extent both pathways contribute to genome maintenance and cell survival in a developing organism exposed to UV light. Here, we show that eukaryotic NER, initiated by two distinct subpathways, is well conserved in the nematode Caenorhabditis elegans. In C. elegans, involvement of TCR and GGR in the UV-induced DNA damage response changes during development. In germ cells and early embryos, we find that GGR is the major pathway contributing to normal development and survival after UV irradiation, whereas in later developmental stages TCR is predominantly engaged. Furthermore, we identify four ISWI/Cohesin and four SWI/SNF family chromatin remodeling factors that are implicated in the UV damage response in a developmental stage dependent manner. These in vivo studies strongly suggest that involvement of different repair pathways and chromatin remodeling proteins in UV-induced DNA repair depends on developmental stage of cells.

Next Generation Sequencing of miRNAs – Strategies, Resources and Methods

miRNAs constitute a family of small RNA species that have been demonstrated to play a central role in regulating gene expression in many organisms. With the advent of next generation sequencing, new opportunities have arisen to identify and quantify miRNAs and elucidate their function. The unprecedented sequencing depth reached by next generation sequencing technologies makes it possible to get a comprehensive miRNA landscape but also poses new challenges for data analysis. We provide an overview of strategies used for miRNA sequencing, public miRNA resources, and useful methods and tools that are available for data analysis.

Insights from the worm: The C. elegans model for innate immunity

The nematode worm Caenorhabditis elegans comprises an ancestral immune system. C. elegans recognizes and responds to viral, bacterial, and fungal infections. Components of the RNA interference machinery respond to viral infection, while highly conserved MAPK signaling pathways activate the innate immune response to bacterial infection. C. elegans has been particularly important for exploring the role of innate immunity in organismal stress resistance and the regulation of longevity. Also functions of neuronal sensing of infectious bacteria have recently been uncovered. Studies on nematode immunity can be instructive in exploring innate immune signaling in the absence of specialized immune cells and adaptive immunity.

p53 in the DNA-Damage-Repair Process

The cells in the human body are continuously challenged by a variety of genotoxic attacks. Erroneous repair of the DNA can lead to mutations and chromosomal aberrations that can alter the functions of tumor suppressor genes or oncogenes, thus causing cancer development. As a central tumor suppressor, p53 guards the genome by orchestrating a variety of DNA-damage-response (DDR) mechanisms. Already early in metazoan evolution, p53 started controlling the apoptotic demise of genomically compromised cells. p53 plays a prominent role as a facilitator of DNA repair by halting the cell cycle to allow time for the repair machineries to restore genome stability. In addition, p53 took on diverse roles to also directly impact the activity of various DNA-repair systems. It thus appears as if p53 is multitasking in providing protection from cancer development by maintaining genome stability.