Monica Gostissa

DNA Repair, Genome Stability, and Aging

Aging can be defined as progressive functional decline and increasing mortality over time. Here, we review evidence linking aging to nuclear DNA lesions: DNA damage accumulates with age, and DNA repair defects can cause phenotypes resembling premature aging. We discuss how cellular DNA damage responses may contribute to manifestations of aging. We review Sir2, a factor linking genomic stability, metabolism, and aging. We conclude with a general discussion of the role of mutant mice in aging research and avenues for future investigation.

Homeodomain-interacting protein kinase-2 phosphorylates p53 at Ser 46 and mediates apoptosis.

Phosphorylation of p53 at Ser 46 was shown to regulate p53 apoptotic activity. Here we demonstrate that homeodomain-interacting protein kinase-2 (HIPK2), a member of a novel family of nuclear serine/threonine kinases, binds to and activates p53 by directly phosphorylating it at Ser 46. HIPK2 localizes with p53 and PML-3 into the nuclear bodies and is activated after irradiation with ultraviolet. Antisense inhibition of HIPK2 expression reduces the ultraviolet-induced apoptosis. Furthermore, HIPK2 and p53 cooperate in the activation of p53-dependent transcription and apoptotic pathways. These data define a new functional interaction between p53 and HIPK2 that results in the targeted subcellular localization of p53 and initiation of apoptosis.

Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1

The growth‐suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin‐like protein SUMO‐1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO‐1. Unlike ubiquitin, attachment of SUMO‐1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO‐1 to wild‐type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO‐1 conjugation provides a novel mechanism to regulate p53 activity.

The prolyl isomerase Pin1 reveals a mechanism to control p53 functions after genotoxic insults

The tumour suppressor p53 is important in the cell decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 post-translational modifications and association with other proteins have been implicated in the regulation of its stability and transcriptional activities. Here we report that, on DNA damage, p53 interacts with Pin1, a peptidyl-prolyl isomerase, which regulates the function of many proteins involved in cell cycle control and apoptosis. The interaction is strictly dependent on p53 phosphorylation, and requires Ser 33, Thr 81 and Ser 315. On binding, Pin1 generates conformational changes in p53, enhancing its transactivation activity. Stabilization of p53 is impaired in UV-treated Pin1-/- cells owing to its inability to efficiently dissociate from Mdm2. As a consequence, a reduced p53-dependent response was detected in Pin1-/- cells, and this correlates with a diminished transcriptional activation of some p53-regulated genes. Our results suggest that, following stress-induced phosphorylation, p53 needs to form a complex with Pin1 and to undergo a conformational change to fulfil its biological roles.

Regulation of p53 activity in nuclear bodies by a specific PML isoform

Covalent modification of the promyelocytic leukaemia protein (PML) by SUMO‐1 is a prerequisite for the assembly of nuclear bodies (NBs), subnuclear structures disrupted in various human diseases and linked to transcriptional and growth control. Here we demonstrate that p53 is recruited into NBs by a specific PML isoform (PML3) or by coexpression of SUMO‐1 and hUbc9. NB targeting depends on the direct association of p53, through its core domain, with a C‐terminal region of PML3. The relocalization of p53 into NBs enhances p53 transactivation in a promoter‐specific manner and affects cell survival. Our results indicate the existence of a cross‐talk between PML‐ and p53‐dependent growth suppression pathways, implying an important role for NBs and their resident proteins as modulators of p53 functions.

Genome-wide Translocation Sequencing Reveals Mechanisms of Chromosome Breaks and Rearrangements in B Cells

Whereas chromosomal translocations are common pathogenetic events in cancer, mechanisms that promote them are poorly understood. To elucidate translocation mechanisms in mammalian cells, we developed high-throughput, genome-wide translocation sequencing (HTGTS). We employed HTGTS to identify tens of thousands of independent translocation junctions involving fixed I-SceI meganuclease-generated DNA double-strand breaks (DSBs) within the c-myc oncogene or IgH locus of B lymphocytes induced for activation-induced cytidine deaminase (AID)-dependent IgH class switching. DSBs translocated widely across the genome but were preferentially targeted to transcribed chromosomal regions. Additionally, numerous AID-dependent and AID-independent hot spots were targeted, with the latter comprising mainly cryptic I-SceI targets. Comparison of translocation junctions with genome-wide nuclear run-ons revealed a marked association between transcription start sites and translocation targeting. The majority of translocation junctions were formed via end-joining with short microhomologies. Our findings have implications for diverse fields, including gene therapy and cancer genomics.

Excision of reprogramming transgenes improves the differentiation potential of iPS cells generated with a single excisable vector.

The residual presence of integrated transgenes following the derivation of induced pluripotent stem (iPS) cells is highly undesirable. Here we demonstrate efficient derivation of iPS cells free of exogenous reprogramming transgenes using an excisable polycistronic lentiviral vector. A novel version of this vector containing a reporter fluorochrome allows direct visualization of vector excision in living iPS cells in real time. We find that removal of the reprogramming vector markedly improves the developmental potential of iPS cells and significantly augments their capacity to undergo directed differentiation in vitro. We further propose that methods to efficiently excise reprogramming transgenes with minimal culture passaging, such as those demonstrated here, are critical since we find that iPS cells may acquire chromosomal abnormalities, such as trisomy of chromosome 8, similar to embryonic stem cells after expansion in culture. Our findings illustrate an efficient method for the generation of transgene‐free iPS cells and emphasize the potential beneficial effects that may result from elimination of integrated reprogramming factors. In addition, our results underscore the consequences of long‐term culture that will need to be taken into account for the clinical application of iPS cells. STEM CELLS 2010;28:64–74

Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease.

Osteosarcoma is the most common primary malignant tumor of bone. Analysis of familial cancer syndromes and sporadic cases has strongly implicated both p53 and pRb in its pathogenesis; however, the relative contribution of these mutations to the initiation of osteosarcoma is unclear. We describe here the generation and characterization of a genetically engineered mouse model in which all animals develop short latency malignant osteosarcoma. The genetically engineered mouse model is based on osteoblast-restricted deletion of p53 and pRb. Osteosarcoma development is dependent on loss of p53 and potentiated by loss of pRb, revealing a dominance of p53 mutation in the development of osteosarcoma. The model reproduces many of the defining features of human osteosarcoma including cytogenetic complexity and comparable gene expression signatures, histology, and metastatic behavior. Using a novel in silico methodology termed cytogenetic region enrichment analysis, we demonstrate high conservation of gene expression changes between murine osteosarcoma and known cytogentically rearranged loci from human osteosarcoma. Due to the strong similarity between murine osteosarcoma and human osteosarcoma in this model, this should provide a valuable platform for addressing the molecular genetics of osteosarcoma and for developing novel therapeutic strategies.

Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters.

ABSTRACT In response to DNA damage, p53 activates G1/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G2/M transition. Like most G2/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the αC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)—HDAC1 before HDAC4 and HDAC5—and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G2/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G2/M transition, and (iii) delineate a new role for PCAF in cell cycle control.

Mechanisms that Promote and Suppress Chromosomal Translocations in Lymphocytes

Recurrent chromosomal translocations are characteristic features of many types of cancers, especially lymphomas and leukemias. Several basic mechanistic factors are required for the generation of most translocations. First, DNA double-strand breaks (DSBs) must be present simultaneously at the two participating loci. Second, the two broken loci must either be in proximity or be moved into proximity to be joined. Finally, cellular DNA repair pathways must be available to join the two broken loci to complete the translocation. These mechanistic factors can vary in different normal and mutant cells and, as a result, substantially influence the frequency at which particular translocations are generated in a given cell type. Ultimately, however, appearance of recurrent oncogenic translocations in tumors is, in most cases, strongly influenced by selection for the translocated oncogene during the tumorigenesis process. In this review, we discuss in depth the factors and pathways that contribute to the generation of translocations in lymphocytes and other cell types. We also discuss recent findings regarding mechanisms that underlie the appearance of recurrent translocations in tumors.