Yansong Gu

Developmental defects and p53 hyperacetylation in Sir2 homolog (SIRT1)-deficient mice

SIRT1 is a mammalian homolog of the Saccharomyces cerevisiae chromatin silencing factor Sir2. Dominant-negative and overexpression studies have implicated a role for SIRT1 in deacetylating the p53 tumor suppressor protein to dampen apoptotic and cellular senescence pathways. To elucidate SIRT1 function in normal cells, we used gene-targeted mutation to generate mice that express either a mutant SIRT1 protein that lacks part of the catalytic domain or has no detectable SIRT1 protein at all. Both types of SIRT1 mutant mice and cells had essentially the same phenotypes. SIRT1 mutant mice were small, and exhibited notable developmental defects of the retina and heart, and only infrequently survived postnatally. Moreover, SIRT1-deficient cells exhibited p53 hyperacetylation after DNA damage and increased ionizing radiation-induced thymocyte apoptosis. In SIRT1-deficient embryonic fibroblasts, however, p53 hyperacetylation after DNA damage was not accompanied by increased p21 protein induction or DNA damage sensitivity. Together, our observations provide direct evidence that endogenous SIRT1 protein regulates p53 acetylation and p53-dependent apoptosis, and show that the function of this enzyme is required for specific developmental processes.

Growth retardation and leaky SCID phenotype of Ku70-deficient mice

Ku70, Ku80, and DNA-PKcs are subunits of the DNA-dependent protein kinase (DNA-PK), an enzyme implicated in DNA double-stranded break repair and V(D)J recombination. Our Ku70-deficient mice were about 50% the size of control littermates, and their fibroblasts were ionizing radiation sensitive and displayed premature senescence associated with the accumulation of nondividing cells. Ku70-deficient mice lacked mature B cells or serum immunoglobulin but, unexpectedly, reproducibly developed small populations of thymic and peripheral alpha/beta T lineage cells and had a significant incidence of thymic lymphomas. In association with B and T cell developmental defects, Ku70-deficient cells were severely impaired for joining of V(D)J coding and recombination signal sequences. These unanticipated features of the Ku70-deficient phenotype with respect to lymphocyte development and V(D)J recombination may reflect differential functions of the three DNA-PK components.

Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

SIRT1 and other NAD-dependent deacetylases have been implicated in control of cellular responses to stress and in tumorigenesis through deacetylation of important regulatory proteins, including p53 and the BCL6 oncoprotein. Hereby, we describe the identification of a compound we named cambinol that inhibits NAD-dependent deacetylase activity of human SIRT1 and SIRT2. Consistent with the role of SIRT1 in promoting cell survival during stress, inhibition of SIRT1 activity with cambinol during genotoxic stress leads to hyperacetylation of key stress response proteins and promotes cell cycle arrest. Treatment of BCL6-expressing Burkitt lymphoma cells with cambinol as a single agent induced apoptosis, which was accompanied by hyperacetylation of BCL6 and p53. Because acetylation inactivates BCL6 and has the opposite effect on the function of p53 and other checkpoint pathways, the antitumor activity of cambinol in Burkitt lymphoma cells may be accomplished through a combined effect of BCL6 inactivation and checkpoint activation. Cambinol was well tolerated in mice and inhibited growth of Burkitt lymphoma xenografts. Inhibitors of NAD-dependent deacetylases may constitute novel anticancer agents.

Telomere dysfunction and Atm deficiency compromises organ homeostasis and accelerates ageing

Ataxia-telangiectasia (A-T) results from the loss of ataxia-telangiectasia mutated (Atm) function and is characterized by accelerated telomere loss, genomic instability, progressive neurological degeneration, premature ageing and increased neoplasia incidence. Here we evaluate the functional interaction of Atm and telomeres in vivo. We examined the impact of Atm deficiency as a function of progressive telomere attrition at both the cellular and whole-organism level in mice doubly null for Atm and the telomerase RNA component (Terc). These compound mutants showed increased telomere erosion and genomic instability, yet they experienced a substantial elimination of T-cell lymphomas associated with Atm deficiency. A generalized proliferation defect was evident in all cell types and tissues examined, and this defect extended to tissue stem/progenitor cell compartments, thereby providing a basis for progressive multi-organ system compromise, accelerated ageing and premature death. We show that Atm deficiency and telomere dysfunction act together to impair cellular and whole-organism viability, thus supporting the view that aspects of A-T pathophysiology are linked to the functional state of telomeres and its adverse effects on stem/progenitor cell reserves.

Genetic interactions between ATM and the nonhomologous end-joining factors in genomic stability and development

DNA ligase IV (Lig4) and the DNA-dependent protein kinase (DNA-PK) function in nonhomologous end joining (NHEJ). However, although Lig4 deficiency causes late embryonic lethality, deficiency in DNA-PK subunits (Ku70, Ku80, and DNA-PKcs) does not. Here we demonstrate that, similar to p53 deficiency, ataxia-telangiectasia-mutated (ATM) gene deficiency rescues the embryonic lethality and neuronal apoptosis, but not impaired lymphocyte development, associated with Lig4 deficiency. However, in contrast to p53 deficiency, ATM deficiency enhances deleterious effects of Lig4 deficiency on growth potential of embryonic fibroblasts (MEFs) and genomic instability in both MEFs and cultured progenitor lymphocytes, demonstrating significant differences in the interplay of p53 vs. ATM with respect to NHEJ. Finally, in dramatic contrast to effects on Lig4 deficiency, ATM deficiency causes early embryonic lethality in Ku- or DNA-PKcs-deficient mice, providing evidence for an NHEJ-independent role for the DNA-PK holoenzyme.

SIRT1 is dispensable for function of hematopoietic stem cells in adult mice

SIRT1 is an NAD(+)-dependent histone deacetylase implicated in the establishment of the primitive hematopoietic system during mouse embryonic development. However, investigation of the role of SIRT1 in adult hematopoiesis has been complicated by the high perinatal mortality of SIRT1-deficient mice (SIRT1(-/-)). We performed a comprehensive in vivo study of the hematopoietic stem cell (HSC) compartment in adult SIRT1(-/-) mice and show that, apart from anemia and leukocytosis in older mice, the production of mature blood cells, lineage distribution within hematopoietic organs, and frequencies of the most primitive HSC populations are comparable to those of wild-type littermate controls. Furthermore, we show that SIRT1-deficient BM cells confer stable long-term reconstitution in competitive repopulation and serial transplantation experiments. The results of the present study rule out an essential physiologic role for cell-autonomous SIRT1 signaling in the maintenance of the adult HSC compartment in mice.