The p53 protein is a major mediator of the cellular response to genotoxic stress and is a crucial suppressor of tumor formation. In a variety of organisms, p53 and its paralogs, p63 and p73, each encode multiple protein isoforms through alternative splicing, promoters, and translation start sites. The function of these isoforms in development and disease are still being defined. Here, we evaluate the apoptotic potential of multiple isoforms of the single p53 gene in the genetic model Drosophila melanogaster. Most previous studies have focused on the p53A isoform, but it has been recently shown that a larger p53B isoform can induce apoptosis when overexpressed. It has remained unclear, however, whether one or both isoforms are required for the apoptotic response to genotoxic stress. We show that p53B is a much more potent inducer of apoptosis than p53A when overexpressed. Overexpression of two newly identified short isoforms perturbed development and inhibited the apoptotic response to ionizing radiation. Analysis of physiological protein expression indicated that p53A is the most abundant isoform, and that both p53A and p53B can form a complex and co-localize to sub-nuclear compartments. In contrast to the overexpression results, new isoformspecific loss-of-function mutants indicated that it is the shorter p53A isoform, not full-length p53B, that is the primary mediator of pro-apoptotic gene transcription and apoptosis after ionizing radiation. Together, our data show that it is the shorter p53A isoform that mediates the apoptotic response to DNA damage, and further suggest that p53B and shorter isoforms have specialized functions.

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ␤-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ␤-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ␤-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ␤-catenin leading to reduced tran- IntroductionFanconi anemia (FA) is the most common inherited bone marrow failure syndrome. The majority of patients develop bone marrow failure within the first decade of life. Patients are also at high risk for the development of acute myeloid leukemia and solid tumors of the head and neck and gastrointestinal tract. The disease is caused by loss-of-function of any 1 of 15 FANC genes. [1][2][3][4][5][6] The nuclear core complex includes FANCA, B, C, E, F, G, L, and M. 1 FANCL is a ring type E3 ubiquitin ligase that monoubiquitinates FANCD2 and FANCI, which enhances their function at sites of DNA damage. [7][8][9][10] The core complex scaffold is essential for facilitating the activity of FANCL because loss of any one of the core proteins results in loss of FANCL E3 ubiquitin ligase function. 1 Although FANC proteins are known to execute a normal DNA damage response to crosslinking agents, emerging evidence points to alternative functions for these proteins in hematopoietic stem cells (HSCs), and the loss of these alternative functions may represent a driving force behind the common FA complications of myelodysplasia and acute myeloid leukemia. [11][12][13][14][15][16] Functional defects in FA HSCs exist, including decreased repopulating ability, reduced numbers of HSCs, defective homing capacity, tumor necrosis factor (TNF)-␣ hypersensitivity, and limited replicative and survival potential compared with normal HSCs. [17][18][19][20][21][22][23][24][25][26] Collectively these studies support the argument that the FA pathway has a role in maintaining the HSC pool and regulating stem cell fitness. 15 However, the molecular mechanisms underlying such HSC defects have not been well characterized.In light of the well established role of TNF-␣ in the pathogenesis of marrow failure and leukemia in Fancc-deficient mice, [24][25][26] we first sought mechanistic insights by comparing the transcriptomal response to TNF-␣ of Fancc-deficient Kit ϩ /Sca1 ϩ /Lin Ϫ (KSL) marrow cells to the transcriptomal response of wild-type (WT) KSL cells. Ontological analysis of gene expression changes in...

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. In this study, we use integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). Our evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2F1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, our results indicate that the status of a CycA—Myb-MuvB—AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease.

SummaryCells switch to polyploid endoreplication cycles during development, wound healing, and cancer. We used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between developmental and induced endoreplicating cells (devECs and iECs). We found that while only devECs had a dampened E2F1 transcriptome, repression of a Cyclin A - Myb-MuvB - Aurora B mitotic network promoted endoreplication in both devECs and iECs. Cyclin A associated with and activated Myb-MuvB to induce transcription of mitotic genes, with expression of one, Aurora B, being key for mitotic commitment. Knockdown of Cyclin A, Myb, Aurora B, or downstream cytokinetic proteins induced distinct types of endoreplication, suggesting that repression of different mitotic network steps may explain the known diversity of polyploid cycles. These findings reveal how remodeling of a mitotic network promotes polyploid cycles that contribute to development, wound healing, and cancer.

The Fanconi anemia pathway supports hematopoietic stem cell survival in response to inflammatory and metabolic stress. We show that polyubiquitination and proteasome degradation of FANCL is inhibited by Akt1 activation, revealing a potentially important mechanism for the maintenance of stem cell function.