PAX2 mutations cause renal-coloboma syndrome (RCS), a rare multi-system developmental abnormality involving optic nerve colobomas and renal abnormalities. End-stage renal failure is common in RCS, but the mechanism by which PAX2 mutations lead to renal failure is unknown. PAX2 is a member of a family of developmental genes containing a highly conserved 'paired box' DNA-binding domain, and encodes a transcription factor expressed primarily during fetal development in the central nervous system, eye, ear and urogenital tract. Presently, the role of PAX2 during kidney development is poorly understood. To gain insight into the cause of renal abnormalities in patients with PAX2 mutations, kidney anomalies were analyzed in patients with RCS, including a large Brazilian kindred in whom a new PAX2 mutation was identified. In a total of 29 patients, renal hypoplasia was the most common congenital renal abnormality. To determine the direct effects of PAX2 mutations on kidney development fetal kidneys of mice carrying a Pax2 (1Neu)mutation were examined. At E15, heterozygous mutant kidneys were approximately 60% of the size of wild-type littermates, and the number of nephrons was strikingly reduced. Heterozygous 1Neu mice showed increased apoptotic cell death during fetal kidney development, but the increased apoptosis was not associated with random stochastic inactivation of Pax2 expression in mutant kidneys; Pax2 was shown to be biallelically expressed during kidney development. These findings support the notion that heterozygous mutations of PAX2 are associated with increased apoptosis and reduced branching of the ureteric bud, due to reduced PAX2 dosage during a critical window in kidney development.

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.

The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell (HSC) self renewal and myeloid differentiation. Gfi1 null mice eventually die of HSC exhaustion, and Gfi1−/− HSC are not competitive in transplantation assays. Here we show that Gfi1 is a master regulator of microRNAs and that transcriptional control of a microRNA gene is critical for Gfi1-directed stem cell competitiveness and myelopoiesis. First, we show that the gene encoding miR21 is a direct transcriptional target of Gfi1. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal Gfi1 binding to specific DNA sequences upstream of the miR21 stem loop. Moreover, the expression of Gfi1 and miR21 is reciprocal in 1) wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), and 3) during treatment-induced differentiation of human myeloid leukemia cell lines. Forced expression of Gfi1 lowers miR21 levels in wild type Lin− bone marrow cells and human cancer cell lines. Knockdown of Gfi1 expression with shRNA in human cancer cell lines increases miR21 expression. Moreover, conditional deletion of Gfi1 induces miR21 expression in primary murine hematopoietic cells, including sorted CMP and GMP. Thus, Gfi1 transcriptionally regulates miR21 in both human and murine hematopoietic cells. Interestingly, we find that the Ski oncoprotein/transcriptional corepressor is a direct target of miR-21. Subsequently, Ski is dramatically reduced in Gfi1−/− HSC and in wild type bone marrow Lin− cells forced to express miR21. Gfi1 may repress miR21 to maintain functional competence. Specifically, we find that Ski is a previously undescribed Gfi1 corepressor. Both endogenous Ski and Gfi1 physically interact. Synthetic Ski and Gfi1 proteins reveal that the interaction is both strong and specific. Chromatin immunoprecipitation reveals Ski and Gfi1 occupy several Gfi1 target genes. However, Ski function is critical as a corepressor on only a subset of Gfi1 target genes. To determine the importance of Ski corepression to Gfi1 induced biology, we examined two well established phenotypes of Gfi1 loss of function; HSC competitiveness and myelopoiesis. When Gfi1−/− embryonic stem cells are injected into a wild type blastocyst, they do not participate in hematopoiesis. Similarly, we find that when Ski−/− embryonic stem cells are injected into a blastocyst, they infrequently participate in hematopoeisis. Next, because Ski−/− animals die at or before birth, we examined the fitness of Ski−/− fetal liver HSC. In competitive transplantation assays, Ski−/− fetal liver HSC were significantly impaired in reconstitution compared to congenic wild-type competitor fetal-liver HSC. Moreover, Ski null HSC generated significantly less myeloid progeny. Thus, Ski−/− HSC display a partial phenocopy of Gfi1−/− hematopoiesis. We conclude that Gfi1 directly targets miR21 to control the expression of Ski, a corepressor for Gfi1, and that the Gfi1/Ski complex is critical to regulate a subset of Gfi1 target genes important for HSC fitness and myeloid cell production.