and collect data. BHC helped generate Tug1-transgenic mice and edit the manuscript. PAO helped edit the manuscript, and FRD oversaw experiments, prepared the manuscript, and provided guidance on overall project design.

Mitochondrial fission has been linked to the pathogenesis of diabetic nephropathy (DN). However, how mitochondrial fission affects progression of DN in vivo is unknown. Here, we report the effect of conditional podocyte-specific deletion of dynamin-related protein 1 (Drp1), an essential component of mitochondrial fission, on the pathogenesis and progression of DN. Inducible podocyte-specific deletion of Drp1 in diabetic mice decreased albuminuria and improved mesangial matrix expansion and podocyte morphology. Ultrastructure analysis revealed a significant increase in fragmented mitochondria in the podocytes of wild-type diabetic mice but a marked improvement in mitochondrial structure in Drp1-null podocytes of diabetic mice. When isolated from diabetic mice and cultured in high glucose, Drp1-null podocytes had more elongated mitochondria and better mitochondrial fitness associated with enhanced oxygen consumption and ATP production than wild-type podocytes. Furthermore, administration of a pharmacologic inhibitor of Drp1, Mdivi1, significantly blunted mitochondrial fission and rescued key pathologic features of DN in mice. Taken together, these results provide novel correlations between mitochondrial morphology and the progression of DN and point to Drp1 as a potential therapeutic target in DN.

Mice lacking p63 cannot form skin, exhibit craniofacial and skeletal defects, and die soon after birth. The p63 gene regulates a complex network of target genes, and disruption of p63 has been shown to affect the maintenance of epithelial stem cells, the differentiation of keratinocytes, and the preservation of the adhesive properties of stratified epithelium. Here, we show that inactivation of p63 in mice is accompanied by aberrantly increased expression of the Ink4a and Arf tumour suppressor genes. In turn, anomalies of the p63-null mouse affecting the skin and skeleton are partially ameliorated in mice lacking either Ink4a or Arf. Rescue of epithelialization is accompanied by restoration of keratinocyte proliferative capacity both in vivo and in vitro and by expression of markers of squamous differentiation. Thus, in the absence of p63, abnormal upregulation of Ink4a and Arf is incompatible with skin development.

Understanding the heterogeneous genetic mechanisms of tumor initiation in lymphoid leukemias (LL) will lead to improvements in prognostic classification and treatment regimens. In previous studies of mouse leukemias, we showed that retroviral insertion at the Evi32 locus leads to increased expression of Prdm14, a pluripotency gene implicated in the self-renewal capacity of embryonic stem cells and the early stages of breast cancer. Here we show that PRDM14 is also overexpressed in ~25% of human lymphoid neoplasms, with increased frequencies in T-cell acute LL (T-ALL) and hyperdiploid precursor B-cell acute LL (pre-B ALL). To test if Prdm14 overexpression could initiate leukemia, mice were transduced with bone marrow (BM) cells transfected with a Prdm14 expression vector. Lymphoid leukemias developed in 96% of female mice and 42% of male mice. Prior to the onset of leukemia, differentiation of transduced cells was biased up to 1000-fold towards cells with features of common lymphoid progenitors (CLP), and lymphoid differentiation showed a relative block at the pro-B stage. Microarray gene expression analysis of expanded CLP-like cells prior to the onset of leukemia demonstrated upregulation of genes involved in pluripotency, tumor initiation, early B-lineage commitment, Wnt/Ras signaling, and the epithelial-to-mesenchymal transition. Among the dysregulated genes were imprinted genes and non-coding RNAs including Dlk1 and Meg3, which are also key pluripotency mediators. Heightened expression of the estrogen-dependent oncogene, Myb, in tumors suggests a basis for the increased frequency of cancer in female mice. These data provide the first direct evidence for the association of Prdm14 with cancer initiation in an in vivo mouse model and in human lymphoid malignancies, while suggesting mechanisms for Prdm14’s mode of action.

SUMMARYPRDM14 functions in embryonic stem cell (ESC) maintenance to promote the expression of pluripotency-associated genes while suppressing differentiation genes. Expression of PRDM14 is tightly regulated and typically limited to ESCs and primordial germ cells; however, aberrant expression is associated with tumor initiation in a wide variety of human cancers, including breast cancer and leukemia. Here, we describe the generation of a Cre-recombinase-inducible mouse model for the spatial and temporal control of Prdm14 misexpression [ROSA26 floxed-stop Prdm14 (R26PR)]. When R26PR is mated to either of two Cre lines, Mx1-cre or MMTV-cre, mice develop early-onset T-cell acute lymphoblastic leukemia (T-ALL) with median overall survival of 41 and 64 days for R26PR;Mx1-cre and R26PR;MMTV-cre, respectively. T-ALL is characterized by the accumulation of immature single-positive CD8 cells and their widespread infiltration. Leukemia is preceded by a dramatic expansion of cells resembling hematopoietic stem cells and lymphoid-committed progenitors prior to disease onset, accompanied by a blockage in B-cell differentiation at the early pro-B stage. Rapid-onset PRDM14-induced T-ALL requires factors that are present in stem and progenitor cells: R26PR;dLck-cre animals, which express Prdm14 starting at the double-positive stage of thymocyte development, do not develop disease. PRDM14-induced leukemic cells contain high levels of activated NOTCH1 and downstream NOTCH1 targets, including MYC and HES1, and are sensitive to pharmacological inhibition of NOTCH1 with the γ-secretase inhibitor DAPT. Greater than 50% of human T-ALLs harbor activating mutations in NOTCH1; thus, our model carries clinically relevant molecular aberrations. The penetrance, short latency and involvement of the NOTCH1 pathway will make this hematopoietic R26PR mouse model ideal for future studies on disease initiation, relapse and novel therapeutic drug combinations. Furthermore, breeding R26PR to additional Cre lines will allow for the continued development of novel cancer models.

PRDM14 is an epigenetic regulator known for maintaining embryonic stem cell identity and resetting potency in primordial germ cells. However, hematopoietic expression of Prdm14 at supraphysiological levels results in fully penetrant and rapid-onset T-cell acute lymphoblastic leukemia (T-ALL) in the mouse. Here, we show that PRDM14-induced T-ALLs are driven by NOTCH1, a frequently mutated driver of human T-ALL. Notch1 is activated in this murine model via RAG-dependent promoter deletions and subsequent production of truncated, ligand-independent protein from downstream regions of the Notch1 locus. These T-ALLs also have focal changes in H3K4me3 deposition at the Notch1 locus and global increases in both H3K4me1 and H3K4me3. Using a PRDM14-FLAG mouse model, we show that PRDM14 binds within an intron of Notch1 prior to leukemia development. Our data support the idea that PRDM14 binding promotes a chromatin state that allows access of the RAG recombinase complex to cryptic RAG signal sequences embedded at the Notch1 locus. Indeed, breeding into a RAG recombination-deficient background abrogates T-ALL development and prevents Notch1 deletions, while allowing for transient hematopoietic stem cell (HSC)-like pre-leukemia cell expansion. Together, our data suggest that PRDM14 expands a progenitor cell population while promoting a permissive epigenetic state for the creation of driver mutations (here, in Notch1), enabling cancer development through the misappropriation of endogenous cellular DNA recombination machinery.

In this study, we show genetic modifier genes of Tp53 that can exacerbate embryonic abnormalities. Using a mouse model in which CE/J mice were crossed with the Tp53-null 129/Sv (129-Trp53(tm1 Tyj)) mice, a subset of Tp53+/- and -/- male and female embryos died during gestation. Our hypothesis, based on the genotypes of survivors, is that two genetic modifiers and a Tp53 null allele lead to an increase in embryonic lethality. We previously identified a recessive modifier (Mop1) from CE/J mice on chromosome 11 centromeric to Tp53. We have uncovered a dominant modifier (Mop2) from 129/Sv mice telomeric to Tp53. We discovered a polymorphic change (321P-->321S) of Ovca1 within the Mop2 locus of CE/J mice. This polymorphism increased both mRNA and protein levels of OVCA1 in various tissues. CE/J primary cells cultured from different tissues proliferated more rapidly than 129/Sv cells. In addition, CE/J cells cycled while 129/Sv cells had a higher arrest in the G1 phase. Transfection of Ovca1 containing the 321P polymorphism into CE/J cells caused a higher G1 arrest. The pattern of OVCA1 expression also changed from being diffuse throughout the cytoplasm in 129/Sv cells to being punctuate in the cytoplasm of CE/J cells. Tp53+/- abnormal embryos had more proliferating cells than normal embryos, but no obvious difference in differentiated neuronal cells. Tp53-/- small embryos had less differentiated neuronal cells and proliferating cells than normal embryos. Thus, a polymorphism of Ovca1, combined with Mop1, genetically modifies embryonic lethality in Tp53 deficient mice.

The tumor suppressor TP53 is mutated in approximately 70% of Li-Fraumeni syndrome (LFS) families; however, other genes may lead to the predisposition to tumors in other families. We developed a mouse model to search for other tumor suppressors that may be involved in the syndrome. Inbred CE/J mice, which succumb to multiple types of tumors similar to those found in LFS, were crossed with the Trp53-null 129-Trp53tm1Tyj mouse. We monitored the tumor onset and type and found a significant earlier tumor onset in the CE/J:129-Trp53tm1Tyj mice compared with 129-Trp53tm1Tyj mice with a Trp53-null allele. Additionally, in CE/J:129-Trp53tm1Tyj-Trp53+/- mice, the tumors metastasize, which does not occur in other strains of mice. Using simple-sequence length polymorphism analysis for loss of heterozygosity in tumors, we identified a putative tumor suppressor locus within 1 cM on mouse chromosome 11, which encompasses 12 mapped genes.