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1.  A Gnotobiotic Mouse Model Demonstrates that Dietary Fiber Protects Against Colorectal Tumorigenesis in a Microbiota- and Butyrate–Dependent Manner 
Cancer discovery  2014;4(12):1387-1397.
It is controversial whether dietary fiber protects against colorectal cancer because of conflicting results from human epidemiologic studies. However, these studies and mouse models of colorectal cancer have not controlled the composition of gut microbiota, which ferment fiber into short-chain fatty acids such as butyrate. Butyrate is noteworthy because it has energetic and epigenetic functions in colonocytes and tumorsuppressive properties in colorectal-cancer cell lines. We utilized gnotobiotic mouse models colonized with wild-type or mutant strains of a butyrate-producing bacterium to demonstrate that fiber does have a potent tumor-suppressive effect but in a microbiota- and butyrate-dependent manner. Furthermore, due to the Warburg effect, butyrate was metabolized less in tumors where it accumulated and functioned as an HDAC inhibitor to stimulate histone acetylation and affect apoptosis and cell proliferation. To support the relevance of this mechanism in human cancer, we demonstrate that butyrate and histone-acetylation levels are elevated in colorectal adenocarcinomas compared to normal colonic tissues.
PMCID: PMC4258155  PMID: 25266735
2.  The Collaborative Cross as a Resource for Modeling Human Disease: CC011/Unc, a New Mouse Model for Spontaneous Colitis 
Mammalian Genome  2014;25(3-4):95-108.
Inflammatory bowel disease (IBD) is an immune-mediated condition driven by improper responses to intestinal microflora in the context of environmental and genetic background. GWAS in humans have identified many loci associated with IBD, but animal models are valuable for dissecting the underlying molecular mechanisms, characterizing environmental and genetic contributions and developing treatments. Mouse models rely on interventions such as chemical treatment or introduction of an infectious agent to induce disease. Here, we describe a new model for IBD in which the disease develops spontaneously in 20-week-old mice in the absence of known murine pathogens. The model is part of the Collaborative Cross and came to our attention due to a high incidence of rectal prolapse in an incompletely inbred line. Necropsies revealed a profound proliferative colitis with variable degrees of ulceration and vasculitis, splenomegaly and enlarged mesenteric lymph nodes with no discernible anomalies of other organ systems. Phenotypic characterization of the CC011/Unc mice with homozygosity ranging from 94.1 to 99.8 % suggested that the trait was fixed and acted recessively in crosses to the colitis-resistant C57BL/6J inbred strain. Using a QTL approach, we identified four loci, Ccc1, Ccc2,Ccc3 and Ccc4 on chromosomes 12, 14, 1 and 8 that collectively explain 27.7 % of the phenotypic variation. Surprisingly, we also found that minute levels of residual heterozygosity in CC011/Unc have significant impact on the phenotype. This work demonstrates the utility of the CC as a source of models of human disease that arises through new combinations of alleles at susceptibility loci.
Electronic supplementary material
The online version of this article (doi:10.1007/s00335-013-9499-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3960486  PMID: 24487921
3.  Functional Redundancy of SWI/SNF Catalytic Subunits in Maintaining Vascular Endothelial Cells in the Adult Heart 
Circulation research  2012;111(5):e111-e122.
SWI/SNF chromatin-remodeling complexes utilize either BRG1 or BRM as a catalytic subunit to alter nucleosome position and regulate gene expression. BRG1 is required for vascular endothelial cell (VEC) development and embryonic survival, whereas BRM is dispensable.
To circumvent embryonic lethality and study Brg1 function in adult tissues, we utilized conditional gene targeting. To evaluate possible Brg1-Brm redundancy, we analyzed Brg1 mutant mice on wild-type and Brm-deficient backgrounds.
Methods and Results
The inducible Mx1-Cre driver was used to mutate Brg1 in adult mice. These conditional-null mutants exhibited a tissue-specific phenotype and unanticipated functional compensation between Brg1 and Brm. Brg1 single mutants were healthy and had a normal lifespan, whereas Brg1/Brm double mutants exhibited cardiovascular defects and died within one month. BRG1 and BRM were required for the viability of VECs but not other cell types where both genes were also knocked out. The VEC phenotype was most evident in the heart, particularly in the microvasculature of the outer myocardium, and was recapitulated in primary cells ex vivo. VEC death resulted in vascular leakage, cardiac hemorrhage, secondary death of cardiomyocytes due to ischemia, and ventricular dissections.
BRG1-catalyzed SWI/SNF complexes are particularly important in cardiovascular tissues. However, in contrast to embryonic development, where Brm does not compensate, Brg1 is required in adult VECs only when Brm is also mutated. These results demonstrate for the first time that Brm functionally compensates for Brg1 in vivo and that there are significant changes in the relative importance of BRG1- and BRM-catalyzed SWI/SNF complexes during the development of an essential cell lineage.
PMCID: PMC3501986  PMID: 22740088
BRG1; BRM; SWI/SNF; cardiac vascular endothelial cell; ventricular dissection
4.  Inactivation of SNF5 Cooperates with p53 Loss to Accelerate Tumor Formation in Snf5+/-;p53+/- Mice 
Molecular carcinogenesis  2009;48(12):1139-1148.
Malignant rhabdoid tumors (MRTs) are poorly differentiated pediatric cancers that arise in various anatomical locations and have a very poor outcome. The large majority of these malignancies are caused by loss of function of the SNF5/INI1 component of the SWI/SNF chromatin remodeling complex. However, the mechanism of tumor development associated with SNF5 loss remains unclear. Multiple studies have demonstrated a role for SNF5 in the regulation of cyclin D1, p16INK4A and pRbf activities suggesting it functions through the SWI/SNF complex to affect transcription of genes involved in cell cycle control. Previous studies in genetically engineered mouse models (GEMM) have shown that loss of SNF5 on a p53 null background significantly accelerates tumor development. Here, we use established GEMM to further define the relationship between the SNF5 and p53 tumor suppressor pathways. Combined haploinsufficiency of p53 and Snf5 leads to decreased latency for MRTs arising in alternate anatomical locations but not for the standard facial MRTs. We also observed acceleration in the appearance of T-cell lymphomas in the p53+/-;Snf5+/- mice. Our studies suggest that loss of SNF5 activity does not bestow a selective advantage on the p53 spectrum of tumors in the p53+/-;Snf5+/- mice. However, reduced p53 expression specifically accelerated the growth of a subset of MRTs in these mice.
PMCID: PMC3527082  PMID: 19676100
SNF5; p53; malignant rhabdoid tumor; SWI/SNF
5.  The BRG1 Chromatin Remodeler Protects Against Ovarian Cysts, Uterine Tumors, and Mammary Tumors in a Lineage-Specific Manner 
PLoS ONE  2012;7(2):e31346.
The BRG1 catalytic subunit of SWI/SNF-related complexes is required for mammalian development as exemplified by the early embryonic lethality of Brg1 null homozygous mice. BRG1 is also a tumor suppressor and, in mice, 10% of heterozygous (Brg1null/+) females develop mammary tumors. We now demonstrate that BRG1 mRNA and protein are expressed in both the luminal and basal cells of the mammary gland, raising the question of which lineage requires BRG1 to promote mammary homeostasis and prevent oncogenic transformation. To investigate this question, we utilized Wap-Cre to mutate both Brg1 floxed alleles in the luminal cells of the mammary epithelium of pregnant mice where WAP is exclusively expressed within the mammary gland. Interestingly, we found that Brg1Wap-Cre conditional homozygotes lactated normally and did not develop mammary tumors even when they were maintained on a Brm-deficient background. However, Brg1Wap-Cre mutants did develop ovarian cysts and uterine tumors. Analysis of these latter tissues showed that both, like the mammary gland, contain cells that normally express Brg1 and Wap. Thus, tumor formation in Brg1 mutant mice appears to be confined to particular cell types that require BRG1 and also express Wap. Our results now show that such cells exist both in the ovary and the uterus but not in either the luminal or the basal compartments of the mammary gland. Taken together, these findings indicate that SWI/SNF-related complexes are dispensable in the luminal cells of the mammary gland and therefore argue against the notion that SWI/SNF-related complexes are essential for cell survival. These findings also suggest that the tumor-suppressor activity of BRG1 is restricted to the basal cells of the mammary gland and demonstrate that this function extends to other female reproductive organs, consistent with recent observations of recurrent ARID1A/BAF250a mutations in human ovarian and endometrial tumors.
PMCID: PMC3283619  PMID: 22363625
6.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
PMCID: PMC1974845  PMID: 17570480
8.  The Scurfy mutation of FoxP3 in the thymus stroma leads to defective thymopoiesis 
The Journal of Experimental Medicine  2005;202(8):1141-1151.
The Scurfy mutation of the FoxP3 gene (FoxP3sf) in the mouse and analogous mutations in human result in lethal autoimmunity. The mutation of FoxP3 in the hematopoietic cells impairs the development of regulatory T cells. In addition, development of the Scurfy disease also may require mutation of the gene in nonhematopoietic cells. The T cell–extrinsic function of FoxP3 has not been characterized. Here we show that the FoxP3sf mutation leads to defective thymopoiesis, which is caused by inactivation of FoxP3 in the thymic stromal cells. FoxP3 mutation also results in overexpression of ErbB2 in the thymic stroma, which may be involved in defective thymopoiesis. Our data reveal a novel T cell–extrinsic function of FoxP3. In combination, the T cell–intrinsic and –extrinsic defects provide plausible explanation for the severity of the autoimmune diseases in the scurfy mice and in patients who have immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome.
PMCID: PMC2213221  PMID: 16230479
9.  Epiregulin Is Not Essential for Development of Intestinal Tumors but Is Required for Protection from Intestinal Damage 
Molecular and Cellular Biology  2004;24(20):8907-8916.
Epiregulin, an epidermal growth factor family member, acts as a local signal mediator and shows dual biological activity, stimulating the proliferation of fibroblasts, hepatocytes, smooth muscle cells, and keratinocytes while inhibiting the growth of several tumor-derived epithelial cell lines. The epiregulin gene (Ereg) is located on mouse chromosome 5 adjacent to three other epidermal growth factor family members, epigen, amphiregulin, and betacellulin. Gene targeting was used to insert a lacZ reporter into the mouse Ereg locus and to ablate its function. Although epiregulin is broadly expressed and regulated both spatially and temporally, Ereg null mice show no overt developmental defects, reproductive abnormalities, or altered liver regeneration. Additionally, in contrast to previous hypotheses, Ereg deficiency does not alter intestinal cancer susceptibility, as assayed in the ApcMin model, despite showing robust expression in developing tumors. However, Ereg null mice are highly susceptible to cancer-predisposing intestinal damage caused by oral administration of dextran sulfate sodium.
PMCID: PMC517889  PMID: 15456865
10.  The Role of Brg1, a Catalytic Subunit of Mammalian Chromatin-remodeling Complexes, in T Cell Development 
The Journal of Experimental Medicine  2003;198(12):1937-1949.
Mammalian SWI–SNF-related complexes use brahma-related gene 1 (Brg1) as a catalytic subunit to remodel nucleosomes and regulate transcription. Recent biochemical data has linked Brg1 function to genes important for T lymphocyte differentiation. To investigate the role of SWI–SNF-related complexes in this lineage, we ablated Brg1 function in T lymphocytes. T cell–specific Brg1-deficient mice showed profound thymic abnormalities, CD4 derepression at the double negative (DN; CD4− CD8−) stage, and a developmental block at the DN to double positive (CD4+ CD8+) transition. 5′-bromo-2′-deoxyuridine incorporation and annexin V staining establish a role for Brg1 complexes in the regulation of thymocyte cell proliferation and survival. This Brg1-dependent cell survival is specific for developing thymocytes as indicated by the presence of Brg1-deficient mature T lymphocytes that have escaped the developmental block in the thymus. However, reductions in peripheral T cell populations lead to immunodeficiency and compromised health of mutant mice. These results highlight the importance of chromatin-remodeling complexes at different stages in the development of a mammalian cell lineage.
PMCID: PMC2194157  PMID: 14676303
Brg1; SWI–SNF; chromatin; T cell; development
11.  Haploinsufficiency of p18INK4c Sensitizes Mice to Carcinogen-Induced Tumorigenesis 
Molecular and Cellular Biology  2003;23(4):1269-1277.
The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and thereby retains the growth-suppressive function of Rb family proteins. Mutations in the CDK4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice. Whereas loss of function of other INK4 genes in mice leads to little or no tumor development, targeted deletion of p18INK4c causes spontaneous pituitary tumors and lymphoma late in life. Here we show that treatment of p18 null and heterozygous mice with a chemical carcinogen resulted in tumor development at an accelerated rate. The remaining wild-type allele of p18 was neither mutated nor silenced in tumors derived from heterozygotes. Hence, p18 is a haploinsufficient tumor suppressor in mice.
PMCID: PMC141153  PMID: 12556487
12.  Functional Collaboration between Different Cyclin-Dependent Kinase Inhibitors Suppresses Tumor Growth with Distinct Tissue Specificity 
Molecular and Cellular Biology  2000;20(16):6147-6158.
The presence of two families of seven distinct mammalian cyclin-dependent kinase (CDK) inhibitor genes is thought to mediate the complexity of connecting a variety of cellular processes to the cell cycle control pathway. The distinct pattern of tissue expression of CDK inhibitor genes suggests that they may function as tumor suppressors with different tissue specificities. To test this hypothesis, we have characterized two strains of double mutant mice lacking either p18INK4c and p27KIP1 or p18INK4c and p21CIP1/WAF1. Loss of both p18 and p27 function resulted in the spontaneous development by 3 months of age of at least eight different types of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testes, pancreas, duodenum, and stomach. Six of these hyperplastic tissues and tumors were in endocrine organs, and several types of tumors routinely developed within the same animal, a phenotype reminiscent of that seen in combined human multiple endocrine neoplasia syndromes. The p18-p21 double null mice, on the other hand, developed pituitary adenomas, multifocal gastric neuroendocrine hyperplasia, and lung bronchioalveolar tumors later in life. G1 CDK2 and CDK4 kinase activities were increased in both normal and neoplastic tissues derived from mice lacking individual CDK inhibitors and were synergistically stimulated by the simultaneous loss of two CDK inhibitors. This indicates that an increase in G1 CDK kinase activity is a critical step during but is not sufficient for tumor growth. Our results suggest that functional collaborations between distinct CDK inhibitor genes are tissue specific and confer yet another level of regulation in cell growth control and tumor suppression.
PMCID: PMC86090  PMID: 10913196
13.  Growth Retardation, DNA Repair Defects, and Lack of Spermatogenesis in BRCA1-Deficient Mice 
Molecular and Cellular Biology  1999;19(10):7061-7075.
BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of tissues during cell division. The inheritance of a mutant BRCA1 allele dramatically increases a woman’s lifetime risk for developing both breast and ovarian cancers. A number of mouse lines carrying mutations in the Brca1 gene have been generated, and mice homozygous for these mutations generally die before day 10 of embryonic development. We report here the survival of a small number of mice homozygous for mutations in both the p53 and Brca1 genes. The survival of these mice is likely due to additional unknown mutations or epigenetic effects. Analysis of the Brca1−/− p53−/− animals indicates that BRCA1 is not required for the development of most organ systems. However, these mice are growth retarded, males are infertile due to meiotic failure, and the mammary gland of the female mouse is underdeveloped. Growth deficiency due to loss of BRCA1 was more thoroughly examined in an analysis of primary fibroblast lines obtained from these animals. Like p53−/− fibroblasts, Brca1−/− p53−/− cells proliferate more rapidly than wild-type cells; however, a high level of cellular death in these cultures results in reduced overall growth rates in comparison to p53−/− fibroblasts. Brca1−/− p53−/− fibroblasts are also defective in transcription-coupled repair and display increased sensitivity to DNA-damaging agents. We show, however, that after continued culture, and perhaps accelerated by the loss of BRCA1 repair functions, populations of Brca1−/− p53−/− fibroblasts with increased growth rates can be isolated. The increased survival of BRCA1-deficient fibroblasts in the absence of p53, and with the subsequent accumulation of additional growth-promoting changes, may mimic the events that occur during malignant transformation of BRCA1-deficient epithelia.
PMCID: PMC84701  PMID: 10490643

Results 1-13 (13)