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Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer.

To investigate the role of the presumed DNA mismatch repair (MMR) gene Msh2 in genome stability and tumorigenesis, we have generated cells and mice that are deficient for the gene. Msh2-deficient cells have lost mismatch binding and have acquired microsatellite instability, a mutator phenotype, and tolerance to methylating agents. Moreover, in these cells, homologous recombination has lost dependence on complete identity between interacting DNA sequences, suggesting that Msh2 is involved in safeguarding the genome from promiscuous recombination. Msh2-deficient mice display no major abnormalities, but a significant fraction develops lymphomas at an early age. Thus, Msh2 is involved in MMR, controlling several aspects of genome stability; loss of MMR-controlled genome stability predisposes to cancer.

E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements

E12 and E47 are two helix-loop-helix transcription factors that arise by alternative splicing of the E2A gene. Both have been implicated in the regulation of immunoglobulin gene expression. We have now generated E2A (-/-) mice by gene targeting. E2A-null mutant mice fail to generate mature B cells. The arrest of B cell development occurs at an early stage, since no immunoglobulin DJ rearrangements can be detected in homozygous mutant mice. While immunoglobulin germline I mu RAG-1, mb-1, CD19, and lambda 5 transcripts are dramatically reduced in fetal livers of E2A (-/-) mice, B29 and mu degrees transcripts are present, but at lower levels. In addition, we show that Pax-5 transcripts are significantly reduced in fetal livers of E2A (-/-) mice. These data suggest a crucial role for E2A products as central regulators in early B cell differentiation.

Retinoblastoma protein functions as a molecular switch determining white versus brown adipocyte differentiation

Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient (Rb–/–) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIα, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb–/–MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se. Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to β3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.

Both E12 and E47 Allow Commitment to the B Cell Lineage

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSα (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSβ (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.

CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA‐PKcs (non‐homologous end‐joining). Genes of the non‐homologous end‐joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.

Flp-mediated tissue-specific inactivation of the retinoblastoma tumor suppressor gene in the mouse

The yeast-derived Flp-frt site-specific DNA recombination system was used to achieve pituitary-specific inactivation of the retinoblastoma (Rb) tumor suppressor gene. Whereas mice carrying only frt sites in both alleles of Rb remain tumor free, tumorigenesis ensues when the Flp recombinase is expressed. The rate of tumorigenesis in these mice depends both on the expression level of the Flp recombinase and on the presence of frt sites in one or both Rb alleles. This permitted a more accurate definition of the consecutive steps in pituitary tumorigenesis. Our study illustrates the potential of this approach for studying sporadic cancer in a defined mouse model.

Adenosine-deaminase-deficient mice die perinatally and exhibit liver-cell degeneration, atelectasis and small intestinal cell death

We report the generation and characterization of mice lacking adenosine deaminase (ADA). In humans, absence of ADA causes severe combined immunodeficiency. In contrast, ADA–deficient mice die perinatally with marked liver–cell degeneration, but lack abnormalities in the thymus. The ADA substrates, adenosine and deoxyadenosine, are increased in ADA–deficient mice. Adenine deoxyribonucleotides are only modestly elevated, whereas S–adenosylhomocysteine hydrolase activity is reduced more than 85%. Consequently, the ratio of S–adenosylhomocysteine (AdoMet) to S–adenosyl homocysteine (AdoHcy) is reduced threefold in liver. We conclude that ADA plays a more critical role in murine than human fetal development. The murine liver pathology may be due to AdoHcy–mediated inhibition of AdoMet–dependent transmethylation reactions.

Pocket Protein Complexes Are Recruited to Distinct Targets in Quiescent and Proliferating Cells

ABSTRACT Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells.

Msh3 is a limiting factor in the formation of intergenerational CTG expansions in DM1 transgenic mice

The CTG repeat involved in myotonic dystrophy is one of the most unstable trinucleotide repeats. However, the molecular mechanisms underlying this particular form of genetic instability—biased towards expansions—have not yet been completely elucidated. We previously showed, with highly unstable CTG repeat arrays in DM1 transgenic mice, that Msh2 is required for the formation of intergenerational and somatic expansions. To identify the partners of Msh2 in the formation of intergenerational CTG repeat expansions, we investigated the involvement of Msh3 and Msh6, partners of Msh2 in mismatch repair. Transgenic mice with CTG expansions were crossed with Msh3- or Msh6-deficient mice and CTG repeats were analysed after maternal and paternal transmissions. We demonstrated that Msh3 but not Msh6 plays also a key role in the formation of expansions over successive generation. Furthermore, the absence of one Msh3 allele was sufficient to decrease the formation of expansions, indicating that Msh3 is rate-limiting in this process. In the absence of Msh6, the frequency of expansions decreased only in maternal transmissions. However, the significantly lower levels of Msh2 and Msh3 proteins in Msh6 -/- ovaries suggest that the absence of Msh6 may have an indirect effect.