Genetic evidence for the involvement of mismatch repair proteins,PMS2 and MLH3, in a late step of homologous recombination

Homologous recombination (HR) repairs DNA double-strand breaks using intact homologous sequences as template DNA. Broken DNA and intact homologous sequences form joint molecules (JMs), including Holliday junctions (HJs), as HR intermediates. HJs are resolved to form crossover and noncrossover products. A mismatch repair factor, MLH3 endonuclease, produces the majority of crossovers during meiotic HR, but it remains elusive whether mismatch repair factors promote HR in nonmeiotic cells. We disrupted genes encoding the MLH3 and PMS2 endonucleases in the human B cell line, TK6, generating null MLH3^−/− and PMS2^−/− mutant cells. We also inserted point mutations into the endonuclease motif of MLH3 and PMS2 genes, generating endonuclease death MLH3^DN/DN and PMS2^EK/EK cells. MLH3^−/− and MLH3^DN/DN cells showed a very similar phenotype, a 2.5-fold decrease in the frequency of heteroallelic HR-dependent repair of restriction enzyme–induced double-strand breaks. PMS2^−/− and PMS2^EK/EK cells showed a phenotype very similar to that of the MLH3 mutants. These data indicate that MLH3 and PMS2 promote HR as an endonuclease. The MLH3^DN/DN and PMS2^EK/EK mutations had an additive effect on the heteroallelic HR. MLH3^DN/DN/PMS2^EK/EK cells showed normal kinetics of γ-irradiation–induced Rad51 foci but a significant delay in the resolution of Rad51 foci and a 3-fold decrease in the number of cisplatin-induced sister chromatid exchanges. The ectopic expression of the Gen1 HJ re-solvase partially reversed the defective heteroallelic HR of MLH3^DN/DN/PMS2^EK/EK cells. Taken together, we propose that MLH3 and PMS2 promote HR as endonucleases, most likely by processing JMs in mammalian somatic cells.     

Handcuffing intrinsically disordered regions in Mlh1–Pms1 disrupts mismatch repair

The DNA mismatch repair (MMR) factor Mlh1–Pms1 contains long intrinsically disordered regions (IDRs) whose exact functions remain elusive. We performed cross-linking mass spectrometry to identify interactions within Mlh1–Pms1 and used this information to insert FRB and FKBP dimerization domains into their IDRs. Baker''s yeast strains bearing these constructs were grown with rapamycin to induce dimerization. A strain containing FRB and FKBP domains in the Mlh1 IDR displayed a complete defect in MMR when grown with rapamycin. but removing rapamycin restored MMR functions. Strains in which FRB was inserted into the IDR of one MLH subunit and FKBP into the other subunit were also MMR defective. The MLH complex containing FRB and FKBP domains in the Mlh1 IDR displayed a rapamycin-dependent defect in Mlh1–Pms1 endonuclease activity. In contrast, linking the Mlh1 and Pms1 IDRs through FRB-FKBP dimerization inappropriately activated Mlh1–Pms1 endonuclease activity. We conclude that dynamic and coordinated rearrangements of the MLH IDRs both positively and negatively regulate how the MLH complex acts in MMR. The application of the FRB-FKBP dimerization system to interrogate in vivo functions of a critical repair complex will be useful for probing IDRs in diverse enzymes and to probe transient loss of MMR on demand.     

Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I-SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.     

Conservation of functional asymmetry in the mammalian MutLα ATPase

The DNA mismatch repair (MMR) protein dimer MutLα is comprised of the MutL homologues MLH1 and PMS2, which each belong to the family of GHL ATPases. These ATPases undergo functionally important conformational changes, including dimerization of the NH₂-termini associated with ATP binding and hydrolysis. Previous studies in yeast and biochemical studies with the mammalian proteins established the importance of the MutLα ATPase for overall MMR function. Additionally, the studies in yeast demonstrated a functional asymmetry between the contributions of the Mlh1 and Pms1 ATPase domains to MMR that was not reflected in the biochemical studies. We investigated the effect of mutating the highly conserved ATP hydrolysis and Mg²⁺ binding residues of MLH1 and PMS2 in mammalian cell lines. Amino acid substitutions in MLH1 intended to impact either ATP binding or hydrolysis disabled MMR, as measured by instability at microsatellite sequences, to an extent similar to MLH1-null mutation. Furthermore, cells expressing these MLH1 mutations exhibited resistance to the MMR-dependent cytotoxic effect of 6-thioguanine (6-TG). In contrast, ATP hydrolysis and binding mutants of PMS2 displayed no measurable increase in microsatellite instability or resistance to 6-TG. Our findings suggest that, in vivo, the integrity of the MLH1 ATPase domain is more critical than the PMS2 ATPase domain for normal MMR functions. These in vivo results are in contrast to results obtained previously in vitro that showed no functional asymmetry within the MutLα ATPase, highlighting the differences between in vivo and in vitro systems.     

Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

The Huntington''s disease gene (HTT) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington''s disease Hdh^Q111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh^Q111) than on a 129 background (129.Hdh^Q111). Linkage mapping in (B6x129).Hdh^Q111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh^Q111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh^Q111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.     

An intact Pms2 ATPase domain is not essential for male fertility

The DNA mismatch repair (MMR) machinery in mammals plays critical roles in both mutation avoidance and spermatogenesis. Meiotic analysis of knockout mice of two different MMR genes, Mlh1 and Mlh3, revealed both male and female infertility associated with a defect in meiotic crossing over. In contrast, another MMR gene knockout, Pms2 (Pms2^ko/ko), which contained a deletion of a portion of the ATPase domain, produced animals that were male sterile but female fertile. However, the meiotic phenotype of Pms2^ko/ko males was less clear-cut than for Mlh1- or Mlh3-deficient meiosis. More recently, we generated a different Pms2 mutant allele (Pms2^cre), which results in deletion of the same portion of the ATPase domain. Surprisingly, Pms2^cre/cre male mice were completely fertile, suggesting that the ATPase domain of Pms2 is not required for male fertility. To explore the difference in male fertility, we examined the Pms2 RNA and found that alternative splicing of the Pms2^cre allele results in a predicted Pms2 containing the C-terminus, which contains the Mlh1-interaction domain, a possible candidate for stabilizing Mlh1 levels. To study further the basis of male fertility, we examined Mlh1 levels in testes and found that whereas Pms2 loss in Pms2^ko/ko mice results in severely reduced levels of Mlh1 expression in the testes, Mlh1 levels in Pms2^cre/cre testes were reduced to a lesser extent. Thus, we propose that a primary function of Pms2 during spermatogenesis is to stabilize Mlh1 levels prior to its critical crossing over function with Mlh3.     

Prediction and Testing of Biological Networks Underlying Intestinal Cancer

Colorectal cancer progresses through an accumulation of somatic mutations, some of which reside in so-called “driver” genes that provide a growth advantage to the tumor. To identify points of intersection between driver gene pathways, we implemented a network analysis framework using protein interactions to predict likely connections – both precedented and novel – between key driver genes in cancer. We applied the framework to find significant connections between two genes, Apc and Cdkn1a (p21), known to be synergistic in tumorigenesis in mouse models. We then assessed the functional coherence of the resulting Apc-Cdkn1a network by engineering in vivo single node perturbations of the network: mouse models mutated individually at Apc (Apc^1638N+/−) or Cdkn1a (Cdkn1a^−/−), followed by measurements of protein and gene expression changes in intestinal epithelial tissue. We hypothesized that if the predicted network is biologically coherent (functional), then the predicted nodes should associate more specifically with dysregulated genes and proteins than stochastically selected genes and proteins. The predicted Apc-Cdkn1a network was significantly perturbed at the mRNA-level by both single gene knockouts, and the predictions were also strongly supported based on physical proximity and mRNA coexpression of proteomic targets. These results support the functional coherence of the proposed Apc-Cdkn1a network and also demonstrate how network-based predictions can be statistically tested using high-throughput biological data.     

Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice

Constitutive β-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (Apc^Min) develop intestinal adenomas. Here, the crossing of Apc^Min with cyclin D1^−/− mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of Apc^Min/cyclin D1^+/− mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.     

Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.CROSSING over during meiosis not only generates variation but is also important for providing the necessary interactions between homologous chromosomes that ensure correct segregation at division I of meiosis. Recombination is initiated by the production of programmed double-strand breaks (DSBs), catalyzed by the covalently attached Spo11p (Bergerat et al. 1997; Keeney et al. 1997), aided by a number of proteins (reviewed in Keeney and Neale 2006). DSBs are made at a much higher frequency than crossovers, and designation of only a subset to yield crossovers is thought to occur during early stages of DSB repair (Borner et al. 2004). At least two distinct pathways contribute to the production of crossover events in Saccharomyces cerevisiae. The major pathway is dependent on Msh4p/Msh5p and the mismatch repair proteins Mlh1p and Mlh3p (Ross-MacDonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Wang et al. 1999; Abdullah et al. 2004) and the second pathway is dependent on Mus81p/Mms4p endonuclease (de los Santos et al. 2001, 2003).Mitotic mismatch repair (MMR) is the process by which mutations that arise during DNA replication and recombination are recognized and removed (reviewed in Kolodner 1996; Harfe and Jinks-Robertson 2000). Msh2p forms a heterodimer with Msh6p (MutSα) to repair base–base mismatches and small insertions and/or deletions and with Msh3p (MutSβ) to repair large insertions and/or deletions (reviewed in Jiricny 2006). Mlh1p forms heterodimers with Pms1p, Mlh2p, and Mlh3p to coordinate the removal of these mismatches (Prolla et al. 1994; Wang et al. 1999). Mlh1p/Pms1p (MutLα) are involved in the repair of all types of mismatches in combination with MutSα and MutSβ, and in the absence of either protein a mutator phenotype is observed (Habraken et al. 1997, 1998). Mlh1p/Mlh2p (MutLβ) and Mlh1p/Mlh3p (MutLγ) are involved in the MutSβ pathway only, which repairs frameshift mutations caused by insertions or deletions. Consequently mlh3Δ mutants only exhibit a weak mutator phenotype, due to a lesser involvement in mismatch repair and a partial overlap in function with Pms1p (Flores-Rozas and Kolodner 1998; Harfe et al. 2000).Although the MutL homologs interact primarily through their C-terminal domains (Pang et al. 1997; Ban and Yang 1998), it is thought that the N-terminal domains must also interact for the complex to be fully functional (Ban and Yang 1998). Binding of ATP causes the proteins to undergo conformational changes, which are essential for the interaction between the N termini (Ban et al. 1999; Tran and Liskay 2000; Sacho et al. 2008). ATP hydrolysis and subsequent release of ADP is required to allow the protein complex to return to its initial state, completing the cycle so that the subunits are ready to bind ATP again if required. Using mutants of MLH1 and PMS1 that are presumed to be defective for ATP binding and/or ATP hydrolysis, it has been shown that both of these functions are essential for fully effective mismatch repair (Tran and Liskay 2000). However, the ATP binding and ATP hydrolysis mutants of PMS1 exhibited lower mitotic mutation rates than the corresponding MLH1 ATPase mutants, suggesting that there is functional asymmetry within the Mlh1p/Pms1p heterodimer (Tran and Liskay 2000; Hall et al. 2002). Another example of the asymmetry in the contributions of these subunits to function can be seen in assays that measure recombination between diverged sequences (homeologous recombination). The Mlh1p ATPase activity has been shown to be more important for the suppression of homeologous recombination than Pms1p ATPase activity (Welz-Voegele et al. 2002). This functional asymmetry is supported by in vitro biochemical analysis that demonstrated Pms1p has a lower ATP binding affinity than Mlh1p (Hall et al. 2002).As mentioned above, Mlh1p/Mlh3p function in the Msh4p/Msh5p pathway for meiotic recombination (Hunter and Borts 1997; Santucci-Darmanin et al. 2000). The Msh4p/Msh5p complex is thought to act in the stabilization of Holliday junction intermediates to allow their resolution in a crossover configuration (Snowden et al. 2004). The Mlh1p/Mlh3p complex has been suggested to act in the resolution of these structures, either directly or indirectly. Human Pms2 and its yeast homolog, Pms1p, have been shown to possess a latent endonuclease activity, conferred by a motif that is conserved among some of the MutL homologs, including Mlh3p (Kadyrov et al. 2006, 2007). Mutations in the DHQA(X)₂E(X)₄E motif in yeast MLH3 cause defects in both mismatch repair and meiotic recombination equivalent to mlh3Δ, suggesting that Mlh3p may also possess an endonuclease activity that is important for the generation of crossovers (Nishant et al. 2008).ATP binding by Mlh1p has been shown to be important for both of its meiotic functions (crossing over and repair of heteroduplex DNA) (Pang et al. 1997; Tran and Liskay 2000; Hoffmann et al. 2003). In contrast, the ATP hydrolysis mutant mlh1-E31A/mlh1-E31A appears to have no effect on meiotic recombination (Tran and Liskay 2000; Hoffmann et al. 2003). This may partly be explained by in vitro studies demonstrating that this mutant exhibits a low level of ATPase activity (Hall et al. 2002).The meiotic functions of MLH1 can be functionally separated as shown by mutating the same residue, G98, to different amino acids (Hoffmann et al. 2003). The residue G98 is situated in the ATPase motif in the GFRGEAL box (GYRGDAL in Mlh3p), which forms the lid of the ATP binding pocket. Mutations in this motif are predicted to affect ATP binding and/or heterodimerization with Pms1p (Ban and Yang 1998; Ban et al. 1999). Mutating the residue G98 in the ATP binding lid to alanine resulted in defective repair of heteroduplex DNA while crossing over was unaffected, but when the same residue was mutated to valine both mismatch repair and crossover functions were defective (Hoffmann et al. 2003). The mlh1-G98V mutant disrupts the interaction of Mlh1p with Pms1p, while mlh1-G98A does not (Pang et al. 1997). This may contribute to the difference observed in the effect on crossing over as Mlh1p is thought to interact with Pms1p and Mlh3p through the same residues (Wang et al. 1999; Kondo et al. 2001). Consequently if the interaction with Pms1p is affected then it is likely that the interaction with Mlh3p is also disrupted.We constructed mlh3 mutants corresponding to the ATP binding and ATP hydrolysis mutants of mlh1 to explore the role of Mlh3p in meiotic recombination. We also constructed mlh3-G97A and mlh3-G97V mutants, equivalent to the mlh1-G98A/V pair that has been shown to differentially affect the mitotic and meiotic functions of Mlh1p. All mutants were assayed for mitotic mismatch repair, meiotic heteroduplex repair, crossing over, and chromosome segregation.     

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.     

Characterization of a Highly Conserved Binding Site of Mlh1 Required for Exonuclease I-Dependent Mismatch Repair

Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K_d values ranging from 8.1 to 17.4 μM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.     

MUS81 Generates a Subset of MLH1-MLH3–Independent Crossovers in Mammalian Meiosis

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81^−/− animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.     

Haploinsufficiency of c-Met in cd44-/- mice identifies a collaboration of CD44 and c-Met in vivo

Recent evidence has shown that the activation of receptor tyrosine kinases is not only dependent on binding of their ligands but in addition requires adhesion molecules as coreceptors. We have identified CD44v6 as a coreceptor for c-Met in several tumor and primary cells. The CD44v6 ectodomain is required for c-Met activation, whereas the cytoplasmic tail recruits ERM proteins and the cytoskeleton into a signalosome complex. Here we demonstrate that c-Met (and hepatocyte growth factor and Gab1) is haploinsufficient in a cd44^−/− background, as the cd44^−/−; met^+/− (and cd44^−/−; hgf^+/− and cd44^−/−; gab1^+/−) mice die at birth. They have impaired synaptic transmission in the respiratory rhythm-generating network and alterations in the phrenic nerve. These results are the first genetic data showing that CD44 and c-Met collaborate in vivo and that they are involved in synaptogenesis and axon myelination in the central and peripheral nervous systems.     

Fas/CD95 Deficiency in ApcMin/+ Mice Increases Intestinal Tumor Burden

Background

Fas, a member of the tumor necrosis family, is responsible for initiating the apoptotic pathway when bound to its ligand, Fas-L. Defects in the Fas-mediated apoptotic pathway have been reported in colorectal cancer.

Methodology/Principal Findings

In the present study, a variant of the Apc^Min/+ mouse, a model for the human condition, Familial Adenomatous Polyposis (FAP), was generated with an additional deficiency of Fas (Apc^Min/+/Fas^lpr) by cross-breeding Apc^Min/+ mice with Fas deficient (Fas^lpr) mice. One of the main limitations of the Apc^Min/+ mouse model is that it only develops benign polyps. However, Apc^Min/+/Fas^lpr mice presented with a dramatic increase in tumor burden relative to Apc^Min/+ mice and invasive lesions at advanced ages. Proliferation and apoptosis markers revealed an increase in cellular proliferation, but negligible changes in apoptosis, while p53 increased at early ages. Fas-L was lower in Apc^Min/+/Fas^lpr mice relative to Apc^Min/+ cohorts, which resulted in enhanced inflammation.

Conclusions/Significance

This study demonstrated that imposition of a Fas deletion in an Apc^Min/+ background results in a more aggressive phenotype of the Apc^Min/+ mouse model, with more rapid development of invasive intestinal tumors and a decrease in Fas-L levels.     

Genetic Mechanisms in Apc-Mediated Mammary Tumorigenesis

Many components of Wnt/β-catenin signaling pathway also play critical roles in mammary tumor development, yet the role of the tumor suppressor gene APC (adenomatous polyposis coli) in breast oncongenesis is unclear. To better understand the role of Apc in mammary tumorigenesis, we introduced conditional Apc mutations specifically into two different mammary epithelial populations using K14-cre and WAP-cre transgenic mice that express Cre-recombinase in mammary progenitor cells and lactating luminal cells, respectively. Only the K14-cre–mediated Apc heterozygosity developed mammary adenocarcinomas demonstrating histological heterogeneity, suggesting the multilineage progenitor cell origin of these tumors. These tumors harbored truncation mutation in a defined region in the remaining wild-type allele of Apc that would retain some down-regulating activity of β-catenin signaling. Activating mutations at codons 12 and 61 of either H-Ras or K-Ras were also found in a subset of these tumors. Expression profiles of acinar-type mammary tumors from K14-cre; Apc^CKO/+ mice showed luminal epithelial gene expression pattern, and clustering analysis demonstrated more correlation to MMTV-neu model than to MMTV-Wnt1. In contrast, neither WAP-cre–induced Apc heterozygous nor homozygous mutations resulted in predisposition to mammary tumorigenesis, although WAP-cre–mediated Apc deficiency resulted in severe squamous metaplasia of mammary glands. Collectively, our results suggest that not only the epithelial origin but also a certain Apc mutations are selected to achieve a specific level of β-catenin signaling optimal for mammary tumor development and explain partially the colon- but not mammary-specific tumor development in patients that carry germline mutations in APC.     

Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair

The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.