Michael F. Kane

The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer

We have identified a human homolog of the bacterial MutS and S. cerevisiae MSH proteins, called hMSH2. Expression of hMSH2 in E. coli causes a dominant mutator phenotype, suggesting that hMSH2, like other divergent MutS homologs, interferes with the normal bacterial mismatch repair pathway. hMSH2 maps to human chromosome 2p22-21 near a locus implicated in hereditary nonpolyposis colon cancer (HNPCC). A T to C transition mutation has been detected in the -6 position of a splice acceptor site in sporadic colon tumors and in affected individuals of two small HNPCC kindreds. These data and reports indicating that S. cerevisiae msh2 mutations cause an instability of dinucleotide repeats like those associated with HNPCC suggest that hMSH2 is the HNPCC gene.

MEIOTIC PACHYTENE ARREST IN MLH1-DEFICIENT MICE

Germ line mutations in DNA mismatch repair genes including MLH1 cause hereditary nonpolyposis colon cancer. To understand the role of MLH1 in normal growth and development, we generated mice that have a null mutation of this gene. Mice homozygous for this mutation show a replication error phenotype, and extracts of these cells are deficient in mismatch repair activity. Homozygous mutant males show normal mating behavior but have no detectable mature sperm. Examination of meiosis in these males reveals that the cells enter meiotic prophase and arrest at pachytene. Homozygous mutant females have normal estrous cycles and reproductive and mating behavior but are infertile. The phenotypes of the mlh1 mutant mice are distinct from those deficient in msh2 and pms2. The different phenotypes of the three types of mutant mice suggest that these three genes may have independent functions in mammalian meiosis.

Olig2-Regulated Lineage-Restricted Pathway Controls Replication Competence in Neural Stem Cells and Malignant Glioma

Recent studies have identified stem cells in brain cancer. However, their relationship to normal CNS progenitors, including dependence on common lineage-restricted pathways, is unclear. We observe expression of the CNS-restricted transcription factor, OLIG2, in human glioma stem and progenitor cells reminiscent of type C transit-amplifying cells in germinal zones of the adult brain. Olig2 function is required for proliferation of neural progenitors and for glioma formation in a genetically relevant murine model. Moreover, we show p21(WAF1/CIP1), a tumor suppressor and inhibitor of stem cell proliferation, is directly repressed by OLIG2 in neural progenitors and gliomas. Our findings identify an Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells.

Mutation in the mismatch repair gene Msh6 causes cancer susceptibility

Mice carrying a null mutation in the mismatch repair gene Msh6 were generated by gene targeting. Cells that were homozygous for the mutation did not produce any detectable MSH6 protein, and extracts prepared from these cells were defective for repair of single nucleotide mismatches. Repair of 1, 2, and 4 nucleotide insertion/deletion mismatches was unaffected. Mice that were homozygous for the mutation had a reduced life span. The mice developed a spectrum of tumors, the most predominant of which were gastrointestinal tumors and B- as well as T-cell lymphomas. The tumors did not show any microsatellite instability. We conclude that MSH6 mutations, like those in some other members of the family of mismatch repair genes, lead to cancer susceptibility, and germline mutations in this gene may be associated with a cancer predisposition syndrome that does not show microsatellite instability.

Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression

Flap endonuclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1. Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli (Apc) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.

Local regulation of gene expression by lncRNA promoters, transcription and splicing

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA–protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes.

Mutation in Rpa1 results in defective DNA double-strand break repair, chromosomal instability and cancer in mice

Most cancers have multiple chromosomal rearrangements; the molecular mechanisms that generate them remain largely unknown. Mice carrying a heterozygous missense change in one of the DNA-binding domains of Rpa1 develop lymphoid tumors, and their homozygous littermates succumb to early embryonic lethality. Array comparative genomic hybridization of the tumors identified large-scale chromosomal changes as well as segmental gains and losses. The Rpa1 mutation resulted in defects in DNA double-strand break repair and precipitated chromosomal breaks as well as aneuploidy in primary heterozygous mutant mouse embryonic fibroblasts. The equivalent mutation in yeast is hypomorphic and semidominant and enhanced the formation of gross chromosomal rearrangements in multiple genetic backgrounds. These results indicate that Rpa1 functions in DNA metabolism are essential for the maintenance of chromosomal stability and tumor suppression.

The central nervous system restricted transcription factor Olig2 opposes p53 responses to genotoxic damage in neural progenitors and malignant glioma

High-grade gliomas are notoriously insensitive to radiation and genotoxic drugs. Paradoxically, the p53 gene is structurally intact in the majority of these tumors. Resistance to genotoxic modalities in p53-positive gliomas is generally attributed to attenuation of p53 functions by mutations of other components within the p53 signaling axis, such as p14(Arf), MDM2, and ATM, but this explanation is not entirely satisfactory. We show here that the central nervous system (CNS)-restricted transcription factor Olig2 affects a key posttranslational modification of p53 in both normal and malignant neural progenitors and thereby antagonizes the interaction of p53 with promoter elements of multiple target genes. In the absence of Olig2 function, even attenuated levels of p53 are adequate for biological responses to genotoxic damage.

Systematic mapping of functional enhancer–promoter connections with CRISPR interference

CRISPR screens illuminate enhancer function The noncoding regions around a gene that control the transcription of the protein-coding region are difficult to identify. Leveraging a CRISPR interference system (CRISPRi), Fulco et al. identified enhancer-promoter connections to map specific noncoding regions affecting gene regulation for the GATA1 and MYC loci (see the Perspective by Einstein and Yeo). Going forward, such CRISPRi-mapping can be used to evaluate promoter-enhancer screens functionally in an unbiased way. Science, this issue p. 769; see also p. 705 Functional mapping of noncoding elements with CRISPR interference may predict enhancer function. Gene expression in mammals is regulated by noncoding elements that can affect physiology and disease, yet the functions and target genes of most noncoding elements remain unknown. We present a high-throughput approach that uses clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) to discover regulatory elements and identify their target genes. We assess >1 megabase of sequence in the vicinity of two essential transcription factors, MYC and GATA1, and identify nine distal enhancers that control gene expression and cellular proliferation. Quantitative features of chromatin state and chromosome conformation distinguish the seven enhancers that regulate MYC from other elements that do not, suggesting a strategy for predicting enhancer–promoter connectivity. This CRISPRi-based approach can be applied to dissect transcriptional networks and interpret the contributions of noncoding genetic variation to human disease.

Identification and Characterization of the Escherichia coli RecT Protein, a Protein Encoded by the recE Region That Promotes Renaturation of Homologous Single-Stranded DNA

Recombination of plasmid DNAs and recombination of bacteriophage lambda red mutants in recB recC sbcA Escherichia coli mutants, in which the recE region is expressed, do not require recA. The recE gene is known to encode exonuclease VIII (exoVIII), which is an ATP-independent exonuclease involved in the RecE pathway of recombination. A 33,000-molecular-weight (MW) protein was observed to be coexpressed with both exoVIII and a truncated version of exoVIII, pRac3 exo, when they were overproduced under the control of strong promoters. We have purified this 33,000-MW protein (p33) and demonstrated by protein sequence analysis that it is encoded by the same coding sequence that encodes the C-terminal 33,000-MW portion of exoVIII. p33 is expressed independently of exoVIII but is probably translated from the same mRNA. p33 was found to bind to single-stranded DNA and also to promote the renaturation of complementary single-stranded DNA. It appears that p33 is functionally analogous to the bacteriophage lambda beta protein, which may explain why RecE pathway recombination does not require recA.