Functional deficiency of the FEN1 gene has been suggested to cause genomic instability and cancer predisposition. We have identified a group of FEN1 mutations in human cancer specimens. Most of these mutations abrogated two of three nuclease activities of flap endonuclease 1 (FEN1). To demonstrate the etiological significance of these somatic mutations, we inbred a mouse line harboring the E160D mutation representing mutations identified in human cancers. Selective elimination of nuclease activities led to frequent spontaneous mutations and accumulation of incompletely digested DNA fragments in apoptotic cells. The mutant mice were predisposed to autoimmunity, chronic inflammation and cancers. The mutator phenotype results in the initiation of cancer, whereas chronic inflammation promotes the cancer progression. The current work exemplifies the approach of studying the mechanisms of individual polymorphisms and somatic mutations in cancer development, and may serve as a reference in developing new therapeutic regimens through the suppression of inflammatory responses.
A Cre recombinase expression cassette was inserted into the X-linked Hprt locus by gene targeting in a mouse embryonic stem (ES) cell line isogenic to strain 129S1/SvImJ (129S1), then the transgene was introduced into 129S1 mice through ES cell chimeras. When females hemizygous for this transgene were mated to males carrying a neomycin selection cassette flanked by loxP sites, the cassette was always excised regardless of Cre inheritance and without detectable mosaicism. The usefulness of this "Cre-deleter" transgenic line is in its efficiency and defined genetic status in terms of mouse strain and location of the transgene.
A ϳ2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.
Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report that (i) TET1/2/3 and TDG can be direct targets of the microRNA miR-26a, (ii) murine TETs, especially TET2 and TDG, are down-regulated in islets during postnatal differentiation, whereas miR-26a is up-regulated, (iii) changes in 5-hydroxymethylcytosine accompany changes in TET mRNA levels, (iv) these changes in mRNA and 5-hydroxymethylcytosine are also seen in an in vitro differentiation system initiated with FACS-sorted adult ductal progenitor-like cells, and (v) overexpression of miR-26a in mice increases postnatal islet cell number in vivo and endocrine/acinar colonies in vitro. These results establish a previously unknown link between miRNAs and TET expression levels, and suggest a potential role for miR-26a and TET family proteins in pancreatic cell differentiation.T en eleven translocation (TET) enzymes and thymine DNA glycosylase (TDG) are implicated in active DNA demethylation (1-3). The three TET family enzymes oxidize 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC), and subsequently to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (1, 2, 4, 5). TDG, a base excision repair glycosylase, replaces 5fC and 5caC with an unmodified cytosine via DNA repair (5, 6). Despite these advances, the molecular mechanisms underlying TETs/ TDG regulation are still not known. In addition, although recent data suggest a role of TET and 5hmC in embryonic stem cells and primordial germ cells (2, 7-12), evidence for enzymatic demethylation by TET enzymes during differentiation of cells of later stages, such as the postnatal and adult stem cells of various organs including pancreas, remains very limited (13-16).MicroRNAs (miRNAs) are an abundant class of small, highly conserved noncoding RNAs that bind the 3′-untranslated regions (UTRs) of protein-coding genes to suppress gene expression. Accumulating data have demonstrated that miRNAs are critical for many developmental and cellular processes, including organogenesis and differentiation (17). However, the role of miRNAs in TET expression and active DNA demethylation remains unclear.Three major cell lineages exist in the adult pancreas-duct, acinar, and endocrine cells. The endocrine pancreas is composed of several hormone-releasing cells, including the insulin-secreting beta cells and glucagon-secreting alpha cells. Many transcription factors are known to control pancreas development (18). For example, the expression of pancreatic and duodenal homeobox 1 (Pdx1) in embryonic foregut region induces pancreas commitment (19,20), and those early progenitor cells have the potential to give rise to all three pancreatic lineages (21,22). Subsequent activation of another transcription factor, neurogenin 3 (N...
Characterization of neural promoter/enhancers is essential for understanding gene regulation during brain development and provides useful genetic tools. However, it relies on the use of transgenic mice. We report a method for the rapid in vivo analysis of neural promoter/enhancers in the developing mouse brain and its application in the isolation of the doublecortin (DCX) promoter/enhancer for genetic labeling of young neurons. In the present study, we demonstrated that reporter genes introduced into the developing mouse cerebral cortex by in utero electroporation can achieve promoter/enhancer-specific patterns of expression. We used the in utero electroporation system to isolate a genomic fragment of the doublecortin gene that can direct reporter expression faithful to doublecortin in young neurons of the cerebral cortex. Finally, we showed that the DCX promoter identified via electroporation could reproduce doublecortin expression in the entire central nervous system in DCX-DsRed-express transgenic mice. The results of our study provide a convenient, reliable, and rapid method for in vivo analysis of neural promoter/enhancers in the developing mouse brain.
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