Proliferation Stage-dependent Expression of DNA Methyltransferase (Dnmt1) in Mouse Small Intestine.

Suetake, Isao; Shi, Liheng; Watanabe, Dai; Nakamura, Masahiko; Tsuji, Shoji

doi:10.1247/csf.26.79

Cited by 23 publications

(9 citation statements)

References 31 publications

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“…Dnmt1 was restricted to the crypts (Supplemental Fig. 1A; Suetake et al 2001), while Dnmt3a was expressed throughout the epithelium, with higher expression in crypts (Supplemental Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

DNA methylation is required for the control of stem cell differentiation in the small intestine

et al. 2014

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The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3-5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.

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Section: Resultsmentioning

confidence: 99%

DNA methylation is required for the control of stem cell differentiation in the small intestine

et al. 2014

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“…Protein extracts were then separated on 6.5% polyacrylamide gel by electrophoresis and transferred onto nitrocellulose membrane. After blocking with 4% bovine serum albumin (BSA) solution in TBST buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1% Tween 20) for 1 h at room temperature, the membrane was incubated with primary antibodies, 64B1466 monoclonal anti-Dnmt3a antibody (1 : 250 dilution; Imgenex), polyclonal anti-Dnmt1 antibody (1 : 5000 dilution) (Takagi et al, 1995;Suetake et al, 2001), or C-11 polyclonal antiactin antibody (1 : 2000 dilution; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. After incubation with secondary antibodies conjugated to horseradish peroxidase (1 : 5000 dilution; ICN), detection of horseradish peroxidase activity was performed using ECL Western blotting detection reagents (Amersham Pharmacia).…”

Section: Immunoblotting Analysismentioning

confidence: 99%

De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2′-deoxycytidine

et al. 2005

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The deoxycytidine analog 5-aza-2 0 -deoxycitidine (5-azadC) is a potent chemotherapeutic agent effective against selective types of cancer. The molecular mechanism by which 5-aza-dC induces cancer cell death, however, is not fully understood. It has been accepted that the mechanism of toxicity is due to the covalent binding between the DNA methyltransferase (Dnmt) and 5-aza-dC-substituted DNA. In order to define which member of the Dnmt family plays a dominant role in the cytotoxicity, we examined the effect of 5-aza-dC on cell growth and apoptosis in various Dnmt null mutant embryonic stem (ES) cells. Of interest, Dnmt3a-Dnmt3b double null ES cells were highly resistant to 5-aza-dC when compared to wild type, Dnmt3a null, Dnmt3b null, or Dnmt1 null ES cells. The cellular sensitivity to 5-aza-dC correlated well with the expression status of Dnmt3 in both undifferentiated and differentiated ES cells. When exogenous Dnmt3a or Dnmt3b was expressed in double null ES cells, the sensitivity to 5-aza-dC was partially restored. These results suggest that the cytotoxic effect of 5-aza-dC may be mediated primarily through Dnmt3a and Dnmt3b de novo DNA methyltransferases. Further, the ability to form Dnmt-DNA adducts was similar in Dnmt1 and Dnmt3, and the expression level of Dnmt3 was not higher than that of Dnmt1 in ES cells. Therefore, Dnmt3-DNA adducts may be more effective for inducing apoptosis than Dnmt1-DNA adducts. These results imply a therapeutic potential of 5-aza-dC to cancers expressing Dnmt3.

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“…Anti-mouse Dnmt1 antibodies were raised against amino-terminal 118 amino acid sequence (anti-amino-terminal antibodies) and carboxyl-terminal sequence (anti-carboxyl-terminal antibodies) of Dnmt1 as described elsewhere (Takagi et al, 1995;Suetake et al, 2001). Monoclonal antibody reactive with 5-methyl-2'-deoxycytidine, FMC9, was used to detect methylated DNA (Mizugaki et al, 1996).…”

Section: Antibodiesmentioning

confidence: 99%

“…Dnmt1 was immunoprecipitated, electrophoresed, Western blotted, and detected as described elsewhere (Suetake et al, 2001).…”

Section: Immunoprecipitationmentioning

confidence: 99%

“…Although there are a few exceptions to Dnmt1 being highly expressed in non-proliferating cells (Mertineit et al, 1998;Inano et al, 2000), the expression level of Dnmt1 is basically under the control of the cell cycle (Szyf et al, 1991;Liu et al, 1996;Suetake et al, 1998;Suetake et al, 2001), that is, it is high under proliferative conditions and low at postmitotic stage. Since it is reported that Dnmt1 interacts with PCNA (Chuang et al, 1997), a marker for proliferating cells, we next detected the expression of PCNA in embryonic testis.…”

Section: Proliferation Stage-dependent Expression Of Dnmt1 In Testismentioning

confidence: 99%

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Expression of DNA Methyltransferase (Dnmt1) in Testicular Germ Cells during Development of Mouse Embryo.

Sakai

Suetake

Itoh

et al. 2001

Cell Struct. Funct.

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ABSTRACT. The DNA methylation pattern is reprogrammed in embryonic germ cells. In female germ cells, the short-form DNA methyltransferase Dnmt1, which is an alternative isoform specifically expressed in growing oocytes, plays a crucial role in maintaining imprinted genes. To evaluate the contribution of Dnmt1 to the DNA methylation in male germ cells, the expression profiles of Dnmt1 in embryonic gonocytes were investigated. We detected a significant expression of Dnmt1 in primordial germ cells in 12.5-14.5 day postcoitum (dpc) embryos. The expression of Dnmt1 was downregulated after 14.5 dpc after which almost no Dnmt1 was detected in gonocytes prepared from 18.5 dpc embryos. The short-form Dnmt1 also was not detected in the 16.5-18.5 dpc gonocytes. On the other hand, Dnmt1 was constantly detected in Sertoli cells at 12.5-18.5 dpc. The expression profiles of Dnmt1 were similar to that of proliferating cell nuclear antigen (PCNA), a marker for proliferating cells, suggesting that Dnmt1 was specifically expressed in the proliferating male germ cells. Inversely, genomewide DNA methylation occurred after germ cell proliferation was arrested, when the Dnmt1 expression was downregulated. The present results indicate that not Dnmt1 but some other type of DNA methyltransferase contributes to the creation of DNA methylation patterns in male germ cells.

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Proliferation Stage-dependent Expression of DNA Methyltransferase (Dnmt1) in Mouse Small Intestine.

Cited by 23 publications

References 31 publications

DNA methylation is required for the control of stem cell differentiation in the small intestine

DNA methylation is required for the control of stem cell differentiation in the small intestine

De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2′-deoxycytidine

Expression of DNA Methyltransferase (Dnmt1) in Testicular Germ Cells during Development of Mouse Embryo.

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