Biomedical subjects

Nemanja Rodić

Publications and source records attributed to Nemanja Rodić.

4 recordsLinked to original sources

PubMed2006

CpG sites preferentially methylated by Dnmt3a in vivo.

Dnmt3a and Dnmt3b are two major de novo DNA methyltransferases essential for embryonic development in mammals. It has been shown that Dnmt3a and Dnmt3b have distinct substrate preferences for certain genomic loci, including major and minor satellite repeats. However, the exact target CpG sites where Dnmt3a and Dnmt3b catalyze DNA methylation remains largely unknown. To identify a CpG site that is specifically methylated by Dnmt3a or Dnmt3b, we screened methylated genomic loci by methylation sensitive restriction fingerprinting using genomic DNA from wild-type, Dnmt3a null, Dnmt3b null, and Dnmt3a-Dnmt3b double null ES cells. Interestingly, one of the CpG sites was preferentially methylated in wild-type and Dnmt3b null ES cells but not in Dnmt3a null or Dnmt3a-Dnmt3b double null ES cells, suggesting that the site-specific methylation was Dnmt3a-dependent. Sequencing results revealed that the isolated CpG site is located within the 1st exon of the G isoform of fibroblast growth factor (Fgf-1.G) on mouse chromosome 18. Exogenous expression of Dnmt3a but not Dnmt3b in the double null ES cells restored DNA methylation of this CpG site. When we examined alternative transcription initiation sites, we determined that another CpG site in the 5'-flanking region of the Fgf-1.A isoform was also methylated specifically by Dnmt3a. Using chimeric constructs between Dnmt3a and Dnmt3b, we further determined that the NH(2)-terminal regulatory domain of Dnmt3a was responsible for establishing its substrate specificity. These results indicate that certain CpG sites within the Fgf-1 gene locus are preferentially methylated by Dnmt3a but not by Dnmt3b. Selective methylation by a specific member of Dnmt3 may therefore play a role in the orchestration of gene expression during embryonic development.

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PubMed2005

DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells.

The capacity for cellular differentiation is governed not only by the repertoire of available transcription factors but by the accessibility of cis-regulatory elements. Studying changes in epigenetic modifications during stem cell differentiation will help us understand how cells maintain or lose differentiation potential. We investigated changes in DNA methylation during the transition of pluripotent embryonic stem cells (ESCs) into differentiated cell types. Using a methylation-sensitive restriction fingerprinting method, we identified a novel adenine nucleotide (ADP/ATP) translocase gene, Ant4, that was selectively hypomethylated and expressed in undifferentiated mouse ESCs. In contrast to other pluripotent stem cell-specific genes such as Oct-4 and Nanog, the Ant4 gene was readily derepressed in differentiated cells after 5-aza-2'-deoxycytidine treatment. Moreover, expression of de novo DNA methyltransferases Dnmt3a and Dnmt3b was essential for repression and DNA methylation of the Ant4 gene during ESC differentiation. Although the deduced amino acid sequence of Ant4 is highly homologous to the previously identified Ant isoforms, the expression of Ant4 was uniquely restricted to developing gametes in adult mice, and its promoter hypomethylation was observed only in testis. Additionally, Ant4 was expressed in primordial germ cells. These data indicate that Ant4 is a pluripotent stem cell- and germ cell-specific isoform of adenine nucleotide translocase in mouse and that DNA methylation plays a primary role in its transcriptional silencing in somatic cells.

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PubMed2005

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

The deoxycytidine analog 5-aza-2'-deoxycitidine (5-aza-dC) 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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PubMed2004

Cell fusion and reprogramming: resolving our transdifferences.

Bone marrow-derived cells (BMDCs) can contribute to the regeneration of diverse adult tissues, including brain, liver and heart, following bone marrow transplantation. These unexpected events were initially considered a result of transdifferentiation of BMDCs, supporting the emerging idea of extended plasticity of adult stem cells. Although studies have now clearly demonstrated that spontaneous cell fusion, rather than transdifferentiation, was the primary cause for unexpected cell fate-switches of BMDCs into hepatocytes, Purkinje cells and cardiac myocytes in vivo.

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