Limits...
DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

No MeSH data available.


Related in: MedlinePlus

MUC1-C induces LINE-1 and CDH1 promoter DNA methylation(A) Genomic DNA from the indicated BT-549 cells was analyzed using the LINE-1 global DNA methylation ELISA kit. The results are expressed as percentage 5-mC (mean±SD of 3 determinations) based on extrapolation from the methylated and unmethylated genomic standards provided in the kit. (B) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of 4 CpG positions in the LINE-1 element. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 4 positions as compared to that obtained in BT-549/CshRNA cells (assigned a value of 100%). The asterisk (*) denotes a p value of <0.05. (C) Genomic DNA from BT-549 cells was subjected to immunoprecipitation of methylated DNA (MeDIP) and the precipitates were analyzed by qPCR of the CDH1 promoter (positions −213 to −116). The results (mean±SD of 3 determinations) are expressed as relative fold enrichment compared to that obtained from MUC1shRNA expressing cells (assigned a value of 1)(left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of the indicated CpG sites in the CDH1 promoter. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 6 CpG sites. The hashtag (#) and asterisk (*) denote p values of >0.05 and <0.05, respectively. (E) Schema depicting the proposed MUC1-C-induced expression of DNMT1 and DNMT3b, and thereby DNA methylation in human cancer cells. MUC1-C induces an autoinductive inflammatory circuit involving activation of the TAK1→IKK→NF-κB p65 pathway (21–23). MUC1-C promotes NF-κB p65-mediated induction of DNMT1 and DNMT3b transcription by interacting with NF-κB p65 on their respective promoters and increasing NF-κB p65 occupancy. In turn, MUC1-C links the inflammatory response with induction of DNA methylation and regulation of gene expression. Thus, targeting MUC1-C in cancer cells with silencing or inhibitors suppresses (i) DNMT1 and DNMT3b expression, and (ii) global and promoter-specific DNA methylation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5121097&req=5

Figure 4: MUC1-C induces LINE-1 and CDH1 promoter DNA methylation(A) Genomic DNA from the indicated BT-549 cells was analyzed using the LINE-1 global DNA methylation ELISA kit. The results are expressed as percentage 5-mC (mean±SD of 3 determinations) based on extrapolation from the methylated and unmethylated genomic standards provided in the kit. (B) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of 4 CpG positions in the LINE-1 element. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 4 positions as compared to that obtained in BT-549/CshRNA cells (assigned a value of 100%). The asterisk (*) denotes a p value of <0.05. (C) Genomic DNA from BT-549 cells was subjected to immunoprecipitation of methylated DNA (MeDIP) and the precipitates were analyzed by qPCR of the CDH1 promoter (positions −213 to −116). The results (mean±SD of 3 determinations) are expressed as relative fold enrichment compared to that obtained from MUC1shRNA expressing cells (assigned a value of 1)(left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of the indicated CpG sites in the CDH1 promoter. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 6 CpG sites. The hashtag (#) and asterisk (*) denote p values of >0.05 and <0.05, respectively. (E) Schema depicting the proposed MUC1-C-induced expression of DNMT1 and DNMT3b, and thereby DNA methylation in human cancer cells. MUC1-C induces an autoinductive inflammatory circuit involving activation of the TAK1→IKK→NF-κB p65 pathway (21–23). MUC1-C promotes NF-κB p65-mediated induction of DNMT1 and DNMT3b transcription by interacting with NF-κB p65 on their respective promoters and increasing NF-κB p65 occupancy. In turn, MUC1-C links the inflammatory response with induction of DNA methylation and regulation of gene expression. Thus, targeting MUC1-C in cancer cells with silencing or inhibitors suppresses (i) DNMT1 and DNMT3b expression, and (ii) global and promoter-specific DNA methylation.

Mentions: The above findings invoked the possibility that MUC1-C also functions in regulating DNA methylation in cancer cells. Indeed, silencing MUC1-C in BT-549 (Fig. 4A) and MDA-MB-231 (Fig. S5A) cells was associated with significant decreases in the percentage of 5-mC in Long Interspersed Nucleotide Element-1 (LINE-1) repeats, a surrogate marker of global DNA methylation (29–31). These effects of targeting MUC1-C were confirmed using bisulfite sequencing of LINE-1 repeats (Fig. 4B), further supporting a role for MUC1-C in inducing global DNA methylation. Based on these results and previous work demonstrating that MUC1-C promotes EMT and downregulation of ECAD expression (24; 25), we investigated the involvement of MUC1-C in DNA methylation of the CDH1 promoter. Using immunoprecipitation of methylated DNA (MeDIP) followed by qPCR of the precipitated DNA fragments, we found that silencing MUC1-C in MDA-MB-231 cells is associated with marked decreases in methylation of the CDH1 promoter (Fig. S5B). In addition and in concert with this response, MUC1-C silencing resulted in significant induction of ECAD mRNA and protein (Fig. S5C). Similarly, silencing MUC1-C in BT-549 cells was associated with decreased methylation of the CDH1 promoter and induction of ECAD expression (Fig. 4C). Additionally, bisulfite conversion of genomic DNA and pyrosequencing of the CpG region of the CDH1 promoter (positions −169 to −116) demonstrated that MUC1-C silencing is associated with significant decreases in DNA methylation of multiple CpG sites (Fig. 4D).


DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS
MUC1-C induces LINE-1 and CDH1 promoter DNA methylation(A) Genomic DNA from the indicated BT-549 cells was analyzed using the LINE-1 global DNA methylation ELISA kit. The results are expressed as percentage 5-mC (mean±SD of 3 determinations) based on extrapolation from the methylated and unmethylated genomic standards provided in the kit. (B) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of 4 CpG positions in the LINE-1 element. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 4 positions as compared to that obtained in BT-549/CshRNA cells (assigned a value of 100%). The asterisk (*) denotes a p value of <0.05. (C) Genomic DNA from BT-549 cells was subjected to immunoprecipitation of methylated DNA (MeDIP) and the precipitates were analyzed by qPCR of the CDH1 promoter (positions −213 to −116). The results (mean±SD of 3 determinations) are expressed as relative fold enrichment compared to that obtained from MUC1shRNA expressing cells (assigned a value of 1)(left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of the indicated CpG sites in the CDH1 promoter. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 6 CpG sites. The hashtag (#) and asterisk (*) denote p values of >0.05 and <0.05, respectively. (E) Schema depicting the proposed MUC1-C-induced expression of DNMT1 and DNMT3b, and thereby DNA methylation in human cancer cells. MUC1-C induces an autoinductive inflammatory circuit involving activation of the TAK1→IKK→NF-κB p65 pathway (21–23). MUC1-C promotes NF-κB p65-mediated induction of DNMT1 and DNMT3b transcription by interacting with NF-κB p65 on their respective promoters and increasing NF-κB p65 occupancy. In turn, MUC1-C links the inflammatory response with induction of DNA methylation and regulation of gene expression. Thus, targeting MUC1-C in cancer cells with silencing or inhibitors suppresses (i) DNMT1 and DNMT3b expression, and (ii) global and promoter-specific DNA methylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5121097&req=5

Figure 4: MUC1-C induces LINE-1 and CDH1 promoter DNA methylation(A) Genomic DNA from the indicated BT-549 cells was analyzed using the LINE-1 global DNA methylation ELISA kit. The results are expressed as percentage 5-mC (mean±SD of 3 determinations) based on extrapolation from the methylated and unmethylated genomic standards provided in the kit. (B) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of 4 CpG positions in the LINE-1 element. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 4 positions as compared to that obtained in BT-549/CshRNA cells (assigned a value of 100%). The asterisk (*) denotes a p value of <0.05. (C) Genomic DNA from BT-549 cells was subjected to immunoprecipitation of methylated DNA (MeDIP) and the precipitates were analyzed by qPCR of the CDH1 promoter (positions −213 to −116). The results (mean±SD of 3 determinations) are expressed as relative fold enrichment compared to that obtained from MUC1shRNA expressing cells (assigned a value of 1)(left panel). Lysates from the indicated cells were analyzed by immunoblotting (right panel). (D) Genomic DNA from BT-549/CshRNA (closed bars) and BT-549/MUC1shRNA (open bars) was analyzed by bisulfite conversion, PCR amplification and pyrosequencing of the indicated CpG sites in the CDH1 promoter. The results are expressed as the percentage DNA methylation (mean±SD of 3 determinations) at each of the 6 CpG sites. The hashtag (#) and asterisk (*) denote p values of >0.05 and <0.05, respectively. (E) Schema depicting the proposed MUC1-C-induced expression of DNMT1 and DNMT3b, and thereby DNA methylation in human cancer cells. MUC1-C induces an autoinductive inflammatory circuit involving activation of the TAK1→IKK→NF-κB p65 pathway (21–23). MUC1-C promotes NF-κB p65-mediated induction of DNMT1 and DNMT3b transcription by interacting with NF-κB p65 on their respective promoters and increasing NF-κB p65 occupancy. In turn, MUC1-C links the inflammatory response with induction of DNA methylation and regulation of gene expression. Thus, targeting MUC1-C in cancer cells with silencing or inhibitors suppresses (i) DNMT1 and DNMT3b expression, and (ii) global and promoter-specific DNA methylation.
Mentions: The above findings invoked the possibility that MUC1-C also functions in regulating DNA methylation in cancer cells. Indeed, silencing MUC1-C in BT-549 (Fig. 4A) and MDA-MB-231 (Fig. S5A) cells was associated with significant decreases in the percentage of 5-mC in Long Interspersed Nucleotide Element-1 (LINE-1) repeats, a surrogate marker of global DNA methylation (29–31). These effects of targeting MUC1-C were confirmed using bisulfite sequencing of LINE-1 repeats (Fig. 4B), further supporting a role for MUC1-C in inducing global DNA methylation. Based on these results and previous work demonstrating that MUC1-C promotes EMT and downregulation of ECAD expression (24; 25), we investigated the involvement of MUC1-C in DNA methylation of the CDH1 promoter. Using immunoprecipitation of methylated DNA (MeDIP) followed by qPCR of the precipitated DNA fragments, we found that silencing MUC1-C in MDA-MB-231 cells is associated with marked decreases in methylation of the CDH1 promoter (Fig. S5B). In addition and in concert with this response, MUC1-C silencing resulted in significant induction of ECAD mRNA and protein (Fig. S5C). Similarly, silencing MUC1-C in BT-549 cells was associated with decreased methylation of the CDH1 promoter and induction of ECAD expression (Fig. 4C). Additionally, bisulfite conversion of genomic DNA and pyrosequencing of the CpG region of the CDH1 promoter (positions −169 to −116) demonstrated that MUC1-C silencing is associated with significant decreases in DNA methylation of multiple CpG sites (Fig. 4D).

View Article: PubMed Central - PubMed

ABSTRACT

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-&kappa;B p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells.

No MeSH data available.


Related in: MedlinePlus