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DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression.

Cho MH, Park JH, Choi HJ, Park MK, Won HY, Park YJ, Lee CH, Oh SH, Song YS, Kim HS, Oh YH, Lee JY, Kong G - Nat Commun (2015)

Bottom Line: DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown.DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions.Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, Korea.

ABSTRACT
DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial-mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

No MeSH data available.


Related in: MedlinePlus

c-Myc is required for formation of DOT1L-containing transcriptional activecomplex and recognition of DOT1L on EMT-TFs in regulation of breast CSCactivity.(a) To confirm the binding of DOT1L or c-Myc with the indicatedproteins, co-immunoprecipitation assays were performed using lysates from293T cells transfected with the indicated constructs. (b) Toinvestigate the effect of c-Myc on DOT1L-induced EMT-TF expression, lysatesfrom CON or DOT1L-overexpressing MCF10A cells transfected with control orc-Myc siRNA (si c-Myc) were analysed by immunoblotting. (c)Dependency of EMT-TF regulation by DOT1L on c-Myc was confirmed usingChIP–qPCR assay in c-Myc siRNA-transfected MCF10A cells expressingDOT1L or its control cells. (d) The CSC population after c-Myc siRNAtransfection was measured by FACS. Results in c,d are shown asmeans±s.d. of experiments in triplicate. * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). (e) Aproposed model for the regulation by DOT1L of EMT-TFs. In the absence ofDOT1L, c-Myc forms a repressive complex with DNMTs and HDACs in the E-boxmotif of the promoter regions of Snail, ZEB1 and ZEB2 EMT-TFs, which leadsto DNA methylation at the CpG island and histone deacetylation within thepromoters. When DOT1L is overexpressed, c-Myc interacts directly with theDOT1L complex containing HATs and recruits them to the promoter region ofEMT-TFs. This leads to dissociation of DNMTs and HDACs from the promotersand enrichment of H3K79me and H3 acetylation in the region for activation ofEMT-TF transcription. The enhanced expression of EMT-TFs inducesEMT-associated CSC and metastasis.
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f5: c-Myc is required for formation of DOT1L-containing transcriptional activecomplex and recognition of DOT1L on EMT-TFs in regulation of breast CSCactivity.(a) To confirm the binding of DOT1L or c-Myc with the indicatedproteins, co-immunoprecipitation assays were performed using lysates from293T cells transfected with the indicated constructs. (b) Toinvestigate the effect of c-Myc on DOT1L-induced EMT-TF expression, lysatesfrom CON or DOT1L-overexpressing MCF10A cells transfected with control orc-Myc siRNA (si c-Myc) were analysed by immunoblotting. (c)Dependency of EMT-TF regulation by DOT1L on c-Myc was confirmed usingChIP–qPCR assay in c-Myc siRNA-transfected MCF10A cells expressingDOT1L or its control cells. (d) The CSC population after c-Myc siRNAtransfection was measured by FACS. Results in c,d are shown asmeans±s.d. of experiments in triplicate. * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). (e) Aproposed model for the regulation by DOT1L of EMT-TFs. In the absence ofDOT1L, c-Myc forms a repressive complex with DNMTs and HDACs in the E-boxmotif of the promoter regions of Snail, ZEB1 and ZEB2 EMT-TFs, which leadsto DNA methylation at the CpG island and histone deacetylation within thepromoters. When DOT1L is overexpressed, c-Myc interacts directly with theDOT1L complex containing HATs and recruits them to the promoter region ofEMT-TFs. This leads to dissociation of DNMTs and HDACs from the promotersand enrichment of H3K79me and H3 acetylation in the region for activation ofEMT-TF transcription. The enhanced expression of EMT-TFs inducesEMT-associated CSC and metastasis.

Mentions: Because c-Myc was a common regulatory factor for DOT1L-induced SNAIL,ZEB1 and ZEB2 transcription (Fig. 4b andSupplementary Fig. 9a,b), wefurther examined the relationship between c-Myc, DOT1L and histone acetylationand DNA methylation modifiers in EMT-TF regulation. Interestingly, DOT1L boundpreferentially to p300 rather than to HDAC1 and DNMT1 under c-Myc overexpressionconditions (Fig. 5a). Similarly, the binding of c-Myc top300 was increased by DOT1L overexpression, even though c-Myc could bind to boththe HDAC1 and DNMT1 transcriptional repressive complex and p300 active complex.These data implied that DOT1L forms a transcriptionally active complex withc-Myc and p300. Because the different binding of epigenetic factors with DOT1Lprotein was dependent on c-Myc expression, we next examined whether c-Myc wasrequired for DOT1L-mediated epigenetic alteration and subsequent EMT-TFactivation. The knockdown of c-Myc using siRNA blocked the increased EMT-TFexpression caused by DOT1L overexpression in MCF10A cells (Fig.5b). Furthermore, the binding of DOT1L and p300/CBP to the promoterregion of EMT-TFs and dissociation of HDAC1 and DNMT1 from the promoters weredependent on the c-Myc status, as confirmed using ChIP assay after inhibition ofc-Myc expression (Fig. 5c and Supplementary Fig. 10). In the control MCF10Acells having very low DOT1L expression, there were no detectable changes in theexpression of EMT-TFs despite of c-Myc knockdown as assessed by immunoblotting(Fig. 5b), while DNMT1 and HDAC1 were recruited morefirmly to the EMT-TF promoters by c-Myc depletion (Supplementary Fig. 10), indicating therequirement of DOT1L for c-Myc-mediated EMT-TFs upregulation. We also confirmedthat the increased CSC population caused by DOT1L was abolished by c-Mycknockdown using siRNA (Fig. 5d), implying that c-Myc couldaffect the DOT1L-induced breast CSC activation. Collectively, DOT1L is recruitedto EMT-TF promoters such as SNAIL, ZEB1 and ZEB2 together with c-Myc andCBP/p300 co-activator complex for epigenetic transactivation of these EMT-TFswith enrichment of H3K79me and H3ac. These results also suggest that c-Myc isrequired for recognition by DOT1L of target chromatin and formation of atranscriptionally active complex with DOT1L-c-Myc-p300 for epigenetic regulationof EMT-TF-associated cancer stemness and tumour progression.


DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression.

Cho MH, Park JH, Choi HJ, Park MK, Won HY, Park YJ, Lee CH, Oh SH, Song YS, Kim HS, Oh YH, Lee JY, Kong G - Nat Commun (2015)

c-Myc is required for formation of DOT1L-containing transcriptional activecomplex and recognition of DOT1L on EMT-TFs in regulation of breast CSCactivity.(a) To confirm the binding of DOT1L or c-Myc with the indicatedproteins, co-immunoprecipitation assays were performed using lysates from293T cells transfected with the indicated constructs. (b) Toinvestigate the effect of c-Myc on DOT1L-induced EMT-TF expression, lysatesfrom CON or DOT1L-overexpressing MCF10A cells transfected with control orc-Myc siRNA (si c-Myc) were analysed by immunoblotting. (c)Dependency of EMT-TF regulation by DOT1L on c-Myc was confirmed usingChIP–qPCR assay in c-Myc siRNA-transfected MCF10A cells expressingDOT1L or its control cells. (d) The CSC population after c-Myc siRNAtransfection was measured by FACS. Results in c,d are shown asmeans±s.d. of experiments in triplicate. * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). (e) Aproposed model for the regulation by DOT1L of EMT-TFs. In the absence ofDOT1L, c-Myc forms a repressive complex with DNMTs and HDACs in the E-boxmotif of the promoter regions of Snail, ZEB1 and ZEB2 EMT-TFs, which leadsto DNA methylation at the CpG island and histone deacetylation within thepromoters. When DOT1L is overexpressed, c-Myc interacts directly with theDOT1L complex containing HATs and recruits them to the promoter region ofEMT-TFs. This leads to dissociation of DNMTs and HDACs from the promotersand enrichment of H3K79me and H3 acetylation in the region for activation ofEMT-TF transcription. The enhanced expression of EMT-TFs inducesEMT-associated CSC and metastasis.
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f5: c-Myc is required for formation of DOT1L-containing transcriptional activecomplex and recognition of DOT1L on EMT-TFs in regulation of breast CSCactivity.(a) To confirm the binding of DOT1L or c-Myc with the indicatedproteins, co-immunoprecipitation assays were performed using lysates from293T cells transfected with the indicated constructs. (b) Toinvestigate the effect of c-Myc on DOT1L-induced EMT-TF expression, lysatesfrom CON or DOT1L-overexpressing MCF10A cells transfected with control orc-Myc siRNA (si c-Myc) were analysed by immunoblotting. (c)Dependency of EMT-TF regulation by DOT1L on c-Myc was confirmed usingChIP–qPCR assay in c-Myc siRNA-transfected MCF10A cells expressingDOT1L or its control cells. (d) The CSC population after c-Myc siRNAtransfection was measured by FACS. Results in c,d are shown asmeans±s.d. of experiments in triplicate. * and†P<0.05 versus CON/siCON andDOT1L/siCON, respectively (Student's t-test). (e) Aproposed model for the regulation by DOT1L of EMT-TFs. In the absence ofDOT1L, c-Myc forms a repressive complex with DNMTs and HDACs in the E-boxmotif of the promoter regions of Snail, ZEB1 and ZEB2 EMT-TFs, which leadsto DNA methylation at the CpG island and histone deacetylation within thepromoters. When DOT1L is overexpressed, c-Myc interacts directly with theDOT1L complex containing HATs and recruits them to the promoter region ofEMT-TFs. This leads to dissociation of DNMTs and HDACs from the promotersand enrichment of H3K79me and H3 acetylation in the region for activation ofEMT-TF transcription. The enhanced expression of EMT-TFs inducesEMT-associated CSC and metastasis.
Mentions: Because c-Myc was a common regulatory factor for DOT1L-induced SNAIL,ZEB1 and ZEB2 transcription (Fig. 4b andSupplementary Fig. 9a,b), wefurther examined the relationship between c-Myc, DOT1L and histone acetylationand DNA methylation modifiers in EMT-TF regulation. Interestingly, DOT1L boundpreferentially to p300 rather than to HDAC1 and DNMT1 under c-Myc overexpressionconditions (Fig. 5a). Similarly, the binding of c-Myc top300 was increased by DOT1L overexpression, even though c-Myc could bind to boththe HDAC1 and DNMT1 transcriptional repressive complex and p300 active complex.These data implied that DOT1L forms a transcriptionally active complex withc-Myc and p300. Because the different binding of epigenetic factors with DOT1Lprotein was dependent on c-Myc expression, we next examined whether c-Myc wasrequired for DOT1L-mediated epigenetic alteration and subsequent EMT-TFactivation. The knockdown of c-Myc using siRNA blocked the increased EMT-TFexpression caused by DOT1L overexpression in MCF10A cells (Fig.5b). Furthermore, the binding of DOT1L and p300/CBP to the promoterregion of EMT-TFs and dissociation of HDAC1 and DNMT1 from the promoters weredependent on the c-Myc status, as confirmed using ChIP assay after inhibition ofc-Myc expression (Fig. 5c and Supplementary Fig. 10). In the control MCF10Acells having very low DOT1L expression, there were no detectable changes in theexpression of EMT-TFs despite of c-Myc knockdown as assessed by immunoblotting(Fig. 5b), while DNMT1 and HDAC1 were recruited morefirmly to the EMT-TF promoters by c-Myc depletion (Supplementary Fig. 10), indicating therequirement of DOT1L for c-Myc-mediated EMT-TFs upregulation. We also confirmedthat the increased CSC population caused by DOT1L was abolished by c-Mycknockdown using siRNA (Fig. 5d), implying that c-Myc couldaffect the DOT1L-induced breast CSC activation. Collectively, DOT1L is recruitedto EMT-TF promoters such as SNAIL, ZEB1 and ZEB2 together with c-Myc andCBP/p300 co-activator complex for epigenetic transactivation of these EMT-TFswith enrichment of H3K79me and H3ac. These results also suggest that c-Myc isrequired for recognition by DOT1L of target chromatin and formation of atranscriptionally active complex with DOT1L-c-Myc-p300 for epigenetic regulationof EMT-TF-associated cancer stemness and tumour progression.

Bottom Line: DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown.DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions.Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, College of Medicine, Hanyang University, Seoul 133-791, Korea.

ABSTRACT
DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial-mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

No MeSH data available.


Related in: MedlinePlus