Methylated in Dnmt3b KO cells and 77.0% were methylated in the

Methylated in Dnmt3b KO cells and 77.0% were methylated in the Dnmt3a/3b DKO cells. These analyses revealed an inverse relationship between the expression levels of the 4 transcript and the methylation state of both the M4-CGI and the M4-Promoter for the wt and single KO ES cells. For the Dnmt3a/3b DKO cells, this inverse relationship between methylation status and 4 transcript levels appears to hold for the M4- Promoter but not for the intragenic M4-CGI. The M4-Promoter region in the DKO Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 10 cells displayed near wild-types levels of the M4-Promoter methylation and 4 transcript levels whereas these same cells displayed a moderate reduction of methylation in the intragenic M4-CGI. 3.5. The M4-Promoter is a methylation-dependent, germ cell-specific promoter In wt J1 and single Dnmt KO ES cells and in GC-1spg cells treated with 5-azaC the decreased methylation in the promoter corresponds to higher 4 expression again leaving open the possibility that methylation of this region could be participating in regulating gene expression. Therefore, we next wanted to determine Indirubin-3′-oxime site whether DNA methylation of the promoter could alter gene expression. As a first step, the ability of the M4-Promoter to drive gene expression was examined in cultured cells. GC-1spg cells but not NIH 3T3 cells SKI II chemical information transfected with the M4Promoter-containing construct display a 2.0 fold greater normalized firefly luciferase activity compared to cells transfected with the construct lacking a promoter. These results demonstrate that the region -700bp to +200bp region of Atp1a4 possesses promoter activity that is specific to the GC-1spg germ cell line. Next, in order to test the methylation dependence of the M4-Promoter activity, the pGL3Basic-M4-Promoter vector was in vitro methylated using the CpG methyltransferase enzyme . As a control, the pGL3-Basic-M4-Promoter construct was mock methylated under the same conditions in the absence of the methyltransferase enzyme. GC-1spg cells containing the in vitro methylated expression construct displayed decreased normalized luciferase activity compared to cells containing the mock-methylated construct demonstrating that the promoter is methylation-sensitive in this germ cell line. 3.6. The M4-CGI does not function as an alternative promoter As a first step towards determining if the M4-CGI functions as an alternative promoter, this region was assayed for promoter activity in GC-1spg and NIH3T3 cells. GC-1spg and NIH3T3 cells transfected with the pGL3-Basic-M4-CGI construct each displayed equivalent normalized luciferase activity as the cells transfected with the promoter-less pGL3-Basic vector demonstrating that the intragenic M4-CGI does not function as a promoter in either of these cell lines. 3.7. Methylation of the intragenic M4-CGI inhibits transcription elongation efficiency In order to determine if DNA methylation in the intragenic M4-CGI inhibits transcriptional elongation, a tandem reporter construct that has been previously used to quantify transcription elongation through polyadenlyation sequences was employed. In the TAN1 construct, transcription driven by the human PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 cytomegalovirus immediate early gene promoter initiates and proceeds through the firefly luciferase reporter sequence, through the putative transcriptional elongationaltering sequence, and then through the Renilla lucifera.Methylated in Dnmt3b KO cells and 77.0% were methylated in the Dnmt3a/3b DKO cells. These analyses revealed an inverse relationship between the expression levels of the 4 transcript and the methylation state of both the M4-CGI and the M4-Promoter for the wt and single KO ES cells. For the Dnmt3a/3b DKO cells, this inverse relationship between methylation status and 4 transcript levels appears to hold for the M4- Promoter but not for the intragenic M4-CGI. The M4-Promoter region in the DKO Author Manuscript Author Manuscript Author Manuscript Author Manuscript Gene. Author manuscript; available in PMC 2017 January 10. Kumar et al. Page 10 cells displayed near wild-types levels of the M4-Promoter methylation and 4 transcript levels whereas these same cells displayed a moderate reduction of methylation in the intragenic M4-CGI. 3.5. The M4-Promoter is a methylation-dependent, germ cell-specific promoter In wt J1 and single Dnmt KO ES cells and in GC-1spg cells treated with 5-azaC the decreased methylation in the promoter corresponds to higher 4 expression again leaving open the possibility that methylation of this region could be participating in regulating gene expression. Therefore, we next wanted to determine whether DNA methylation of the promoter could alter gene expression. As a first step, the ability of the M4-Promoter to drive gene expression was examined in cultured cells. GC-1spg cells but not NIH 3T3 cells transfected with the M4Promoter-containing construct display a 2.0 fold greater normalized firefly luciferase activity compared to cells transfected with the construct lacking a promoter. These results demonstrate that the region -700bp to +200bp region of Atp1a4 possesses promoter activity that is specific to the GC-1spg germ cell line. Next, in order to test the methylation dependence of the M4-Promoter activity, the pGL3Basic-M4-Promoter vector was in vitro methylated using the CpG methyltransferase enzyme . As a control, the pGL3-Basic-M4-Promoter construct was mock methylated under the same conditions in the absence of the methyltransferase enzyme. GC-1spg cells containing the in vitro methylated expression construct displayed decreased normalized luciferase activity compared to cells containing the mock-methylated construct demonstrating that the promoter is methylation-sensitive in this germ cell line. 3.6. The M4-CGI does not function as an alternative promoter As a first step towards determining if the M4-CGI functions as an alternative promoter, this region was assayed for promoter activity in GC-1spg and NIH3T3 cells. GC-1spg and NIH3T3 cells transfected with the pGL3-Basic-M4-CGI construct each displayed equivalent normalized luciferase activity as the cells transfected with the promoter-less pGL3-Basic vector demonstrating that the intragenic M4-CGI does not function as a promoter in either of these cell lines. 3.7. Methylation of the intragenic M4-CGI inhibits transcription elongation efficiency In order to determine if DNA methylation in the intragenic M4-CGI inhibits transcriptional elongation, a tandem reporter construct that has been previously used to quantify transcription elongation through polyadenlyation sequences was employed. In the TAN1 construct, transcription driven by the human PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 cytomegalovirus immediate early gene promoter initiates and proceeds through the firefly luciferase reporter sequence, through the putative transcriptional elongationaltering sequence, and then through the Renilla lucifera.