Mature sperm. In addition, the mouse Atp1a4 promoter was not identified as a HMR in the whole genome studies discussed above. While these data could be interpreted to indicate that Celgosivir custom synthesis methylation of the promoter region is not important in regulating Atp1a4 expression, it has been documented that some testis-specific genes such as Pgk-2, ApoA1 and Alf, known to be regulated by methylation of CpGs in their promoter region, undergo remethylation of the CpGs present in their promoter region in the mature spermatozoa as part of the sperm maturation process that takes place in the epididymis. Thus it remains possible that hypomethylation of the Atp1a4 promoter is involved in increasing Atp1a4 expression earlier in sperm development and that the individual CpGs in the promoter had been remethylated in the mature sperm as translation is shut down in these cells. Our dual luciferase reporter assay confirms that the activity of the M4- Promoter is methylation dependent in GC1-spg cells. In all strongly suggesting that methylation of the Atp1a4 promoter may play a role in regulating the expression of this gene in the mouse. The analysis of the methylation state of the Atp1a4 promoter in cells at earlier stages of spermatogenesis awaits further study. Expression and methylation analysis in Dnmt KO ES cells suggests that Dnmt3a plays a major role in regulating the expression of Atp1a4. Interestingly, an essential role for Dnmt3a in male germ cell development has been reported, with specific deletion of Dnmt3a, but not Dnmt3b, in male germ cells resulting in azoospermia and loss of methylation at imprinted loci. Thus Dnmt3a may be the important DNA methyltransferase directing the normal development of male germ cells, where at least part of its function is to ensure the proper level of expression of certain germ cell-specific genes like Atp1a4. It is not completely clear why loss of either Dnmt3 isoform alone reduces promoter and CGI methylation and increases Atp1a4 expression whereas loss of both de novo methyltransferases has less effect on methylation and no effect on gene expression. However, it has been reported that, in addition to de novo methylation, Dnmt3a and Dnmt3b may possess deaminase activity and may therefore also be involved in the process of active demethylation. Thus, it is possible that the loss of both Dnmt3a and Dnmt3b actually reduces demethylation which results in increased methylation of the Atp1a4 promoter and the CGI in the Dnmt3a/3b DKO ES cells compared to the Dnmt3a KO or Dnmt3b KO ES cells. 
Studies have suggested that intragenic CGIs are be able to alter transcription by at least three fundamentally different mechanisms: 1) by functioning as an alternative promoters that drives the expression of potential regulatory RNA molecules, 2) by serving as cis regulatory elements that can either enhance or repress gene expression or 3) by altering elongation of their transcripts. To provide a comprehensive analysis of whether the 4 intragenic CGI Relebactam web impacts expression of Atp1a4 and if its function is regulated by methylation, we tested all three of these possibilities. First, the potential role of the M4-CGI in regulating gene expression was assessed. Towards that end, we tested the role of the intragenic M4-CGI as an PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 alternative promoter. Our data demonstrates that the M4-CGI is unable to drive reporter gene expression in GC-1spg cells or NIH3T3 cells and is therefore not a functional promoter in this context. Further analysis of the.Mature sperm. In addition, the mouse Atp1a4 promoter was not identified as a HMR in the whole genome studies discussed above. While these data could be interpreted to indicate that methylation of the promoter region is not important in regulating Atp1a4 expression, it has been documented that some testis-specific genes such as Pgk-2, ApoA1 and Alf, known to be regulated by methylation of CpGs in their promoter region, undergo remethylation of the CpGs present in their promoter region in the mature spermatozoa as part of the sperm maturation process that takes place in the epididymis. Thus it remains possible that hypomethylation of the Atp1a4 promoter is involved in increasing Atp1a4 expression earlier in sperm development and that the individual CpGs in the promoter had been remethylated in the mature sperm as translation is shut down in these cells. Our dual luciferase reporter assay confirms that the activity of the M4- Promoter is methylation dependent in GC1-spg cells. In all strongly suggesting that methylation of the Atp1a4 promoter may play a role in regulating the expression of this gene in the mouse. The analysis of the methylation state of the Atp1a4 promoter in cells at earlier stages of spermatogenesis awaits further study. Expression and methylation analysis in Dnmt KO ES cells suggests that Dnmt3a plays a major role in regulating the expression of Atp1a4. Interestingly, an essential role for Dnmt3a in male germ cell development has been reported, with specific deletion of Dnmt3a, but not Dnmt3b, in male germ cells resulting in azoospermia and loss of methylation at imprinted loci. Thus Dnmt3a may be the important DNA methyltransferase directing the normal development of male germ cells, where at least part of its function is to ensure the proper level of expression of certain germ cell-specific genes like Atp1a4. It is not completely clear why loss of either Dnmt3 isoform alone reduces promoter and CGI methylation and increases Atp1a4 expression whereas loss of both de novo methyltransferases has less effect on methylation and no effect on gene expression. However, it has been reported that, in addition to de novo methylation, Dnmt3a and Dnmt3b may possess deaminase activity and may therefore also be involved in the process of active demethylation. Thus, it 
is possible that the loss of both Dnmt3a and Dnmt3b actually reduces demethylation which results in increased methylation of the Atp1a4 promoter and the CGI in the Dnmt3a/3b DKO ES cells compared to the Dnmt3a KO or Dnmt3b KO ES cells. Studies have suggested that intragenic CGIs are be able to alter transcription by at least three fundamentally different mechanisms: 1) by functioning as an alternative promoters that drives the expression of potential regulatory RNA molecules, 2) by serving as cis regulatory elements that can either enhance or repress gene expression or 3) by altering elongation of their transcripts. To provide a comprehensive analysis of whether the 4 intragenic CGI impacts expression of Atp1a4 and if its function is regulated by methylation, we tested all three of these possibilities. First, the potential role of the M4-CGI in regulating gene expression was assessed. Towards that end, we tested the role of the intragenic M4-CGI as an PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1985460 alternative promoter. Our data demonstrates that the M4-CGI is unable to drive reporter gene expression in GC-1spg cells or NIH3T3 cells and is therefore not a functional promoter in this context. Further analysis of the.