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Circ. Res. 106(9), 1459-67. Deep mRNA Sequencing for In Vivo Functional Analysis of Cardiac Transcriptional Regulators: Application to Gαq 2010

S.J. Matkovich, Y. Zhang, D. Van Booven, and G.W. Dorn, II

Notes: Barcoded adaptors were ligated to cDNA using the Promega LigaFast kit prior to sequencing on an Illumina platform. (4530)

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Nucl. Acids Res. 35, 1245-1256. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene 2007

Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.

Notes: Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed. (3641)

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J. Biol. Chem. 278, 1758-1768. Identification of Dss1 as a 12-O-tetradecanoylphorbol-13-acetate-responsive gene expressed in keratinocyte progenitor cells, with possible involvement in early skin tumorigenesis 2003

Wei, S.-J., Trempus, C.S., Cannon, R.E., Bortner, C.D., and Tennant, R.W.

Notes: The LigaFast™ Rapid DNA Ligation System was used in the subcloning of an insert from a mammalian expression vector into a retroviral vector and also into a cellular reporter vector.  (2610)

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