Screening of human gene promoter activities using transfected-cell arrays

Abstract

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique.

Functional analysis and identification of cis-regulatory elements of human chromosome 21 gene promoters

“…Then, a secondary PCR was performed with Gateway adapter primers (Invitrogen), followed by PEG-8000 precipitation of PCR products. The modified reporter gene vector pZsGreen1-1 and the control plasmid pHcRed1-N1 were used as described before (9). PCR products were cloned into the pZsGreen vector using Gateway BP Clonase II Enzyme Mix (Invitrogen) and transformed into competent TOP10 cells following the manufacturer’s recommendations.…”

“…Samples for array spotting were prepared as previously described (9). Effectene reagent (Qiagen) with Enhancer (Qiagen) was used as transfection agent.…”

“…We recently established a procedure based on transfected-cell arrays for the functional characterization of promoters (9). Here, we expanded this approach to characterize the promoters of the human chromosome 21 (HSA21) genes, since HSA21 serves as a model for pilot genomic studies due to its small size (48 Mb) and because of its association with Down syndrome.…”

Development of a multifunctional luciferase reporters system for assessing endoplasmic reticulum-targeting photosensitive compounds

“…Bioassays utilizing reporter vectors regulated by promoters of cellular stress-responsive gene have been suggested as promising tools to assess photocytotoxic (Cheng et al 2010). A recent study has shown that MCF-7 cells containing a human HSPA1A promoter-driven luciferase reporter could be used for assessing oxidative stress associated with low-dose photodynamic therapy (Zheng et al 2013).…”

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy

“…Bioassays using reporter vectors regulated by cellular stress-responsive gene promoters have been suggested as promising tools for photocytotoxic assessment (Cheng et al 2010). The expression of significant stress responsiveness of HSPA1A is often regarded as a potential biomarker to evaluate oxidative cytotoxicity (Demirovic and Rattan 2012;Xin et al 2012).…”