Engineering synthetic VIM and TCF21 enhancer/promoter plasmids.

The human Vim (A) and Tcf21 (B) genomic locus with H3K27Ac Mark on seven cell lines from ENCODE, alignments of 100 vertebrates, and ENCODE Candidate Cis-Regulatory Elements (cCREs) combined from all cell types. cCREs are labeled according to the regulatory signatures: Red box, promoter-like signature; orange box, proximal enhancer-like signature; yellow box, distal enhancer-like signature. All data was extracted from USCS Genome Browser. (A) Construction strategy of pGL4.14(Vim, GLuc-WPRE-SpA). Two Vim mRNA sequenes (ENST00000544301.7 and ENST00000224237.9) are registered and there are two promoter-like signatures (red box) in cCREs. PCR 2.5 kbp including both promoter-like signatures, first exon and intron of ENST00000544301.7, second exon of ENST00000544301.7 (this is a first exon of ENST00000224237.9), and the annotated translation start site (TISS), then insert into Eco31I site of a pGL4.14 reporter vector. The secreted luciferase GLuc is expressed downstream of the inserted enhancer/promoter candidates. In addition, synthetic polyadenylation signal and RNA polymerase II transcriptional pause signal from the human α2 globin gene are upstream of the candidate sequences to suppress transcriptions from the vector backbone Amp and ori. (B) Construction strategy of pGL4.14(Tcf21, GLuc-WPRE-SpA). Two Tcf21 mRNAs (ENST00000367882.5 and ENST00000237316.3) are registered and two antisense RNA (TARID, ENST00000607033.5 and ENST00000630119.2) also transcribe from the same locus. There are two promoter-like signatures (red box) in cCREs, but one signature (dashed box) may be associated with the antisenses. PCR 0.9 kbp enhancer and Tcf21 promoter-like signiture, 1.6 kbp intragenic enhancer signatures, and the annotated TISS, then insert 0.9 kbp into Eco31I site and 1.6 kbp into BglII site of the pGL4.14.

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