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. 2017 Aug 30;45(20):11658–11672. doi: 10.1093/nar/gkx762

Figure 5.

Figure 5.

The LZA element functioned in human cells. (A) HEK293T cells were cultured in growth medium containing 0 or 40 μM TPEN for 3 h, and RNA was analyzed by qRT-PCR. Values are the ratio of mRNA levels at 40 μM TPEN/ 0 μM TPEN, an indication of transcriptional activation and are the average of three biological replicates and the standard deviation. In response to zinc deficiency, transcripts of the control gene Heat Shock Protein 90 Alpha Family Class B Member 1 (HSPCB) did not accumulate, whereas transcripts of ZIP2 and ZIP13 displayed significant accumulation (*P < 0.01). (B) Diagrams (not to scale) of the SV40 promoter (green) driving expression of the coding region of Firefly luciferase protein (yellow). The zipt-2.3 promoter region extended from −2199 to the ATG start codon (black line) and was cloned upstream of the SV40 promoter. Boxes show the LZA1 element (blue) and the LZA2 element (red); the LZA2 element was mutated by randomizing the order of 24 base pairs (MutLZA2). (C) Plasmids were co-transfected with a plasmid expressing renilla luciferase as a control for transfection efficiency into HEK293T cells. Forty-eight hours post-transfection, cells were incubated with 0 or 40 μM TPEN for 3 h and extracts were analyzed for firefly and renilla luciferase enzyme activity. Values are the ratio of normalized firefly luciferase enzyme activity at 40 μM TPEN/0 μM TPEN, an indication of transcriptional activation and are the average of three biological replicates and the standard deviation (*P < 0.01). (D) Sequence alignments of the LZA elements in the promoter regions of Caenorhabditis elegans genes zipt-7.1, zipt-2.1 and zipt-2.3, which were used to generate the LZA weight matrix, the promoter regions of human ZIP2 and ZIP13, and the intron of human ZIP13, which were identified in gene-specific searches for LZA elements. Identical residues are highlighted in black. Location is the position of the first base pair of the LZA element relative to the ATG start codon, and +/− refers to the orientation relative to the DNA strand that is transcribed.