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. 2020 Oct 6;48(19):10890–10908. doi: 10.1093/nar/gkaa832

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — The LTR12C repeats upstream of GBP2 and GBP5 act as promoters. (A) Promoter and enhancer elements overlapping with the LTR12C repeats upstream of GBP2 (top) and GBP5 (bottom). Chromosomal positions are indicated on top, GBP genes are shown in dark blue, LTR12C repeats are highlighted in pink and promoter/enhancer sequences identified by GeneHancer (66) are shown in light blue. Histone modifications frequently found near promoters (H3K4Me3) or enhancer elements (H3K4Me1, H3K27Ac) are shown as three individual tracks and were obtained from the Encyclopedia of DNA Elements (ENCODE) via the UCSC browser (98). An overlay of data obtained from seven different cell types (GM12878, H1-hESC, HSMM, HUVEC, K562, NHEK, NHLF) is shown. The tracks show data from the Bernstein lab at the Broad Institute, as part of the ENCODE Consortium. (B) LTR12C repeats upstream of GBP2 or GBP5 were inserted into enhancer (left) or promoter (middle, right) reporter vectors. Schematic vector maps are shown on top. In the left and middle panels, HEK293T cells were co-transfected with the indicated reporter vectors expressing Gaussia luciferase (GLuc) or firefly luciferase (FLuc) and control vector. Two days post transfection, reporter luciferase activity was determined and normalized to the activity of the control luciferase. In the right panel, primary CD4⁺ T cells were electroporated with the indicated reporter vectors expressing blue fluorescent protein (BFP). Two days post electroporation, the mean fluorescence intensity (MFI) of BFP cells was quantified by flow cytometry. In (B), mean values of 3–18 independent experiments ± SEM are shown (*P < 0.05; *** P < 0.001). (C) The overall structure of the consensus LTR12C (1577 nt) is schematically illustrated on top. At the bottom, partial LTR12C sequences upstream of the globin locus, GBP2 and GBP5 were aligned with the consensus sequence. Two pairs of TATA box and initiator (Itr) sequence are indicated by blue and violet frames, respectively. The 5′ TATA box (TATA I) and initiator (Itr I) have previously been shown to initiate transcription in the LTR12C repeat upstream of the globin locus.