Fig 2: In/del binding transcription factor protein. A, schematic illustration of the double-stranded DNA probes for EMSA (gel electrophoresis mobility shift assay) in the region upstream of the HTRA1 activity coding region. Linking c-fos transcription factor to in/del-6. Profile of the c-fos transcription factor-binding sequence (ID: MA0099.3 from the JASPAR database). B, EMSA was performed to determine the in/del binding activity protein. In total, 50 μl nuclear protein from 661W cells was incubated with 100 pmol biotin-labeled Double-stranded DNA probes and analyzed on a 7.5% EMSA gel. Bands of interest were cut out and processed for LC-MS/MS analysis. C, Gtf2i-DNA probes binding test. The binding ability between Gtf2i and the in/del region was confirmed by WB. In/del DNA probes 6 and 7 were detected by an anti-Gtf2i antibody. Detection of Lamin A/C was used as an internal control. D, gene structure of Murine Gtf2i. Coding exons are depicted as black boxes and noncoding exons are in blank boxes. Five isoforms signify various alternatively spliced isoforms with exon 9, 10, 11, and 12 indicated. In 661W cells, there are only isoforms β and δ present by TA cloning (E, F). G, Gtf2i β/δ -DNA probes binding test. Both Gtf2i β and δ bind to the in/del-6 probe by WB. H, expression of Gtf2i isoform β and δ in nuclear or cytoplasmic extracts. Vector of Gtf2i isoform β or δ was transfected into 661W cells, followed by WB. Gtf2i isoform β was expressed in both nuclear and cytoplasmic extracts. However, isoform δ was only detected in nuclear but cytoplasmic extracts.

Fig 3: Promoter recognition by the TFII-I family.TFII-I (in red) and BEN (in blue) possess distinct promoter recognition properties in ESCs and embryonic craniofacial tissues (ETs). First, the majority of ESC promoters occupied by TFII-I become vacant in ETs (a); second, a large number of ESC promoters recognized by BEN recruit both transcription factors to the same site in ETs (b); third, the ESC promoters occupied by TFII-I and BEN are still recognized by both transcription factors in ETs, predominantly in the same sequence, although some sites lost their binding completely (d); and fourth, the promoters active in ETs recruit more BEN than TFII-I (e). The black numbers on the right indicate percentage expected from the random distribution of TFII-I or BEN binding. The green numbers indicate the observed distribution significantly deviated from the random distribution (chi-squared test).

Fig 4: Target recognition by TFII-I factors. (A) Chromatin recognition by TFII-I transcription factors in ESCs and embryonic craniofacial tissues (ETs). (1), the majority of TFII-I-bound ESC promoters tend to lose TFII-I binding in ETs. (2), the majority of BEN-bound ESC promoters recruit TFII-I de novo in ETs. (3), most of TFII-I and BEN-bound ESC promoters continue to retain these factors in ETs. (4), the promoters active in ETs recruit more BEN than TFII-I. (B) The feed-forward model explains the lack of correlation between the promoter binding by TFII-I and expression of the corresponding genes (stage 1, ESCs). Transcription factors (TFs) activated by TFII-I at stage 2 (ETs) can also recognize the TFII-I target sites and together they could initiate expression of stage-specific genes and additionally activate chromatin-modifying genes Ezh2 and Nsd1. These epigenetic factors mark novel target promoters (genes F and G) for repression or activation (e.g. repressive mark H3K27me3 and active mark H3K36me3). Stage 1, embryonic stem cells; stage 2, embryonic tissues.

Fig 5: Function analysis for Gtf2i and HTRA1.A, HTRA1 in/del and normal with or without Gtf2i β/δ vectors were transfected in HEK-293, 661W and COS-7 cells, respectively, following ELISA. Presence of the in/del significantly enhanced HTRA1 secretion and Gtf2i β/δ promoted enhanced efficiency with indel-HTRA1 but not the normal-HTRA1 in all cell lines. B, secretion of HTRA1 in human iPSCs derived from AMD patients. iPSCs were derived from individuals with normal versus in/del transcription regulators, followed by ELISA. Transfection of 661W cells with the Gtf2i β or δ expression vectors significantly enhanced in/del-Luciferase transcription comparing with normal. Representative WB (C) and immunocytochemistry (D) results are shown. Throughout, the results are expressed as the mean ± SEM. The p value was obtained by Student's t test.