Evaluation of the transcriptional repression potential of different KRAB domains.

Heterologous luciferase reporter assays using fusions between the indicated KRAB domains and the Gal4-DNA-binding domain (Gal4). Results of 3–5 independend experiments (n = 3–5, see charts). Asterisks denote statistical significance in a two-tailed paired T-test (one asterisk in brackets means p<0.055; one asterisk p<0.05, two asterisks p<0.01 and three asterisk p<0.001); A: Illustration of assay; only the firefly luciferase reporter carries upstream Gal4 DNA-binding sites (Gal4-DBS) while the Renilla luciferase does not and is used for normalization. B: Assay in human HeLa cells, comparing Gal4 as baseline (set to 1) with its fusions to the indicated KRAB domains/subdomains. C: KRAB-B domain swapping experiment in human HeLa cells, switching the ZNF10-B domain to XFIN-A and vice versa. D: Same experiment as C, but done in Xenopus laevis A6 cells. E: Testing of various N-terminal parts of PRDM9 in human HeLa cells, numbers designate amino acid positions in the full-length protein. PRDM9 domain abbreviations: SSXRD = SSX repression domain motif (PFAM PF09514; [78]); PR/SET = derivative of SET doman , (Drosophila Su(var)3–9, Enhancer-of-zeste and Trithorax; PFAM PF00856 [79].