Figure 1.
Comparative depiction of the KRAB domain sequences of ZNF10, XFIN, PRDM7 and PRDM9.
Alignment of KRAB-A (A) or KRAB-B (B) subdomains and comparison to the respective human and frog HMM models. The sequences were derived from NCBI Refseq database entries for human ZNF10/Kox1 (ZNF10; NP_056209), Xenopus laevis XFIN (XFIN-ref; NP_001095247), human PRDM7 (NP_001091643) and human PRDM9 (NP_064612). The corrected XFIN-A sequences were derived by in-silico translation from GenBank EU277665 (labeled XFIN-A corr; see text for details). Brackets with an asterisk denote amino acid groups whose mutation have been shown to disrupt transcriptional repression, whereas those with open circle denote positions where mutation had not much effect [11]. Arrowheads point to amino acids that might be responsible for observed functional differences (see text). The right arrow marks the methionine that has been considered the start of the XFIN protein in the database reference sequence. The consensus reflects amino acids in at least 60% of the molecules at each position. HMMER scores against respective human or Xenopus HMM matrices are given to the right of each sequence. The HMM matrices are visualized as HMM-Logos [57] at the bottom of each sub-figure. Note, that the amino acid positions in the logo are aligned with the ones in the sequence alignments.
Figure 2.
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].
Figure 3.
Intracellular distribution of ectopically expressed Gal4-KRAB fusion proteins and colocalization analysis with endogenous TRIM28 in human HeLa cells.
24-transfection of the indicated Gal4 effector constructs cells were fixed and stained for Gal4 (shown in green) and TRIM28 (red). Each row shows the cells of the same image pane. Arrowheads point to some of the telltale prominent foci showing accumulated Gal4 and TRIM28 proteins. Bar = 10 µm.
Figure 4.
Analysis of the nuclear/cytoplasmic compartmentalization of Gal4-KRAB fusion proteins containing a strong nuclear export sequence (NES).
Cells were transfected with the indicated Gal4 fusion proteins, fixed and stained for Gal4 and (in human cells) for TRIM28. Cells with Gal4 expression were manually scored under the fluorescence microscope to contain excess nuclear (Nuc>Cyt), excess cytoplasmic (Nuc<Cyt) or similar distributions (Nuc≈Cyt) and counted. A: Example fluorescence images from human HeLa cells (corresponding channels for Gal4 and TRIM28 staining of the same image pane one below the other). Arrowheads mark some of the foci with the telltale simultaneous Gal4-KRAB and TRIM28 accumulation. Bar = 10 µm. B: Quantification for human HeLa cells (7–9 independent experiments for each construct; between 134 and 442 cells counted per experiment) 24 hours post-transfection and Xenopus laevis A6 cells (4–7 independent experiments, between 154 and 447 cells counted) 48 hours after transfection. Two-sided T-tests looking at the data between ZNF10-KRAB-AB and XFIN-KRAB-AB Gal 4 fusions indicated p-values of p = 1.7×10−6 (HeLa) and p = 0.23 (A6), respectively. Exact numbers of scored cells and complete statistical analysis can be found in Table S3.
Figure 5.
Cellular interaction analysis of different KRAB domains with the endogenous TRIM28 protein in human HeLa cells.
24-KRAB fusion constructs, extracts were analyzed by immunoprecipitation (IP) with GST antibodies followed by Western blotting to look for shared complexes with cellular TRIM28 protein. An extract sample (ex) was run as positive control for Western blot immunostaining. The upper part of the blot was probed with anti-TRIM28, the lower part with anti-GST antibodies. A: Example gel for the ZNF10-AB versus A only and ZNF10-PP-AB analysis. B, D: Quantitative evaluation of the blot signals from 3 independent experiments. Columns and numbers indicate relative TRIM28/GST-fusion ratios (ZNF-AB result set to 100%). C: Example gel for the comparison of ZNF10-AB, XFIN-AB and the B subdomain swaps. Statistical significance tested using a one sample 2-tailed T-test. One asterisk p<0.05, two asterisks p<0.01, three asterisks p<0.001.
Figure 6.
Characterization of KRAB-domains in EPC fish cells.
A: Example of indirect immunofluorescence analysis of the nuclear/cytoplasmic compartmentalization of Gal4-KRAB-NES proteins alone (a–c, Gal4-staining) or after co-transfection with human TRIM28 (d–f, Gal4-staining; g–i, TRIM28-staining; corresponding panes one below the other). Bar = 10 µm. B: Quantification of the nuclear/cytoplasmic compartmentalization experiments (5–10 independent experiments for each construct, between 105 and 301 cells counted per experiment) 48 hours after transfection. Exact numbers and statistical analysis are given in Table S3. C: Transcriptional repression potential of different KRAB domains measured by heterologous luciferase reporter assays using Gal4-KRAB effector constructs as in Figure 2. Filled bars represent experiments without exogenous human TRIM28, open bars those with co-transfection of TRIM28. The Gal4 result of each experiment in absence of TRIM28 was used as baseline and set to 1 and four independent experiments were run. Statistical significance of a 2-tailed paired T-test is indicated by one asterisk (p<0.05), two asterisks (p<0.01) and three asterisks (p<0.001).
Figure 7.
Protein alignments of human TRIM28 with the putative orthologs from Xenopus and lobe-finned fish species.
Sequences are designated according to their database accession numbers (NCBI Refseq or ENSEMBL) where available and a species prefix (hsap = Homo sapiens; xlae = Xenopus laevis; xtro = Xenopus tropicalis; pann = Protopterus annectens (lungfish); lcha = Latimeria chalumnae (coelacanth)). The lungfish sequences do not bear official identifiers yet and are named arbitrarily here. They were obtained as described in Materials and Methods (Bioinformatics section). The original transcript sequences are provided in Table S2. The consensus sequence under the alignments is based on the occurence of the shown amino acids in at least 60% of the molecules. Reciprocal BLASTp of the frog and fish sequences against human sequence databases resulted in human TRIM28 as the best hit and thus supported the ortholog assignment (data not shown). Boxes around and labels under the sequence alignments delineate the domain organization based on human TRIM28 [80].