Born N , Thiesen HJ , Lorenz P .

	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].
	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.

Abrink, Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 beta. 2001, Pubmed

Abrink, Conserved interaction between distinct Krüppel-associated box domains and the transcriptional intermediary factor 1 beta. 2001, Pubmed
Amemiya, The African coelacanth genome provides insights into tetrapod evolution. 2013, Pubmed
Andreazzoli, RNA binding properties and evolutionary conservation of the Xenopus multifinger protein Xfin. 1993, Pubmed , Xenbase
Bellefroid, The evolutionarily conserved Krüppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. 1991, Pubmed , Xenbase
Birtle, Meisetz and the birth of the KRAB motif. 2006, Pubmed
Boudinot, Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish. 2011, Pubmed
Carey, An amino-terminal fragment of GAL4 binds DNA as a dimer. 1989, Pubmed
Corsinotti, Global and stage specific patterns of Krüppel-associated-box zinc finger protein gene expression in murine early embryonic cells. 2013, Pubmed
De Lucchini, A Xenopus multifinger protein, Xfin, is expressed in specialized cell types and is localized in the cytoplasm. 1991, Pubmed , Xenbase
Deuschle, Tetracycline-reversible silencing of eukaryotic promoters. 1995, Pubmed
Ding, SysZNF: the C2H2 zinc finger gene database. 2009, Pubmed
Dominski, A novel zinc finger protein is associated with U7 snRNP and interacts with the stem-loop binding protein in the histone pre-mRNP to stimulate 3'-end processing. 2002, Pubmed , Xenbase
Eddy, Profile hidden Markov models. 1998, Pubmed
Emerson, Adaptive evolution in zinc finger transcription factors. 2009, Pubmed
Fleischer, PML-associated repressor of transcription (PAROT), a novel KRAB-zinc finger repressor, is regulated through association with PML nuclear bodies. 2006, Pubmed
Frietze, ZNF274 recruits the histone methyltransferase SETDB1 to the 3' ends of ZNF genes. 2010, Pubmed
Frietze, Genomic targets of the KRAB and SCAN domain-containing zinc finger protein 263. 2010, Pubmed
Gentry, A functional interaction between the p75 neurotrophin receptor interacting factors, TRAF6 and NRIF. 2004, Pubmed
Grondin, The KRAB zinc finger gene ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix. 1996, Pubmed
Henderson, A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. 2000, Pubmed
Huang, The PR domain of the Rb-binding zinc finger protein RIZ1 is a protein binding interface and is related to the SET domain functioning in chromatin-mediated gene expression. 1998, Pubmed
Huntley, A comprehensive catalog of human KRAB-associated zinc finger genes: insights into the evolutionary history of a large family of transcriptional repressors. 2006, Pubmed
Ito, Role of zinc finger structure in nuclear localization of transcription factor Sp1. 2009, Pubmed
Itokawa, KAP1-independent transcriptional repression of SCAN-KRAB-containing zinc finger proteins. 2009, Pubmed
Ivanov, PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing. 2007, Pubmed
Jheon, Characterization of a novel KRAB/C2H2 zinc finger transcription factor involved in bone development. 2001, Pubmed
Li, A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. 2008, Pubmed
Lim, A KRAB-related domain and a novel transcription repression domain in proteins encoded by SSX genes that are disrupted in human sarcomas. 1998, Pubmed
Looman, A novel Krüppel-Associated Box identified in a panel of mammalian zinc finger proteins. 2004, Pubmed
Lorenz, Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation. 2001, Pubmed
Lorenz, Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies. 1998, Pubmed
Lorenz, The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues. 2010, Pubmed
Lupo, KRAB-Zinc Finger Proteins: A Repressor Family Displaying Multiple Biological Functions. 2013, Pubmed
Mannini, Structure/function of KRAB repression domains: structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses. 2006, Pubmed
Margolin, Krüppel-associated boxes are potent transcriptional repression domains. 1994, Pubmed
Mark, Comparative analysis of KRAB zinc finger proteins in rodents and man: evidence for several evolutionarily distinct subfamilies of KRAB zinc finger genes. 1999, Pubmed
Medugno, The Krüppel-like zinc-finger protein ZNF224 represses aldolase A gene transcription by interacting with the KAP-1 co-repressor protein. 2005, Pubmed
Messerschmidt, Trim28 is required for epigenetic stability during mouse oocyte to embryo transition. 2012, Pubmed
Mingot, Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors. 2009, Pubmed
Moosmann, Transcriptional repression by RING finger protein TIF1 beta that interacts with the KRAB repressor domain of KOX1. 1996, Pubmed
Mysliwiec, Characterization of zinc finger protein 496 that interacts with Jumonji/Jarid2. 2007, Pubmed
Nielsen, Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. 1999, Pubmed
Nowick, A prominent role of KRAB-ZNF transcription factors in mammalian speciation? 2013, Pubmed
Peng, Reconstitution of the KRAB-KAP-1 repressor complex: a model system for defining the molecular anatomy of RING-B box-coiled-coil domain-mediated protein-protein interactions. 2000, Pubmed
Peng, Biochemical analysis of the Kruppel-associated box (KRAB) transcriptional repression domain. 2000, Pubmed
Peng, The structurally disordered KRAB repression domain is incorporated into a protease resistant core upon binding to KAP-1-RBCC domain. 2007, Pubmed
Pengue, Repression of transcriptional activity at a distance by the evolutionarily conserved KRAB domain present in a subfamily of zinc finger proteins. 1994, Pubmed , Xenbase
Ponting, What are the genomic drivers of the rapid evolution of PRDM9? 2011, Pubmed
Pudney, Establishment of a cell line (XTC-2) from the South African clawed toad, Xenopus laevis. 1973, Pubmed , Xenbase
Quenneville, The KRAB-ZFP/KAP1 system contributes to the early embryonic establishment of site-specific DNA methylation patterns maintained during development. 2012, Pubmed
Rowe, KAP1 controls endogenous retroviruses in embryonic stem cells. 2010, Pubmed
Ruiz i Altaba, Xfin: an embryonic gene encoding a multifingered protein in Xenopus. 1987, Pubmed , Xenbase
Ryan, KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. 1999, Pubmed
Sadowski, GAL4 fusion vectors for expression in yeast or mammalian cells. 1992, Pubmed
Schultz, SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. 2002, Pubmed
Schuster-Böckler, HMM Logos for visualization of protein families. 2004, Pubmed
Shannon, Differential expansion of zinc-finger transcription factor loci in homologous human and mouse gene clusters. 2003, Pubmed
Shibata, TRIM28 is required by the mouse KRAB domain protein ZFP568 to control convergent extension and morphogenesis of extra-embryonic tissues. 2011, Pubmed
Shin, PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson's disease. 2011, Pubmed
Silver, Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. 1984, Pubmed
Sripathy, The KAP1 corepressor functions to coordinate the assembly of de novo HP1-demarcated microenvironments of heterochromatin required for KRAB zinc finger protein-mediated transcriptional repression. 2006, Pubmed
Tadepally, Evolution of C2H2-zinc finger genes and subfamilies in mammals: species-specific duplication and loss of clusters, genes and effector domains. 2008, Pubmed
Takahashi, Epigenetic regulation of TLR4 gene expression in intestinal epithelial cells for the maintenance of intestinal homeostasis. 2009, Pubmed
Tan, Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells. 2013, Pubmed
Thiesen, From repression domains to designer zinc finger proteins: a novel strategy of intracellular immunization against HIV. 1996, Pubmed
Thiesen, Multiple genes encoding zinc finger domains are expressed in human T cells. 1990, Pubmed , Xenbase
Vaquerizas, A census of human transcription factors: function, expression and evolution. 2009, Pubmed
Venturini, TIF1gamma, a novel member of the transcriptional intermediary factor 1 family. 1999, Pubmed
Vissing, Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins. 1995, Pubmed
Wagner, A broad role for the zinc finger protein ZNF202 in human lipid metabolism. 2000, Pubmed
Wang, Novel activity of KRAB domain that functions to reinforce nuclear localization of KRAB-containing zinc finger proteins by interacting with KAP1. 2013, Pubmed
Wessel, A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. 1984, Pubmed
Winton, Current lineages of the epithelioma papulosum cyprini (EPC) cell line are contaminated with fathead minnow, Pimephales promelas, cells. 2010, Pubmed
Witte, Specific interactions of the autoantigen L7 with multi-zinc finger protein ZNF7 and ribosomal protein S7. 1997, Pubmed
Witzgall, The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression. 1994, Pubmed
Wolf, Embryonic stem cells use ZFP809 to silence retroviral DNAs. 2009, Pubmed
Zuo, Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain. 2012, Pubmed