The Transcriptional Repressive Activity of KRAB Zinc Finger Proteins Does Not Correlate with Their Ability to Recruit TRIM28

<div><p>KRAB domain Zinc finger proteins are one of the most abundant families of transcriptional regulators in higher vertebrates. The prevailing view is that KRAB domain proteins function as potent transcriptional repressors by recruiting TRIM28 and promoting heterochromatin spreading. However, the extent to which all KRAB domain proteins are TRIM28-dependent transcriptional repressors is currently unclear. Our studies on mouse ZFP568 revealed that TRIM28 recruitment by KRAB domain proteins is not sufficient to warrant transcriptional repressive activity. By using luciferase reporter assays and yeast two-hybrid experiments, we tested the ability of ZFP568 and other mouse KRAB domain proteins to repress transcription and bind TRIM28. We found that some mouse KRAB domain proteins are poor transcriptional repressors despite their ability to recruit TRIM28, while others showed strong KRAB-dependent transcriptional repression, but no TRIM28 binding. Together, our results show that the transcriptional repressive activity of KRAB-ZNF proteins does not correlate with their ability to recruit TRIM28, and provide evidence that KRAB domains can regulate transcription in a TRIM28-independent fashion. Our findings challenge the current understanding of the molecular mechanisms used by KRAB domain proteins to control gene expression and highlight that a high percentage of KRAB domain proteins in the mouse genome differ from the consensus KRAB sequence at amino acid residues that are critical for TRIM28 binding and/or repressive activity.</p></div

Mutations in both the first and second KRAB motifs of ZFP568 affect ZFP568 ability to respond to TRIM28 levels.

<p>Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells, in the presence of wild type and mutant versions of GAL4DBD-ZFP568 as indicated. <b>(A)</b> Luciferase expression in the presence of increasing amounts of transfected FLAG-TRIM28. <b>(B)</b> Luciferase expression upon TRIM28 depletion with siRNA. No siRNA and unspecific siRNAs were used as controls. Western blots show levels of TRIM28 as tested with α-TRIM28 antibodies. Luciferase expression is graphed as fold repression normalized to Gal4DBD (empty vector) and relative to wild type (column 1). Error bars represent standard deviation. Asterisks indicate samples which pairwise comparison had a p<0.005 (**). ns, no statistical significance.</p

The <i>chato</i> mutation disrupts ZFP568 repressive activity, but not its ability to bind TRIM28.

<p><b>(A)</b> Domain structure of ZFP568. The red asterisk indicates the location of the <i>chato</i> mutation, which causes a Leu to Pro substitution. The red arrow indicates the position of the early stop codon in <i>P103E09</i> mutants, which results in a truncated protein containing only the first ZFP568 11 amino acids. <b>(B)</b> Sequence alignment of the first and second KRAB motifs of ZFP568 with the Pfam KRAB consensus. Grey boxes indicate residues important for repressive activity as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref011" target="_blank">11</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref025" target="_blank">25</a>]. The red box highlights the conserved Leu residue mutated in <i>chato</i>. <b>(C-E)</b> Whole mount wild type (C), <i>Zfp568</i>^<i>chato</i> (D) and <i>Zfp568</i>^{<i>P103E09</i>} (E) E8.5 embryos. The discontinued line highlights the abnormal U-shape of <i>Zfp568</i> mutants, as compared with the V-shaped profile of wild type embryos. Empty arrowheads point to the yolk sac, which is abnormally ruffled in <i>Zfp568</i> mutants. <b>(F)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells, in the presence of wild type (grey) and a KRAB1 L-P mutant (green) GAL4DBD-ZFP568. <b>(G)</b> Yeast two-hybrid assay for TRIM28 interaction with wild type ZFP568 (upper left) and a KRAB1 Leu-Pro mutant ZFP568 (upper right). T-antigen and p53 interaction was used as positive control. T-antigen and LmnC were used as negative control. <b>(H)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells, in the presence of wild type (grey) and KRAB1 Leu-Pro mutant (green) GAL4DBD-ZFP568, either in the absence (columns 1, 4) or presence of increasing amounts of FLAG-TRIM28 (columns 2–3 & 5–6). Luciferase expression is graphed as fold repression normalized to Gal4DBD (empty vector) and relative to wild type (column 1). Error bars represent standard deviation. Western blots show levels of FLAG-TRIM28 protein for each condition. Asterisks indicate samples which pairwise comparison had a p<0.05 (*) or p<0.005 (**). ns, no statistical significance.</p

The repressive activity of ZFP568 depends on the properties of its KRAB motifs, but not on their number or location within the protein.

<p><b>(A)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells, in the presence of wild type (grey) and mutant versions of GAL4DBD-ZFP568: lacking the second KRAB motif (∆KRAB2), lacking the first KRAB motif (∆KRAB1), with two KRAB1 motifs (KRAB1 KRAB1), with two KRAB2 motifs (KRAB2 KRAB2), or with a mutated KRAB1 motif (KRAB1 L-P). Luciferase expression is graphed as fold repression compared to Gal4DBD (empty vector) and relative to wild type (column 1). Error bars represent standard deviation. <b>(B-D)</b> Yeast two-hybrid assays showing the interaction of TRIM28 with wild type and mutant versions of ZFP568 and ZFP809 as indicated. <b>(E)</b> T-antigen and p53 were used as positive controls. T-antigen and LmnC were used as negative controls. A truncated ZFP568 lacking all KRAB motifs (ZF) was also used as a negative control in panel B (column 6). The pictures show representative yeast colonies for each of the interactions tested, as grown on low stringency media (upper row) and high stringency media (lower row). Asterisks indicate samples which pairwise comparison had a p<0.05 (*) or p<0.005 (**). ns, no statistical significance.</p

Mutations in the second KRAB motif of ZFP568 do not disrupt ZFP568 repressive activity.

<p><b>(A-B)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells, in the presence of wild type (grey) and mutant versions of GAL4DBD-ZFP568 as follows: KRAB1 L-P (green in A), KRAB2 L-P (orange in A), L-P mutations in both KRAB1 & KRAB2 (striped), KRAB1 DV-AA (green in B), KRAB2 DV-AA (orange in B). <b>(C)</b> Yeast two-hybrid assay testing the interaction of TRIM28 with wild type and mutant versions of ZFP568 as indicated. T-antigen and p53 interaction was used as positive control. T-antigen and LmnC were used as negative control. A truncated ZFP568 lacking all KRAB motifs (ZF) was also used as a negative control (column 7). The pictures show representative yeast colonies for each of the interactions tested, as grown on low stringency media (upper row) and high stringency media (lower row). Luciferase expression is graphed as fold repression normalized to Gal4DBD (empty vector) and relative to wild type. Error bars represent standard deviation. Asterisks indicate samples which pairwise comparison had a p<0.05 (*) or p<0.005 (**). ns, no statistical significance.</p

Amino acid differences between the first and second KRAB motifs of ZFP568 influence transcriptional repressive activity.

<p><b>(A)</b> Alignment of the amino acid sequence of the first and second KRAB domains of ZFP568 with a weblogo for all mouse KRAB domains. Residues highlighted in grey indicate residues important for repressive activity as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref011" target="_blank">11</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref025" target="_blank">25</a>]. Residues highlighted in blue and yellow indicate differences between the first and second ZFP568 KRAB motifs that were tested for their effects on transcriptional repressive activity as shown in B and C. (<b>B-C)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells in the presence of <b>(B)</b> mutant versions of GAL4DBD-ZFP568 lacking the second KRAB motif and with KRAB1-to-KRAB2 amino acid substitutions in the indicated KRAB1 domain residues (K4E in column 2, Q12L in column 3, Q22H in column 4, D28A in column 5 & E32D in column 6) and <b>(C)</b> mutant versions of GAL4DBD-ZFP568 lacking the first KRAB motif and with KRAB2-to-KRAB1 amino acid substitutions in the indicated KRAB2 domain residues (D32E in column 3). Mutant versions of GAL4DBD-ZFP568 lacking the second KRAB motif (∆KRAB2, column 1) or the first KRAB motif (∆KRAB1, column 7 in B, column 2 in C) are shown for comparison. Luciferase expression is graphed as fold repression compared to Gal4DBD (empty vector) and relative to ∆KRAB2 (column 1). Error bars represent standard deviation. Asterisks indicate samples which pairwise comparison had a p<0.05 (*) or p<0.005 (**). ns, no statistical significance.</p

Transcriptional repression and TRIM28 binding in selected mouse KRAB Zinc finger proteins.

<p><b>(A)</b> Alignment of KRAB domain sequences of ZFP568 with the indicated mouse KRAB domain proteins. Residues highlighted in grey indicate amino acids important for repressive activity (as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref011" target="_blank">11</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163555#pone.0163555.ref025" target="_blank">25</a>]). Amino acid differences between the first and second ZFP568 KRAB motifs are highlighted in yellow, if they showed an impact in transcriptional repressive activity, or in blue, if they had no impact. Red font was used to highlight critical KRAB residues that constitute non-conservative amino acid substitutions with respect to the KRAB consensus sequence and/or are not highly represented in mouse KRAB domain proteins. <b>(B-C)</b> Quantification of luciferase expression from a 5xUAS-luciferase reporter in HEK293T cells for the indicated mouse Zinc finger proteins, <b>(B)</b> in the absence (-) or presence (+) of transfected FLAG-TRIM28. <b>(C)</b> in wild type and mutant versions that carried point mutations or lacked the KRAB motif as indicated. Luciferase expression is graphed as fold repression compared to Gal4DBD (empty vector) and relative to wild type ZFP568. Error bars represent standard deviation. Asterisks indicate samples which pairwise comparison had a p<0.05 (*) or p<0.005 (**). ns, no statistical significance. <b>(D)</b> Yeast two-hybrid assays showing the interaction of TRIM28 with the indicated mouse KRAB domain proteins. ZFP568 L-P mutant was included for reference. T-antigen and p53 were used as positive controls. T-antigen and LmnC were used as negative controls. A truncated ZFP568 lacking all KRAB motifs was also used as a negative control (column 12). The pictures show representative yeast colonies for each of the interactions tested, as grown on low stringency media (upper row) and high stringency media (lower row).</p

Prostasin is required for the activation of matriptase during placental differentiation.

<p>(A and B) Expression of prostasin (A) and matriptase (B) in placental tissues of wildtype mice at E11.5. Both proteins were expressed in the chorionic (arrows) and labyrinthine (arrowheads) trophoblasts. (C) Western blot detection of active prostasin in the fetal part of the placenta of wildtype (<i>Prss8^+/+</i> and <i>St14^+/+</i>, lanes 2, 4, and 6), prostasin-deficient (<i>St14^+/+;Prss8^−/−</i>) (lane 1), and matriptase-deficient (<i>St14^−/−;Prss8^+/+</i>) (lanes 3 and 5) embryos at E11.5 after immunoprecipitation with anti-mouse HAI-1 antibodies. Immunoprecipitated proteins in lanes 5 and 6 were acid-exposed to dissociate prostasin-HAI-1 complexes, and then incubated with PN-1 prior to western blot analysis. Positions of bands corresponding to active prostasin, prostasin/PN-1 complex, as well as non-specific signals of IgG heavy and light chains are indicated on the right. Positions of molecular weight markers (kDa) are shown on the left. (D) Omission of anti-HAI-1 antibody resulted in loss of detectable prostasin (compare lanes 1 and 2), indicating that the detected prostasin formed complexes with HAI-1. (E) Quantification of the relative amount of active prostasin in wildtype and matriptase placentae by densitometric scanning of prostasin western blots of HAI-1 immunoprecipitated material from (<i>Prss8^−/−;St14^+/+</i>, N = 3, <i>Prss8^+/+;St14^−/−</i>, N = 3, and <i>Prss8^+/+;St14^+/+</i>, N = 6). Data are shown as mean ± standard deviation (N.S., not significant). (F and G) Western blot detection of active matriptase in the fetal part of the placenta at E11.5 (F) after anti-HAI-1 immunoprecipitation, and in the epidermis of newborn skin (G) of wildtype (<i>Prss8^+/+</i> and <i>St14^+/+</i>) (F, lanes 1, and 3, and G, lane 2), prostasin-deficient (<i>St14^+/+;Prss8^−/−</i>) (F, lane 4 and G, lane 1), and matriptase-deficient (<i>St14^−/−;Prss8^+/+</i>) (F, lane 2, and G, lane 3) embryos. A 30 kDa band representing the active serine protease domain of matriptase (Mat SPD) was present in extracts from wildtype (lanes 1 and 3 in F), but not in matriptase- (lane 2 in F) or prostasin-deficient (lane 4 in F) placenta. Zymogen (Mat FL) and active (Mat SPD) forms of matriptase were detected in extracts from both wildtype and prostasin-deficient, but not matriptase-deficient epidermis. (H–H″) Immunohistochemical staining of matriptase in control <i>Prss8⁺</i> (H) and prostasin-deficient <i>Prss8^−/−</i> (H′) placenta at E11.5. Specificity of staining of chorionic and labyrinthine trophoblasts (examples with arrows) is shown by the absence of staining of corresponding cells in <i>St14^−/−</i> placenta (H″). Insets in H and H′ are parallel sections stained with prostasin antibodies. Open arrowheads in H–H″ show examples of non-specific staining. Scale bars: A, B, H, H′, and H″, 50 µm.</p

Neural tube defects and embryonic lethality in HAI-2–deficient mice are not dependent on PAR-2, and combined PAR-1 and matriptase deficiency does not phenocopy combined PAR-1 and PAR-2 deficiency.

<p>(A) Distribution of <i>Spint2</i> genotypes at E9.5 in PAR-2-expressing (<i>F2rl1^+/+</i> or <i>F2rl1^+/−</i>, blue bars) and PAR-2-deficient (<i>F2rl1^−/−</i>, green bars) offspring from interbred <i>Spint2^+/−,F2rl1^+/−</i> mice. No <i>Spint2^−/−</i> embryos were detected irrespective of PAR-2 expression. (B) Frequency of exencephaly observed in HAI-2 and PAR-2-sufficient (<i>Spint2⁺;F2rl1⁺</i> N = 366), PAR-2-deficient (<i>Spint2⁺;F2rl1^−/−</i>, N = 164), HAI-2-deficient (<i>Spint2^−/−,F2rl1⁺</i>, N = 18), and PAR-2 and HAI-2 double- (<i>Spint2^−/−;F2rl1^−/−</i>, N = 12) deficient embryos extracted at E9.5–E11.5. Loss of PAR-2 activity fails to correct neural tube defects in HAI-2-deficient embryos. (C) Distribution of <i>St14</i> alleles at E11.5–15.5 in PAR-1-expressing (<i>F2r^+/+</i> or <i>F2r^+/−</i>, blue bars) and PAR-1-deficient (<i>F2r^−/−</i>, green bars) embryos from interbred <i>St14</i>^+/−<i>;F2r^+/−</i> mice. Loss of PAR-1 activity does not affect embryonic survival of matriptase-deficient mice. (D) Frequency of exencephaly (Ex), spina bifida (SB), and curly tail (CT) in E9.5–18.5 embryos with different levels of expression of PAR-1 (<i>F2r⁺</i> or <i>F2r^−/−</i>) and matriptase (<i>St14⁺</i>or <i>St14^−/−</i>). A total of 326 embryos were analyzed. Loss of matriptase does not significantly increase the incidence of neural tube defects in PAR-1-deficient embryos. (E) Comparison of the severity of exencephaly in HAI-2-deficient (<i>Spint2^−/−</i>, N = 29) and PAR-1 and PAR-2 double-deficient (<i>F2r^−/−;F2rl1^−/−</i>, N = 39) embryos. 95% of affected <i>F2r^−/−;F2rl1^−/−</i> embryos exhibited exencephaly that was confined to hindbrain region of the cranium (HB only, green bars), with the remaining 5% extending to the midbrain region (MB-HB, blue bars). In contrast, only 10% of exencephalies observed in <i>Spint2^−/−</i>-deficient mice were confined to the hindbrain, with 59% extended to midbrain, and 31% to forebrain region (FB-HB, red bars). (F–G′) Ventral (F and G) and dorsal (F′ and G′) view of non-affected control (F and F′) and affected PAR-1 and PAR-2 double-deficient (<i>F2r^−/−;F2rl1^−/−</i>) (G and G′) embryos at E9.5. The initial stages of neural tube closure all appear to be unaffected by the combined absence of PAR-1 and PAR-2. (H–J) Appearance of control (H) and PAR-1 and PAR-2 double-deficient embryos with exencephaly (I and J) at E14.5. Exencephaly in 95% of the affected PAR-1 and PAR-2 double-deficient embryos was restricted to hindbrain region (HB, two-sided arrow in I) and extended to midbrain (MB-HB, two-sided arrow in J) in only 5% of the cases. (K and K′) Ventral (K) and dorsal (K′) view of the macroscopic appearance of HAI-2-deficient (<i>Spint2^−/−</i>) embryos at E9.5. Divergence of neural folds (arrows) and defects in neural tube closure extending from forebrain region to cervix are obvious. Open arrowheads show normal formation of medial hinge points. (L) Macroscopic appearance of a HAI-2-deficient embryo with exencephaly at E14.5. 90% of embryos presented with exencephaly that included at least midbrain and hindbrain regions of the developing cranium. (M) Histological appearance (nuclear fast red staining) of PAR-1 and PAR-2 double-deficient embryo with exencephaly at E9.5. Defined medial (arrow) and dorsolateral (arrowheads) hinge points are clearly visible. Scale bar: 150 µm.</p

Reduced prostasin activity restores placental development and embryonic survival of HAI-1–deficient mice.

<p>(A) Distribution of genotypes of born offspring of intercrossed <i>Spint1</i>^+/−<i>;Prss8^fr/−</i> mice. No <i>Spint1^−/−</i> mice expressing one or two wildtype prostasin alleles (<i>Prss8^+/+</i> or <i>Prss8^+/fr</i>, blue bars) were identified, while <i>Spint1^−/−</i> embryos carrying two mutant prostasin alleles (<i>Prss8^fr/fr</i>, green bars) were found in near-expected frequency. (B–G) Representative low (B–D) and high (E–G) magnification images showing the histological appearance of H&E-stained placental tissues of (<i>Spint1⁺;Prss8⁺</i>) (B and E), (<i>Spint1^−/−;Prss8⁺</i>) (C and F), and (<i>Spint1^−/−;Prss8^fr/fr</i>) (D and G) embryos at E11.5. The thickness of the placental labyrinth (two-sided arrows between the dotted lines in B–D), as well as the number of fetal vessels (E–G, arrows) and lacunae filled with maternal blood (E–G, arrowheads) within the labyrinth is markedly reduced in prostasin-sufficient (C and F), but not in prostasin-deficient (D and G) <i>Spint1^−/−</i> embryos, when compared to the controls (B and E). (H, I) Quantification of the maximum thickness of the labyrinth layer (H) and the number of fetal vessels in the placental labyrinth (I) of <i>Spint1⁺;Prss8⁺</i>, <i>Spint1⁺;Prss8^fr/fr</i>, <i>Spint1^−/−;Psrr8⁺</i>, and <i>Spint1^−/−;Psrr8^fr/fr</i> embryos at E11.5. The thickness of the labyrinth and fetal vessel density were strongly diminished in HAI-1-deficient mice but completely restored in HAI-1-deficient mice with low prostasin activity. (J) Outward appearance of one-year-old <i>Spint1^−/−;Prss8^fr/fr</i> and littermate <i>Spint1⁺;Prss8⁺</i> mice. ***, p<0.0001, Student's t-Test, two tailed. Scale bars: B–D, 100 µm; E–G, 25 µm.</p