Transcription factor YY1 functions as a PcG protein in vivo

Abstract

Polycomb group (PcG) proteins function as high molecular weight complexes that maintain transcriptional repression patterns during embryogenesis. The vertebrate DNA binding protein and transcriptional repressor, YY1, shows sequence homology with the Drosophila PcG protein, pleiohomeotic (PHO). YY1 might therefore be a vertebrate PcG protein. We used Drosophila embryo and larval/imaginal disc transcriptional repression systems to determine whether YY1 repressed transcription in a manner consistent with PcG function in vivo. YY1 repressed transcription in Drosophila, and this repression was stable on a PcG-responsive promoter, but not on a PcG-non-responsive promoter. PcG mutants ablated YY1 repression, and YY1 could substitute for PHO in repressing transcription in wing imaginal discs. YY1 functionally compensated for loss of PHO in pho mutant flies and partially corrected mutant phenotypes. Taken together, these results indicate that YY1 functions as a PcG protein. Finally, we found that YY1, as well as Polycomb, required the co-repressor protein CtBP for repression in vivo. These results provide a mechanism for recruitment of vertebrate PcG complexes to DNA and demonstrate new functions for YY1.

Fig. 1. (A) Transgenic constructs. The top construct shows the YY1 cDNA (Park and Atchison, 1991) (black rectangle) under control of the armadillo promoter at the 5′ side, with the hsp70 poly(A) site on the 3′ side (Muller et al., 1995). The same expression plasmid was also used for making the arm-pho transgene (Brown et al., 1998) (second construct). The third and fourth constructs show the YY1 cDNA (black rectangle) fused to the GAL4 DNA binding domain (sequences 1–147, cross hatched rectangle) (Bushmeyer et al., 1995) under control of either the hunchback promoter or the hsp70 promoter on the 5′ side and the hsp70 poly(A) site on the 3′ side (Muller, 1995). (B) Strategy for crosses to determine the repression activity of GALYY1 in transgenic flies. The reporter construct (Muller, 1995) in transgenic fly strain BGUZ is shown at top. The expression pattern of this gene in embryos (i.e. throughout the embryo) is shown at the right, with the ‘A’ denoting anterior, and the ‘P’ posterior ends of the embryo. The GALYY1 effector plasmid expression pattern in the anterior half of embryos is shown at the right by black shading. Anticipated pattern of expression of embros from a cross between the reporter and effector transgenic lines (should repression occur), is shown at the bottom. LacZ expression will only be observed in the posterior ends of the embryos. (C) YY1 represses transcription in Drosophila embryos. LacZ expression in embryos (blue color) is shown in the parental reporter line (BGUZ; left panel) and embryos derived from crosses with two independent hbGALYY1 transgenic lines (middle and right panels). In each cross, LacZ expression is observed to be repressed in the anterior half of the embryo in either 10 h (middle panel) or 6 h (right panel) embryos. (D) The GAL DNA binding domain alone does not repress the BGUZ reporter. LacZ expression is shown in embryos of a cross between transgene lines hbGAL and BGUZ.

Fig. 2. Transcriptional repression by YY1 is stable. The BGUZ reporter line was crossed with the hsp70GALYY1 line. Embryos were either untreated, or heat shocked at 37°C for 45 min at various times, and embryos were processed for staining 16 h after laying.

Fig. 3. Early embryonic YY1 expression cannot repress the GAL4-NP6 gene. (A) Strategy for potential repression using the GAL4-NP6 reporter line. A synthetic NP6 enhancer and minimal heat shock promoter yields the expression pattern shown at the right. Anticipated potential repression with hbGALYY1 is shown at the bottom. (B) Transient YY1 expression from the hb promoter does not lead to repression of the GAL4-NP6 reporter. Similar staining patterns were observed either in the absence (left) or presence (right) of hbGALYY1.

Fig. 4. YY1 expressed late in embryonic development can repress GAL4-NP6 expression. Embryos from hsp70GALYY1 X GAL4-NP6 crosses were heat shocked at various times, harvested at 18 h after laying, then processed for LacZ staining. Only YY1 induced at 15 h was able to strongly repress GAL4-NP6 activity.

Fig. 5. YY1 transcriptional repression requires PcG function and can occur at both embryonic and larval stages. Embryos were collected from the BGUZ parent (A), the BGUZ hbGALYY1 recombinant chromosome line (B and C), and the recombinant chromosome line in various PcG homozygous mutant backgrounds (D–J). Embryos were collected at 16 h [or 6 h; (C)] and processed for LacZ staining. Blue staining indicates LacZ expression and light colored areas indicate repression of LacZ expression by GALYY1. The source of each embryo is shown at the right. (K) GALYY1 can compensate for PHO in wing imaginal discs. A diagram of the recombinant chromosome containing the hsp70GALYY1 and PBX-PRE-IDE-LacZ transgenes is shown at the top. This recombinant chromosome was crossed into either a wild type or a pho^–/– (pho¹/pho¹) mutant background and developing larvae were either untreated or heat shocked twice daily. In the pho^–/– crosses 18% of larvae are expected to contain the recombinant chromosome and the pho¹/pho¹ alleles to yield derepression of the reporter gene and resultant LacZ expression. As expected, 13 out of 75 larvae (17%) yielded wing imaginal discs that expressed LacZ (a representative positive wing disc is shown in the middle panel). The half of the larvae from the same cross that were heat shocked to induce GALYY1 expression showed dramatically different results. Only a single larvae out of 83 (1%) yielded wing discs staining positive for LacZ (a representative of the 82 negative imaginal discs is shown). The single positive larvae likely represents an organism in which the two transgenes became unlinked during the second cross due to absence of the balancer chromosome.

Fig. 6. GALYY1 and PHO can partially correct phenotypic defects in pho¹/pho^cv mutant flies. (A) PHO and YY1 partially correct segmentation defects. Wild-type, pho¹/pho^cv mutant, arm-pho-bearing and hsp70GALYY1-bearing pho¹/pho^cv flies are shown. Wild-type male flies are darkly pigmented on the tergites of the last two posterior segments (segments 5 and 6, left panel). Mutant flies show posterior transformation of the segmentation pattern that results in pigmentation on tergite 4 (and sometimes tergites 3 and 2; see arrows pointing to extra pigment). In addition, males lack segment A7 (the posterior-most segment) and transformation of A6 towards A7 can be detected as a smaller A6 which causes the male genitalia to protrude more than in wild-type flies (see mutant panel, smaller A6). In pho¹/pho^cv flies bearing the arm-pho transgene, the posterior-most segment is wild type, although pigmentation is still abnormal on tergite number 4 (right panel). In pho¹/pho^cv flies bearing the hsp70GALYY1 transgene, segmentation pattern is almost completely normal, although pigmentation is not complete. (B) The arm-pho and hsp70GALYY1 transgenes can rescue antenna development. Head mount photographs of wild-type, pho¹/pho^cv mutant, arm-pho-corrected and hsp70GALYY1-corrected flies are shown. The arrows point to arista structures. The arista (an appendage of the antenna) is normally bushy and branch like. In pho¹/pho^cv mutants the aristae are either absent, or are poorly developed and clumped (middle panel). In 85–90% of pho¹/pho^cv mutant flies bearing the arm-pho transgene and 95% of flies bearing the hsp70GALYY1 transgene, the aristae were of normal appearance, indicating substantial correction of the mutant phenotype (right panel). (C) The arm-pho and hsp70GALYY1 transgenes can completely correct the sex comb and claw defects found in pho¹/pho^cv mutant flies. Leg mounts are shown of wild-type, pho¹/pho^cv mutant, arm-pho-corrected pho¹/pho^cv mutant and hsp70GALYY1-corrected pho¹/pho^cv mutant flies. Arrows point to sex comb and claw structures. (D) GALYY1 protein is expressed after heat shock of hspGALYY1 transgenic embryos. Embryos from hsp70GALYY1 flies were either untreated or heat shocked for 45 min at 37°C. Western blots of lysates were assayed with either GAL4 or YY1 specific antibodies. The arrow points to the induced GALYY1 band.

Fig. 7. (A) Heterozygous CtBP mutants completely abolished YY1 repression. The GBUZ hbGALYY1 recombinant line was crossed into the either ctbp or groucho mutant backgrounds. Heterozgous CtBP mutants completely abolished YY1 repression, whereas Groucho mutants showed little effect. (B) YY1 physically interacts with CtBP. GST pull-down experiments were performed with recombinant YY1 prepared by in vitro translation, and GST–CtBP prepared in bacteria. The arrow indicates full-length YY1. (C) Heterozygous CtBP mutation abolishes Pc repression. The hbGAL-Pc BGUZ recombinant chromosome was crossed into a heterozygous ctbp mutant background, resulting in loss of Pc repression. (D) Potential mechanism linking YY1, CtBP and the PcG complex. In this model, YY1 binds a homodimer of CtBP that interacts with the PcG complex to recruit the complex to PRE sequences, thereby repressing transcription. Arrows show potential dual functions of CtBP in recruiting PcG proteins and repressing transcription.