Polycomb recruitment to DNA in vivo by the YY1 REPO domain - PubMed (original) (raw)

Comparative Study

. 2006 Dec 19;103(51):19296-301.

doi: 10.1073/pnas.0603564103. Epub 2006 Dec 8.

Affiliations

Comparative Study

Polycomb recruitment to DNA in vivo by the YY1 REPO domain

Frank H Wilkinson et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Polycomb group (PcG) proteins are responsible for maintaining transcriptional repression of developmentally important genes. However, the mechanism of PcG recruitment to specific DNA sequences is poorly understood. Transcription factor YY1 is one of the few PcG proteins with sequence-specific DNA binding activity. We previously showed that YY1 can recruit other PcG proteins to DNA, leading to histone posttranslational modifications and stable transcriptional repression. Using Drosophila transgenic approaches, we identified YY1 sequences 201-226 as necessary and sufficient for PcG transcriptional repression in vivo. When fused to a heterologous DNA-binding domain, this short 26-aa motif was sufficient for transcriptional repression, recruitment of PcG proteins to DNA, and methylation of histone H3 lysine 27. Deletion of this short YY1 motif did not affect transient transcriptional repression but ablated PcG repression, PcG protein recruitment to DNA, and methylation of H3 lysine 27. We propose that this motif be named the REPO domain for its function in recruitment of Polycomb. The REPO domain is well conserved in YY1 orthologs and in related proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Strategy for detecting PcG transcriptional repression by YY1 mutant transgenes. (Top) The BGUZ reporter with the BXD enhancer, GAL4-binding sites, Ubx promoter, and LacZ gene indicated. This reporter gives ubiquitous staining in embryos shown on the right of the figure. (Middle) The general structure of the mutant YY1 effector transgenes driven by the hunchback promoter. The GAL4 DNA-binding domain (residues 1–147) is indicated in red, and the location of the fused YY1 DNA segments is shown in black. These effector transgenes are expressed only in the anterior half of Drosophila embryos (black shading in the embryo at the right). (Bottom) Potential LacZ repression in the anterior end of embryos from crosses between the BGUZ reporter and the GALYY1 transgenes.

Fig. 2.

Fig. 2.

YY1 sequences 201–226 are necessary and sufficient for YY1 PcG repression. YY1 sequences in each effector transgene are diagrammed on the right. Embryos stained for LacZ expression from the BGUZ reporter are shown on the left with dorsal and lateral views of stained embryos. The name of each YY1 effector transgene is indicated on the left.

Fig. 3.

Fig. 3.

YY1 sequences 201–226 are incapable of transient transcriptional repression. (A) YY1 sequences 201–226 are not needed for transient repression in fly embryos. Effector transgenes (indicated on the left) were crossed with the GAL-NP6-LacZ reporter, heat-shocked at 15 h after laying, and then stained for LacZ expression at 18 h. Repression was observed with full-length GALYY1 and GALYY1Δ201–226, but not with GALYY1 201–226. (B and C) YY1 sequences 201–226 are not needed for transient repression in cell culture. Quantities (in nanograms) are indicated for CMV expression (Exp.) plasmids cotransfected with the (YY1)4TKCAT reporter in NIH 3T3 cells. Fold activation is plotted (C) with error bars representing the standard deviation from the mean. (D and E) The REPO domain does not support transient activation or repression. NIH 3T3 cells were cotransfected with GALTKCAT and various doses of either CMVGALYY1 or CMVGALYY1 201–226. CAT data in D, are presented in quantitative form in E.

Fig. 4.

Fig. 4.

YY1 sequences 201–226 are necessary and sufficient for recruitment of PcG proteins to DNA in vivo. (A Top) Schematic of the BGUZ reporter locus indicating the structural elements and the location of the PCR primers for ChIP experiments. (Middle and Bottom) Representative ChIP Southern blot from embryos expressing either the GALYY1Δ201–226, or the GAL 201–226 transgenes. Antibodies are shown above the lanes. Triangles indicate the increase in template concentration used for PCR (2 and 20 ng). (B) Quantitative ChIP data from three independent experiments with full-length GALYY1, GALΔ201–226, and GALYY1 201–226 transgenes. Values were normalized to the level of the GAL4 signal to normalize for equal amounts of GAL fusion protein bound to DNA in vivo. Error bars show the standard deviation from the mean. (C) YY1 recruits HDAC activity, but not H3K27 methyltransferase activity to the GAL-NP6-LacZ reporter. ChIP assays were performed with antibodies shown above the lanes. After PCR of either 2 or 20 ng DNA with primers specific for the GAL-NP6-LacZ reporter, samples were electrophoresed on agarose gels.

Fig. 5.

Fig. 5.

Homology of YY1 REPO domain to similar domains in other proteins. The REPO domain is highly conserved. (Upper) The human YY1 REPO domain (residues 205–226) is shown at the top with homologous sequences from YY1 and YY2 proteins from other species as well as from Drosophila PHO and PHOL shown below. Dashes indicate amino acid identity, and asterisks represent deletions. (Lower) Human YY2 REPO domain is compared with the corresponding YY2 REPO domains from chimpanzee, mouse, rat, and dog. Accession numbers are as follows: human YY1 (AAA59926), chimpanzee YY1 (XP_510162), sheep YY1 (AAT74924), rat YY1 (AAR14688), mouse YY1 (AAH55899), dog YY1 (XP_854514), chicken YY1 (NP_510162), frog YY1 (CAA54777), zebra fish YY1 (AAH71351), puffer fish YY1 (CAG01508), sea urchin YY1 (XP_790188), honey bee YY1 (XP-397280), fly PHO (AAL48765), fly PHOL (NP_648317), human YY2 (AAS68634), chimpanzee YY2 (XP_529235), dog YY2 (XP_868531), and rat YY2 (AAZ38710). The mouse YY2 sequence was taken from Luo et al. (36).

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