A novel family of cofactors that differentially interact with homeoproteins have been identified via a yeast twohybrid screen. The proteins contain a conserved protein kinase domain that is separated from a domain that interacts with homeoproteins and hence are termed homeodomain-interacting protein kinases (HIPKs): HIPK1, HIPK2, and HIPK3. We show that HIPKs are nuclear kinases using GFP-HIPK fusion constructs. The DNA binding activity of the NK-3 homeoprotein is greatly enhanced by HIPK2, but this effect is independent of its phosphorylation by HIPK2. In cultured cells, HIPKs localize to nuclear speckles and potentiate the repressor activities of NK homeoproteins. The co-repressor activity of HIPKs depends on both its homeodomain interaction domain and a co-repressor domain that maps to the N terminus. Thus, HIPKs represent a heretofore undescribed family of co-repressors for homeodomain transcription factors.

[Keywords: Myb; HIPK2; NLK; TAK1; phosphorylation; degradation] Supplemental material is available at http://www.genesdev.org.

Transcriptional repression by sequence-specific DNA binding factors is mediated by the recruitment of a corepressor complex to the promoter region. The NK-3 homeodomain protein is a transcriptional repressor that recruits the nuclear protein kinase, homeodomain interacting protein kinase 2 (HIPK2). Here we show that HIPK2 is a component of a corepressor complex containing Groucho and a histone deacetylase complex. Groucho, like HIPK2, acts as a corepressor for NK-3 and binds to NK-3 and HIPK2. Moreover, HIPK2 appears to regulate the corepressor activity of Groucho. Transcriptional repression by NK-3 and Groucho is relieved by the histone deacetylase inhibitor trichostatin A, and both NK-3 and Groucho directly interact with the histone deacetylase HDAC1 that is associated with mSin3A in vivo. Recruitment of the histone deacetylase complex by NK-3 decreases the acetylated histones that are associated with the target gene promoter. These results indicate that NK-3 represses transcription by recruiting a complex containing Groucho and a histone deacetylase complex that leads to histone modification on chromatin and suggest that HIPK2 may play a regulatory role in the corepressor complex formation.

Four Drosophila melanogaster homeobox genes were found by screening a genomic DNA library with oligodeoxynucleotides that correspond to a conserved amino acid sequence that is part of the putative site of homeobox proteins that recognizes nucleotide sequences in DNA. The amino acid sequences of NK-2, NK-3, and NK-4 homeoboxes are more closely related to one another (59-66% homology) than they are to other Drosophila homeoboxes (28-54% homology), whereas the homeobox ofNK-1 is most closely related, in order of decreasing homology, to muscle segment homeobox, zerknufllt-l, NK-3, and distal-less homeoboxes. Three of the genes, NK-1, NK-3, and NK-4, comprise a cluster of homeobox genes located in the 93E1-5 region of the right arm of the third chromosome, whereas the fourth homeobox gene, NK-2, is located in the lCl-5 region of the X chromosome.Homeobox genes encode DNA-binding proteins that regulate gene expression during development or in the adult (1-4). In most cases, the similarity between different kinds of homeobox proteins extends only over a segment of the protein that consists of 60-61 amino acid residues, the homeodomain, which is thought to be the portion of the protein that recognizes nucleotide sequences in DNA. Homeobox genes are particularly well expressed in nervous system, and the homeobox family of genes encodes the largest set of proteins that regulate gene expression in the nervous system that has been identified thus far (5-9).In this report, we describe four newly discovered, related Drosophila homeobox genes that were detected with oligonucleotide probes corresponding to an amino acid sequence that is thought to be part of the nucleotide sequence recognition site of homeobox proteins. METHODS AND MATERIALSOligodeoxynucleotides. An Applied Biosystems DNA synthesizer 380B was used to synthesize oligodeoxynucleotides. Oligonucleotides with the trityl groups attached were purified by OPC column chromatography and trityl groups then were removed as described by Applied Biosystems. [Y-32P]ATP with a specific activity of6000 Ci/mmol (1 Ci = 37 GBq) (New England Nuclear) was used for phosphorylation of oligodeoxynucleotides catalyzed by T4 polynucleotide kinase (10).Detection and Cloning of Homeobox Genes. A Drosophila melanogaster genomic DNA library in Charon 4A (11) Locations of Genes on Chromosomes. Salivary gland polytene chromosomes were hybridized with EcoRI-cleaved genomic DNA fragments that contained the appropriate homeobox region and had incorporated biotin-16 dUMP in place of some dTMP residues as described (14). Detek 1-HRP kits (Enzo Biochemicals) and the protocol supplied by the manufacturer were used. RESULTS AND DISCUSSIONDetection of Homeobox Genes. The Drosophila genomic DNA library of Maniatis et al. (11) in Charon 4A was screened for recombinants corresponding to homeobox genes with five [32Ploligodeoxynucleotide probe preparations designed to hybridize to highly conserved homeobox nucleotide sequences. The oligonucleotide preparations were 16 or 17 nucleotides long and...