Yongsok Kim

Homeodomain-interacting Protein Kinases, a Novel Family of Co-repressors for Homeodomain Transcription Factors

A novel family of cofactors that differentially interact with homeoproteins have been identified via a yeast two-hybrid 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.

The homeodomain protein NK-3 recruits Groucho and a histone deacetylase complex to repress transcription.

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 mSin3Ain 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.

D-mef2 is a target for Tinman activation during Drosophila heart development

The NK‐type homeobox gene tinman and the MADS box gene D‐mef2 encode transcription factors required for the development and differentiation of the Drosophila heart, and closely related genes regulate cardiogenesis in vertebrates. Genetic analyses indicate that tinman and D‐mef2 act at early and late steps, respectively, in the cardiogenic lineage. However, it is unknown whether regulatory interactions exist between these developmental control genes. We show that D‐mef2 expression in the developing Drosophila heart requires a novel upstream enhancer containing two Tinman binding sites, both of which are essential for enhancer function in cardiac muscle cells. Transcriptional activity of this cardiac enhancer is dependent on tinman function, and ectopic Tinman expression activates the enhancer outside the cardiac lineage. These results define the only known in vivo target for transcriptional activation by Tinman and demonstrate that D‐mef2 lies directly downstream of tinman in the genetic cascade controlling heart formation in Drosophila.

A functional genomic screen for cardiogenic genes using RNA interference in developing Drosophila embryos

Identifying genetic components is an essential step toward understanding complex developmental processes. The primitive heart of the fruit fly, the dorsal vessel, which is a hemolymph-pumping organ, has provided a unique model system to identify cardiogenic genes and to further our understanding of the molecular mechanisms of cardiogenesis. Using RNA interference in developing Drosophila embryos, we performed a genomewide search for cardiogenic genes. Through analyses of the >5,800 genes that cover ≈40% of all predicted Drosophila genes, we identified a variety of genes encoding transcription factors and cell signaling proteins required for different steps during heart development. Analysis of mutant heart phenotypes and identified genes suggests that the Drosophila heart tube is segmentally patterned, like axial patterning, but assembled with regional modules. One of the identified genes, simjang, was further characterized. In the simjang mutant embryo, we found that within each segment a subset of cardial cells is missing. Interestingly, the simjang gene encodes a protein that is a component of the chromatin remodeling complex recruited by methyl-CpG-DNA binding proteins, suggesting that epigenetic information is crucial for specifying cardiac precursors. Together, these studies not only identify key regulators but also reveal mechanisms underlying heart development.

Genetically distinct cardial cells within the Drosophila heart

Summary: Although often viewed as a simple pulsating tube, the Drosophila dorsal vessel is intricate in terms of its structure, cell types, and patterns of gene expression. Two nonidentical groups of cardial cells are observed in segments of the heart based on the differential expression of transcriptional regulators. These include sets of four cell pairs that express the homeodomain protein Tinman (Tin), alternating with groups of two cell pairs that express the orphan steroid hormone receptor Seven Up (Svp). Here we show that these myocardial cell populations are distinct in terms of their formation and gene expression profiles. The Svp‐expressing cells are generated by asymmetric cell divisions of precursor cells based on decreases or increases in their numbers in numb or sanpodo mutant embryos. In contrast, the numbers of Tin‐expressing cardial cells are unchanged in these genetic backgrounds, suggesting they arise from symmetric cell divisions. One function for Svp in the two pairs of cardial cells is to repress the expression of the tin gene and at least one of its targets, the β3 tubulin gene. Further differences in the cells are substantiated by the identification of separable enhancers for D‐mef2 gene transcription in the distinct cardioblast sets. Taken together, these results demonstrate a greater cellular and genetic complexity of the Drosophila heart than previously appreciated. genesis 28:36–43, 2000.

Phosphorylation by the DHIPK2 Protein Kinase Modulates the Corepressor Activity of Groucho

Groucho function is essential for Drosophila development, acting as a corepressor for specific transcription factors that are downstream targets of various signaling pathways. Here we provide evidence that Groucho is phosphorylated by the DHIPK2 protein kinase. Phosphorylation modulates Groucho corepressor activity by attenuating its protein-protein interaction with a DNA-bound transcription factor. During eye development, DHIPK2 modifies Groucho activity, and eye phenotypes generated by overexpression of Groucho differ depending on its phosphorylation state. Moreover, analysis of nuclear extracts fractionated by column chromatography further shows that phospho-Groucho associates poorly with the corepressor complex, whereas the unphosphorylated form binds tightly. We propose that Groucho phosphorylation by DHIPK2 and its subsequent dissociation from the corepressor complex play a key role in relieving the transcriptional repression of target genes regulated by Groucho, thereby controlling cell fate determination during development.

Requirement of the co-repressor homeodomain-interacting protein kinase 2 for ski-mediated inhibition of bone morphogenetic protein-induced transcriptional activation.

Multiple co-repressors such as N-CoR/SMRT, mSin3, and the c-ski proto-oncogene product (c-Ski) mediate the transcriptional repression induced by Mad and the thyroid hormone receptor by recruiting the histone deacetylase complex. c-Ski also binds directly to Smad proteins, which are transcriptional activators in the transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signaling pathways, and inhibits TGF-β/BMP-induced transcriptional activation. However, it remains unknown whether other co-repressor(s) are also involved with Ski in the negative regulation of the TGF-β/BMP signaling pathways. Here, we report that the co-repressor homeodomain-interacting protein kinase 2 (HIPK2) directly binds to both c-Ski and Smad1. HIPK2 efficiently inhibited Smad1/4-induced transcription from the Smad site-containing promoter. A dominant negative form of HIPK2, in which the ATP binding motif in the kinase domain and the putative phosphorylation sites were mutated, enhanced Smad1/4-dependent transcription and the BMP-induced expression of alkaline phosphatase. Furthermore, the c-Ski-induced inhibition of the Smad1/4-dependent transcription was suppressed by a dominant negative form of HIPK2. The HIPK2 co-repressor activity may be regulated by an uncharacterized HIPK2 kinase. These results indicate that HIPK2, together with c-Ski, plays an important role in the negative regulation of BMP-induced transcriptional activation.

Combinatorial control of Drosophila mef2 gene expression in cardiac and somatic muscle cell lineages

Abstract The Drosophila mef2 gene encodes a MADS domain transcription factor required for the differentiation of cardiac, somatic, and visceral muscles during embryogenesis and the patterning of adult indirect flight muscles assembled during metamorphosis. A prerequisite for D-MEF2 function in myogenesis is its precise expression in multiple cell types during development. Novel enhancers for D-mef2 transcription in cardiac and adult muscle precursor cells have been identified and their regulation by the Tinman and Twist myogenic factors have been demonstrated. However, these results suggested the existence of additional regulators and provided limited information on the specification of progenitor cells for different muscle lineages. We have further characterized the heart enhancer and show it is part of a complex regulatory region controlling the activation and repression of D-mef2 transcription in several cell types. The mutation of a GATA sequence in the enhancer changes its specificity from cardial to pericardial cells. Also, the addition of flanking sequences to the heart enhancer results in expression in a new cell type, that being the founder cells of a subset of body wall muscles. As tinman function is required for D-mef2 expression in both the cardial and founder cells, these results define a shared regulatory DNA that functions in distinct lineages due to the combinatorial activity of Tinman and other factors that work through adjacent sequences. The analysis of D-mef2-lacZ fusion genes in mutant embryos revealed that the specification of the muscle precursor cells involved the wingless gene and the activation of a receptor tyrosine kinase signaling pathway.

Desumoylation of homeodomain-interacting protein kinase 2 (HIPK2) through the cytoplasmic-nuclear shuttling of the SUMO-specific protease SENP1

The modification of homeodomain‐interacting protein kinase 2 (HIPK2) by small ubiquitin‐like modifier 1 (SUMO‐1) plays an important role in its targeting into the promyelocytic leukemia body, as well as in its differential interaction with binding partner, but the desumoylation of HIPK2 by SUMO‐specific proteases is largely unknown. In this study, we show that HIPK2 is a desumoylation target for the SUMO‐specific protease SENP1 that shuttles between the cytoplasm and the nucleus. Mutation analyses reveal that SENP1 contains the nuclear export sequence (NES) within the extreme carboxyl‐terminal region, and SENP1 is exported to the cytoplasm in a NES‐dependent manner. Sumoylated HIPK2 are deconjugated by SENP1 both in vitro and in cultured cells, and the desumoylation is enhanced either by the forced translocation of SENP1 into the nucleus or by the SENP1 NES mutant. Concomitantly, desumoylation induces dissociation of HIPK2 from nuclear bodies. These results demonstrate that HIPK2 is a target for SENP1 desumoylation, and suggest that the desumoylation of HIPK2 may be regulated by the cytoplasmic‐nuclear shuttling of SENP1.

Differential interactions of the homeodomain-interacting protein kinase 2 (HIPK2) by phosphorylation-dependent sumoylation

Homeodomain‐interacting protein kinase 2 (HIPK2) interacts with and phosphorylates various transcription factors that are critical regulators of cell fate decisions and apoptosis during development. Here we show that lysine 25 of HIPK2 is the major sumoylation site, both in vitro and in vivo, and that the sumoylation of this site occurs in a phosphorylation‐dependent manner. This became clear with the finding that kinase‐dead HIPK2 (K221R) could not be efficiently sumoylated in vitro. The sumoylation of HIPK2 resulted in the disruption of its interaction with a Groucho corepressor. Consequently, sumoylation inhibited the regulatory activity of HIPK2 on the Groucho‐mediated repression of transcription, whereas not on p53‐mediated transactivation. These results suggest that phosphorylation‐dependent sumoylation enables HIPK2 to drive different target gene transcription by means of differential interactions with its binding partners.