Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos.

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here we describe 'enhancer-FACS-seq' (eFS) for highly parallel identification of active, tissue-specific enhancers in Drosophila melanogaster embryos. Analysis of enhancers identified by eFS as being active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, previously uncharacterized mesodermal transcription factors. Naive Bayes classifiers using transcription factor binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded in them.

Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors.

A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs - including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) - to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.

A machine learning approach for identifying novel cell type-specific transcriptional regulators of myogenesis.

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA-based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type-specific developmental gene expression patterns.

Mdm2 associates with Ras effector NORE1 to induce the degradation of oncoprotein HIPK1.

The Ras effector NORE1 is frequently silenced in primary adenocarcinomas, although the significance of this silencing for tumorigenesis is unclear. Here we show that NORE1 induces polyubiquitination and proteasomal degradation of oncoprotein HIPK1 by facilitating its interaction with the Mdm2 E3 ubiquitin ligase. Endogenous HIPK1 is stabilized in Nore1-deficient mouse embryonic fibroblasts, and depletion of HIPK1 in NORE1-silenced lung adenocarcinoma cells inhibits anchorage-independent cell growth and tumour formation in nude mice. These findings indicate that the control of HIPK1 stability by Mdm2-NORE1 has a major effect on cell behaviour, and epigenetic inactivation of NORE1 enables adenocarcinoma formation in vivo through HIPK1 stabilization.

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.

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation.

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.

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.

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 >5800 genes that cover approximately 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.

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-beta (TGF-beta)/bone morphogenetic protein (BMP) signaling pathways, and inhibits TGF-beta/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-beta/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.

Wnt-1 signal induces phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK.

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.