Kenji Kokura

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.

Ski co‐repressor complexes maintain the basal repressed state of the TGF‐β target gene, SMAD7, via HDAC3 and PRMT5

The products encoded by ski and its related gene, sno, (Ski and Sno) act as transcriptional co‐repressors and interact with other co‐repressors such as N‐CoR/SMRT and mSin3A. Ski and Sno mediate transcriptional repression by various repressors, including Mad, Rb and Gli3. Ski/Sno also suppress transcription induced by multiple activators, such as Smads and c‐Myb. In particular, the inhibition of TGF‐β‐induced transcription by binding to Smads is correlated with the oncogenic activity of Ski and Sno. However, the molecular mechanism by which Ski and Sno mediate transcriptional repression remains unknown. In this study, we report the purification and characterization of Ski complexes. The Ski complexes purified from HeLa cells contained histone deacetylase 3 (HDAC3) and protein arginine methyltransferase 5 (PRMT5), in addition to multiple Smad proteins (Smad2, Smad3 and Smad4). Chromatin immunoprecipitation assays indicated that these components of the Ski complexes were localized on the SMAD7 gene promoter, which is the TGF‐β target gene, in TGF‐β‐untreated HepG2 cells. Knockdown of these components using siRNA led to up‐regulation of SMAD7 mRNA. These results indicate that Ski complexes serve to maintain a TGF‐β‐responsive promoter at a repressed basal level via the activities of histone deacetylase and histone arginine methyltransferase.

Molecular cloning and characterization of neural activity-related RING finger protein (NARF): a new member of the RBCC family is a candidate for the partner of myosin V

Activity‐dependent synaptic plasticity has been thought to be a cellular basis of memory and learning. The late phase of long‐term potentiation (L‐LTP), distinct from the early phase, lasts for up to 6 h and requires de novo synthesis of mRNA and protein. Many LTP‐related genes are enhanced in the hippocampus during pentyrenetetrazol (PTZ)‐ and kainate (KA)‐mediated neural activation. In this study, mice were administered intraperitoneal injections of PTZ 10 times, once every 48 h, and showed an increase in seizure indexes. Genes related to plasticity were efficiently induced in the mouse hippocampus. We used a PCR‐based cDNA subtraction method to isolate genes that are expressed in the hippocampus of repeatedly PTZ‐treated mice. One of these genes, neural activity‐related RING finger protein (NARF), encodes a new protein containing a RING finger, B‐box zinc finger, coiled‐coil (RBCC domain) and β‐propeller (NHL) domain, and is predominantly expressed in the brain, especially in the hippocampus. In addition, KA up‐regulated the expression of NARF mRNA in the hippocampus. This increase correlated with the activity of the NMDA receptor. By analysis using GFP‐fused NARF, the protein was found to localize in the cytoplasm. Enhanced green fluorescent protein‐fused NARF was also localized in the neurites and growth cones in neuronal differentiated P19 cells. The C‐terminal β‐propeller domain of NARF interacts with myosin V, which is one of the most abundant myosin isoforms in neurons. The NARF protein increases in hippocampal and cerebellar neurons after PTZ‐induced seizure. These observations indicated that NARF expression is enhanced by seizure‐related neural activities, and NARF may contribute to the alteration of neural cellular mechanisms along with myosin V.

Heterogeneous nuclear RNA‐ribonucleoprotein F binds to DNA via an oligo(dG)‐motif and is associated with RNA polymerase II

The heterogeneous nuclear ribonucleoprotein F (hnRNP‐F) is one of the constituents of the splicing‐related hnRNP complex. Recent studies suggest that pre‐mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre‐mRNA‐engaged factors might be associated with a promoter.

Identity between rat htf and human xbp-1 genes: determination of gene structure, target sequence, and transcription promotion function for HTF.

Hepatocarcinogenesis-related transcription factor (HTF) was originally isolated from rats in which the expression was enhanced in hepatocellular carcinomas. Rat HTF (rHTF) is structurally similar to human X-box-binding protein-1 (hXBP-1), and both factors are unique in respective genomes. A previous study showed that hXBP-1 mRNA is detectable ubiquitously but is enriched in the human liver as rHTF. In this study, we demonstrated the analogous exon-intron organization and significant sequence homology for rhtf and hxbp-1 genes. Alignment of amino acid sequences of rHTF and hXBP-1 revealed that all the characteristic motifs in rHTF were conserved in hXBP-1. Moreover, Southern blotting patterns provided with the rHTF and hXBP-1 probes were basically the same. These two genes were thus thought to belong to the same evolutional lineage. We determined the consensus binding sequence (CRCGTCA) for rHTF by CASTing, and it was found to be nearly the same as that for hXBP-1. Transactivation ability of rHTF was also demonstrated. The rhtf gene generates two types of mRNAs (2.0 kb and 2.5 kb), both of which encode identical rHTF protein. These transcripts had distinct transcription initiation sites. The 2.0 kb promoter, that was revealed by the transient luciferase assay, contained GC-box and CAAT-box. Sequences around the transcription initiation site for the 2.0 kb transcript were similar in rhtf and hxbp-1 genes. Our observations suggest that HTF is a rat homolog of hXBP-1.

p53 Suppresses c-Myb-induced trans-Activation and Transformation by Recruiting the Corepressor mSin3A

p53 is known to repress transcription of a number of genes, but the mechanism of p53 recruitment to these target genes is unknown. The c-myb proto-oncogene product (c-Myb) positively regulates proliferation of immature hematopoietic cells, whereas p53 blocks cell cycle progression. Here, we demonstrate that p53 inhibits c-Myb-induced transcription and transformation by directly binding to c-Myb. The ability of c-Myb to maintain the undifferentiated state of M1 cells was also suppressed by p53. p53 did not affect the ability of c-Myb to bind to DNA but formed a ternary complex with the corepressor mSin3A and c-Myb. Thus, p53 antagonizes c-Myb by recruiting mSin3A to down-regulate specific Myb target genes.

Gene expression in hepatomas

Gene expression changes in accordance with cell growth, differentiation and carcinogenesis. To elucidate the molecular mechanisms for hepatocarcinogenesis as well as maintenance of normal hepatocytes, it is important to identify the genes that have altered expression with carcinogenesis. We established a new and efficient cDNA subtraction method via two cDNA populations. By using this method along with rat hepatomas made by the Soh‐Farber protocol, we identified a number of genes, some of which are activated in hepatocellular carcinoma (HCC). These genes include ones which code for a transcription factor and a metabolic enzyme. One particular gene can be used as a tumour marker. Our method is beneficial for the isolation of a wide range of HCC‐related genes in rats which, in turn, enables easy identification of their human counterparts. In this review, we describe details of our method and the isolated genes. We also briefly describe transcription factors in the liver.