Search

The eukaryotic polymerase processivity factor, proliferating cell nuclear antigen (PCNA), interacts with many cell cycle-regulator proteins and with proteins involved in the mechanisms of DNA replication and repair. In the present study using two-hybrid analysis with PCNA from rice, Oryza sativa L. cv. Nipponbare (OsPCNA), we found that OsPCNA interacted in vitro and in vivo with rice JUN-activation-domain-binding protein 1 (OsJab1), which is known as COP9/signalsome subunit 5. Both OsPCNA and OsJab1 transcripts were expressed strongly in the shoot apical meristem and weakly in young leaves, flag leaves, ears, roots and root tips. No expression was detected in the mature leaves. The OsPCNA and OsJab1 proteins were expressed and accumulated mostly in the shoot apical meristem and ears, suggesting that OsJab1 is involved in cell proliferation in cooperation with OsPCNA. The role of OsPCNA with OsJab1 in plant DNA proliferation is discussed.

Abstract: The N terminal domain of human apolipoprotein E3 (apoE3-NT) functions as a ligand for members of the low-density lipoprotein receptor (LDLR) family. Whereas lipid-free apoE3-NT adopts a stable four-helix bundle conformation, a lipid binding induced conformational change is required for LDLR recognition. To investigate the role of a leucine zipper motif identified in the helix bundle on lipid binding activity, three leucine residues in helix 2 (Leu63, Leu71 and Leu78) were replaced by alanine. Recombinant “leucine to alanine” (LA) apoE3-NT was produced in E. coli, isolated and characterized. Stability studies revealed a transition midpoint of guanidine hydrochloride induced denaturation of 2.7 M and 2.1 M for wild type (WT) and LA apoE3-NT, respectively. Results from fluorescent dye binding assays revealed that, compared to WT apoE3-NT, LA apoE3-NT has an increased content of solvent exposed hydrophobic surfaces. In phospholipid vesicle solubilization assays, LA apoE3-NT was more effective than WT apoE3-NT at inducing a time-dependent decrease in dimyristoylphosphatidylglycerol vesicle light scattering intensity. Likewise, in lipoprotein binding assays, LA apoE3-NT protected human low-density lipoprotein from phospholipase C induced aggregation to a greater extent than WT apoE3-NT. On the other hand, LA apoE3-NT and WT apoE3-NT were equivalent in terms of their ability to bind a soluble LDLR fragment. The results suggest that the leucine zipper motif confers stability to the apoE3-NT helix bundle state and may serve to modulate lipid binding activity of this domain and, thereby, influence the conformational transition associated with manifestation of LDLR binding activity. [Copyright &y& Elsevier]

Abstract: The homeodomain protein Hex can function either as a transcriptional repressor or activator in animals. Recent reports have indicated that Hex is involved in liver development. However, its target genes and interacting proteins are largely unknown. We found that Hex functionally interacted with hepatocyte nuclear factor (HNF) 1α to further stimulate its activity using reporter gene containing multiple copies of HNF1α-binding site of the L-type pyruvate kinase (L-PK) gene promoter or natural L-PK promoter. This stimulation required the homeodomain and the acidic carboxyl-terminal region of Hex. Over-expression of Hex in primary cultured hepatocytes resulted in stimulation of the L-PK gene expression. Glutathione S-transferase pull-down assay and co-immunoprecipitation revealed that Hex physically interacted with HNF1α in mammalian cells through the homeodomain of Hex and POU-homeodomain of HNF1α. Since HNF1α is an important liver-enriched transcription factor involved in liver differentiation, Hex may contribute to liver differentiation through interaction with HNF1α. [Copyright &y& Elsevier]

Hex is one of the homeobox genes suggested to be important for hematopoietic cell differentiation. However, its biological function and mechanism of transcriptional regulation in hematopoietic cells remain elusive. We have identified the regulatory region necessary for transcription of the mouse Hex gene in K562 leukemia cells through transient reporter assays involving various deletion mutants. This region, comprising +775 to +1177 in the first intron, had enhancer-like properties and showed high activity in other hematopoietic cell lines such as U937, HEL, and RAW264.7, but little activity in other Hex-expressing cell lines such as MH1C1 and H4IIE hepatoma cells, suggesting that this region functions as a hematopoietic cell-specific enhancer-like element. Binding site mutation of hematopoietic transcription factors, such as GATAs and c-Myb present in the enhancer-like element, significantly decreased the luciferase reporter gene expression in K562 cells. Electrophoretic mobility shift assays showed that GATA-1, GATA-2, or c-Myb actually binds to three of these putative binding sites, and also suggested that several unidentified factors might interact with the enhancer-like element. Overexpression of GATA-1, GATA-2, or c-Myb stimulated the enhancer-like activity via these three binding sites. Thus, we conclude that Hex expression in hematopoietic cells is mainly regulated by GATA-1, GATA-2, and c-Myb via this intronic enhancer-like element. [ABSTRACT FROM PUBLISHER]

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb - Cre / Hhex loxP / loxP ) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases. • Hhex is involved in bile duct development and cyst formation through growth control. • Liver progenitor cells may participate in cyst formation. • Cyst cells have unbalanced expression of liver-enriched transcription factors. • Hhex knockout mice may be a good animal model for hepatic cystic disease. [ABSTRACT FROM AUTHOR]

Abstract: Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between −4687 and −4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus. [Copyright &y& Elsevier]

Abstract: Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between −163 and −32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (−66 to −50 and −93 to −78), a sterol regulatory element (−101 to −110), and two nuclear factor-Y (NF-Y) binding sites (−23 to −19 and −131 to −127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription. [Copyright &y& Elsevier]