👤 Yoku Hayakawa

Liver lobules are typically subdivided into 3 metabolic zones: zones 1, 2, and 3. However, the contribution of zonal differences in hepatocytes to liver regeneration, as well as to carcinogenic susceptibility, remains unclear. We developed a new method for sustained genetic labelling of zone 3 hepatocytes and performed fate tracing to monitor these cells in multiple mouse liver tumour models. We first examined changes in the zonal distribution of the Wnt target gene Hepatocytes receiving Wnt/β-catenin signalling from their microenvironment have high neoplastic potential, and Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma. Lineage tracing revealed that zone 3 hepatocytes residing in the pericentral niche have high neoplastic potential. Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling broadly exist across all hepatic zones and significantly contribute to liver tumorigenesis as well as liver regeneration. Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma. Show less

Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in the biosynthesis of monounsaturated fatty acids, and the expression of the Scd1 gene is induced by the intake of the lipogenic sugar fructose. We examined the effects of a high-fructose diet on hepatic acetylation of histones H3 and H4 and the binding of carbohydrate response element-binding protein (ChREBP) on the Scd1 gene promoter in rats. Rats were fed a control diet or a high-fructose diet for 10 days. The intake of a high-fructose diet significantly increased histone H3 and H4 acetylation and ChREBP binding to the Scd1 gene promoter as well as the amount of triglyceride and the expression of the Scd1 gene. These results suggest that short-term intake of high fructose upregulates expression of Scd1 by enhancing acetylation of histones H3 and H4 and binding of ChREBP at the Scd1 promoter. Show less

ELOVL fatty acid elongase 6 (ELOVL6) is a long-chain fatty acid elongase, and the hepatic expression of the Elovl6 gene and accumulation of triglycerides (TG) are enhanced by long-term high-fructose intake. Fatty acid synthesis genes, including Elovl6, are regulated by lipogenic transcription factors, sterol regulatory element-binding protein 1c (SREBP-1c) and carbohydrate-responsive element-binding protein (ChREBP). In addition, carbohydrate signals induce the expression of fatty acid synthase not only via these transcription factors but also via histone acetylation. Since a major lipotrope, myo-inositol (MI), can repress short-term high-fructose-induced fatty liver and the expression of fatty acid synthesis genes, we hypothesized that MI might influence SREBP-1c, ChREBP, and histone acetylation of Elovl6 in fatty liver induced by even short-term high-fructose intake. This study aimed to investigate whether dietary supplementation with MI affects Elovl6 expression, SREBP-1 and ChREBP binding, and acetylation of histones H3 and H4 at the Elovl6 promoter in short-term high-fructose diet-induced fatty liver in rats. Rats were fed a control diet, high-fructose diet, or high-fructose diet supplemented with 0.5% MI for 10 days. This study showed that MI supplementation reduced short-term high-fructose diet-induced hepatic expression of the Elovl6 gene, ChREBP binding, but not SREBP-1 binding, and acetylation of histones H3 and H4 at the Elovl6 promoter. Show less

Dietary supplementation with the major lipotrope myo-inositol (MI) potently reduces triglyceride (TG) content and expression levels of the fatty acid synthesis genes, for example, fatty acid synthase (FASN), in rat nonalcoholic fatty liver induced by high-fructose diet. Fatty acid synthesis genes are regulated by the carbohydrate-responsive element-binding protein (ChREBP) that exists in 2 isoforms: ChREBP-α and ChREBP-β. The gene encoding the latter isoform is more responsive to fructose. Because MI repressed the induction of fatty acid synthesis gene expression by high-fructose diet, we hypothesized that MI may reduce binding of ChREBP to the carbohydrate response elements (ChoREs) in the ChREBP-β gene as well as in fatty acid synthesis genes in the liver. Rats were fed high-glucose, high-fructose, or high-fructose diets supplemented with MI (0.05% and 0.25%) for 2 weeks. Hepatic TG content and expression levels of the glucose-6-phosphate dehydrogenase, malic enzyme 1, FASN, acetyl-CoA carboxylase alpha, S14, and ChREBP-β were remarkably elevated in rats fed with high fructose compared with the corresponding levels in high-glucose group. Notably, elevated values of these parameters in high-fructose group were reduced by MI. Similarly, high-fructose-induced ChREBP binding to the ChoREs of the ChREBP-β and FASN genes was nominally decreased by MI. This study showed that treatment with MI reduced elevated TG content and expression of genes related to fatty acid synthesis, such as FASN and ChREBP-β, in rat nonalcoholic fatty liver induced by high-fructose diet. Furthermore, MI treatment nominally decreased increased binding of ChREBP to the ChoREs of ChREBP-β and FASN genes. Show less

Three subtypes of HP1, a conserved non-histone chromosomal protein enriched in heterochromatin, have been identified in humans, HP1alpha, beta and gamma. In the present study, we utilized a Drosophila system to characterize human HP1 functions. Over-expression of HP1beta in eye imaginal discs caused abnormally patterned eyes, with reduced numbers of ommatidia, and over-expression of HP1gamma in wing imaginal discs caused abnormal wings, in which L4 veins were gapped. These phenotypes were specific to the HP1 subtypes and appear to reflect suppressed gene expression. To determine the molecular domains of HP1 required for each specific phenotype, we constructed a series of chimeric molecules with HP1beta and HP1gamma. Our data show that the C-terminal chromo shadow domain (CSD) of HP1gamma is necessary for HP1gamma-type phenotype, whereas for the HP1beta-type phenotype both the chromo domain and the CSD are required. These results suggest human HP1 subtypes use different domains to suppress gene expression in Drosophila cells. Show less

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes. Show less

Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation. Three subtypes of HP1, namely HP1alpha, beta, and gamma, have been identified in humans. In this study, using yellow fluorescent protein (YFP) fusion constructs, we examined the intracellular localization of human HP1 subtypes during the cell cycle. During interphase, all three HP1 subtypes were localized to centromeric heterochromatin and to promyelocytic leukemia (PML) nuclear bodies. Different preferences, however, were observed among the subtypes: during interphase HP1beta localized most preferentially to centromeric heterochromatin, whereas HP1alpha and gamma were more preferentially localized to PML nuclear bodies. During metaphase, only HP1alpha, was localized to the centromere. We thus determined which molecular domains of HP1 were necessary for their intracellular localization. Our results showed that the C-terminal fragment (amino acid residues 101-180) of HP1alpha was necessary for localization to the metaphase centromere and the N-terminal fragment (amino acid residues 1-76) of HP1beta was necessary for localization to the interphase centromere. Interestingly, simultaneous observations of residues 101-180 of HP1alpha and residues 1-76 of HP1beta in living HeLa cells revealed that during late prophase, the HP1beta fragment dissociated from centromeric regions and the HP1alpha fragment accumulated in centromeric regions. These results indicate that different specific regions of human HP1alpha and HP1beta mediate localization to metaphase and interphase centromeric regions resulting in association of different subtypes of HP1 with the centromere at different times during the cell cycle. Show less