Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.

The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/ß-catenin signaling. We aimed at investigating EZH2's role in epigenetically regulating Wnt/ß-catenin signaling following bacterial infection. NIH:Swiss outbred and Apc(Min/+) mice were infected with CR (10(8) CFU); BLT1(-/-)Apc(Min/+) mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and ß-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3's occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although ß-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in ß-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and ß-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected Apc(Min/+) mice paralleled changes recorded in BLT1(-/-)Apc(Min/+), AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/ß-catenin-dependent crypt hyperplasia in response to CR infection.

Kank proteins: structure, functions and diseases.

The Kank family of proteins, Kank1-Kank4, are characterized by their unique structure, coiled-coil motifs in the N-terminal region, and ankyrin-repeats in the C-terminal region, with an additional motif, the KN motif, at the N-terminus. Kank1 was obtained by positional cloning of a tumor suppressor gene in renal cell carcinoma, while the other members were found by homology search. The family is involved in the regulation of actin polymerization and cell motility through signaling pathways containing PI3K/Akt and/or unidentified modulators/effectors. Their relationship to diseases such as cancer, and to neuronal and developmental disorders, will be an important subject of future study.

Studies on the synthesis of trans-dihydrodiols of polycyclic aromatic thiaarenes as potential proximate carcinogenic metabolites: first synthesis of trans-10,11-dihydroxy-10,11-dihydroacenaphtho[1, 2-b]benzo[d]thiophene and 6,7-dihydroxy-6,7-dihydronaphtho[1, 2-b]thiophene.

Polyaromatic thiophene compounds are found to occur concomitantly with numerous coal-derived products and shale oils and are suspected mutagens and/or carcinogens. The first synthesis of the two title compounds 9 and 16 has been achieved in five or four steps starting from 8,9-dihydroacenaphtho[1,2-b]benzo[d]thiophene (1) and 7-methoxynaphtho[1,2-b]thiophene (12), respectively. Compound 1 was converted to the cis-diol (11) (via treatment with OsO(4)/pyridine) or to trans-diol (3) [via Prevost reaction (PhCOOAg/I(2)) followed by hydrolysis] in 95-98% yield, respectively. Subsequent dehydration (PTS/benzene) of the diol followed by aromatization of the resulting ketone (5) produced the phenolic compound 6 in 97% yield. Oxidation of the phenol with phenyl iododiacetate followed by hydrolysis of the o-quinone monoketal 7 gave the o-quinone (8) in 86% yield. Stereoselective reduction of 8 with NaBH(4)/EtOH under oxygen afforded trans-10,11-dihydroxy-10,11-dihydroacenaphtho[1,2-b]benzo[d]thi oph ene(9) (orange yellow solid) in 55% yield. Compound 16 was obtained as a colorless solid, through the stereoselective reduction of the o-quinone 15 (with NaBH(4)), which in turn was prepared from 12 following the protocol of functional group transformation of methoxy --> phenol --> o-quinone monoketal --> o-quinone, as used in the previous case. The yields for all the steps are very good. The mutagenicity assay of compound 9 and 16 as well as their parent thiaarenes have been performed. The results showed that 9 may not be the proximate carcinogen of acenaphtho[1,2-b]benzo[d]thiophene, while it is likely that compound 16 is one of the possible proximate carcinogens for naphtho[1,2-b]thiophene.

Physiological expression of the gene for PrP-like protein, PrPLP/Dpl, by brain endothelial cells and its ectopic expression in neurons of PrP-deficient mice ataxic due to Purkinje cell degeneration.

Recently, a novel gene encoding a prion protein (PrP)-like glycoprotein, PrPLP/Dpl, was identified as being expressed ectopically by neurons of the ataxic PrP-deficient (PRNP(-/-)) mouse lines exhibiting Purkinje cell degeneration. In adult wild-type mice, PrPLP/Dpl mRNA was physiologically expressed at a high level by testis and heart, but was barely detectable in brain. However, transient expression of PrPLP/Dpl mRNA was detectable by Northern blotting in the brain of neonatal wild-type mice, showing maximal expression around 1 week after birth. In situ hybridization paired with immunohistochemistry using anti-factor VIII serum identified brain endothelial cells as expressing the transcripts. Moreover, in the neonatal wild-type mice, the PrPLP/Dpl mRNA colocalized with factor VIII immunoreactivities in spleen and was detectable on capillaries in lamina propria mucosa of gut. These findings suggested a role of PrPLP/Dpl in angiogenesis, in particular blood-brain barrier maturation in the central nervous system. Even in the ataxic Ngsk PRNP(-/-) mice, the physiological regulation of PrPLP/Dpl mRNA expression in brain endothelial cells was still preserved. This strongly supports the argument that the ectopic expression of PrPLP/Dpl in neurons, but not deregulation of its physiological expression in endothelial cells, is involved in the neuronal degeneration in ataxic PRNP(-/-) mice.

Recognition of flexible peptides in water by transition metal complexes.

This paper describes the design, synthesis, and evaluation of transition metal complexes capable of recognizing flexible histidine-containing peptides in aqueous medium (25 mM HEPES buffer, pH = 7.0, 25 degrees C). When the pattern of metal ions on a complex matches with the pattern of histidine moieties on the peptide, strong interaction (K = 1.2 x 10(6) M-1) can be achieved. The complex was highly selective (> 200:1) in discriminating similar flexible peptides differing only by one glycine unit.

Identification of a novel nuclear localization signal in Sam68.

Sam68, a nuclear RNA binding protein, binds to Src and is phosphorylated at tyrosine residues in an M-phase specific manner. Here we identified a stretch of 24 amino acid residues in the COOH-terminal portion of Sam68 which function as a nuclear localization signal. This signal sequence bears no apparent homology to any other known nuclear localization sequence. However, this sequence was found to contain a motif, PPXXR (P, Pro; R, Arg), which is conserved in various RNA binding proteins including hnRNP proteins. Replacement of Arg in this motif with Ala abolished the nuclear accumulation of a GFP fusion protein, suggesting that this residue is important in translocating the protein to the nucleus.

MCC, a cytoplasmic protein that blocks cell cycle progression from the G0/G1 to S phase.

The MCC gene was isolated from the human chromosome 5q21 by positional cloning and was found to be mutated in several colorectal tumors. In this study, we prepared specific antibodies and detected the MCC gene product as a cytoplasmic 100-kDa phosphoprotein in mouse NIH3T3 cells. Immunoelectron microscopic analysis showed that the MCC protein is associated with the plasma membrane and membrane organelles in mouse intestinal epithelial cells and neuronal cells. The amount of the MCC protein remained constant during the cell cycle progression of NIH3T3 cells, while its phosphorylation state changed markedly in a cell cycle-dependent manner, being weakly phosphorylated in the G0/G1 and highly phosphorylated during the G1 to S transition. Overexpression of the MCC protein blocked the serum-induced cell cycle transition from the G1 to S phase, whereas a mutant MCC, initially identified in a colorectal tumor, did not exhibit this activity. These results suggest that the MCC protein may play a role in the signaling pathway negatively regulating cell cycle progression.

Atypical clinical presentation of ataxia telangiectasia.

Ataxia telangiectasia (AT) is an autosomal recessive disorder characterized by progressive cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency with recurrent infections, IgA and IgE deficiency, and increased incidence of malignancies. The pathognomonic biological abnormalities consist of spontaneous chromosomal instability resulting in a high in vivo occurrence of cells with translocations, especially involving chromosomes 7 and 14, and a relative insensitivity of DNA replication in vitro to radiation exposure. We report on a patient with the biological hallmarks of AT but with atypical clinical manifestations. Although progressive cerebellar ataxia was present, the neurological picture was broader than that usually seen in AT and included peripheral polyneuropathy and spinal atrophy. On the other hand, telangiectasias, recurrent infections, malignancies, IgA deficiency, or other immunological abnormalities were not present. This illustrates that the clinical picture of AT is broad and nonspecific, and highlights the diagnostic value of cytogenetic analysis and studies of radioresistance of DNA synthesis.

Tricho-rhino-phalangeal syndrome type I (TRP I) due to an apparently balanced translocation involving 8q24.

Tricho-rhino-phalangeal (TRP) syndromes type I and II are caused by a defective gene located on chromosome 8q24.1. We report a family with 2 sibs affected with TRP type I in combination with an apparently balanced chromosome (8;18) translocation involving 8q24.11. It is very likely that the 8q24 translocation breakpoint is physically linked to the TRP gene(s), thereby facilitating future efforts to clone the TRP gene(s).