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BBA - Gene Structure and Expression (v.1769, #3)

Editorial Board (pp. ii).

The ins and outs of ATP-dependent chromatin remodeling in budding yeast: Biophysical and proteomic perspectives by Joke J.F.A. van Vugt; Michael Ranes; Coen Campsteijn; Colin Logie (pp. 153-171).

ATP-dependent chromatin remodeling is performed by multi-subunit protein complexes. Over the last years, the identity of these factors has been unveiled in yeast and many parallels have been drawn with animal and plant systems, indicating that sophisticated chromatin transactions evolved prior to their divergence. Here we review current knowledge pertaining to the molecular mode of action of ATP-dependent chromatin remodeling, from single molecule studies to genome-wide genetic and proteomic studies. We focus on the budding yeast versions of SWI/SNF, RSC, DDM1, ISWI, CHD1, INO80 and SWR1.

Keywords: Nucleosome; Chromatin; SNF2; STH1; ISW1; ISW2; INO80; SWR1; CHD1; YFR038w

Molecular cloning of two isoforms of Xenopus (Silurana) tropicalis estrogen receptor mRNA and their expression during development by Minoru Takase; Taisen Iguchi (pp. 172-181).

Native estrogens and estrogenic chemicals exert their actions primarily through the nuclear estrogen receptors (ERs). The present study is directed towards providing a molecular basis for detection of estrogenic and antiestrogenic effects of compounds. In the present study, we isolated successfully two isoforms of a full-length ER mRNA from the liver of Xenopus (Silurana) tropicalis, and analyzed their expression in various tissues during the development by RT-PCR method. The isolated full-length ERα and ERβ cDNAs contained a 1755-bp and a 1644-bp coding regions, respectively. The deduced amino acid sequence of X. tropicalis ERα was highly homologous to previously published X. tropicalis ERα, Xenopus laevis ERs (xer3, ERα1, and ERα2), and X. laevis ER-related proteins. The N-terminal region of the X. tropicalis ERα amino acid sequence cloned in the present study was more highly homologous to that of the xer3 amino acid sequence than the previously published X. tropicalis ERα sequence. Some amino acid residues in the D domain were absent in this sequence. In contrast, the deduced amino acid sequence of the C and E/F domains in X. tropicalis ERβ was highly homologous to those in teleosts, quail, and human ERβs. The RT-PCR analysis showed that expression of the two ER transcripts was relatively abundant in the brain, liver, and gonad/kidney complex of 2-month-old froglet, but weakly expressed in the heart, stomach, leg muscle, and back skin. Gonadal sex differentiation occurred histologically in the X. tropicalis tadpole between Nieuwkoop and Faber stages 54 and 59. The ERα transcripts in the brain, liver and gonad/kidney complex, and ERβ in the brain were expressed at low levels in the tadpole at stage 51, but became extremely abundant in the tadpole at stage 60, and remained at relatively high levels in the froglet after metamorphosis. In contrast, ERβ transcript was expressed abundantly in a consistent manner in the liver and gonad/kidney complex from stage 51 through metamorphosis. Expression of the two ER transcripts was recognized at almost the same levels in both sexes during the development, except for relatively lower expression of ERβ in the female gonad after metamorphosis. These results taken together suggest that the brain, liver, and gonad/kidney complex of the X. tropicalis tadpole may be more susceptible to toxic influences by exogenous estrogenic substances after sex differentiation.

Keywords: Estrogen receptor; Endocrine disrupting compound; Xenopus (Silurana) tropicalis; Gonad; Brain; Liver

In vivo footprinting analysis of the Glypican 3 (GPC3) promoter region in neuroblastoma cells by Gino Boily; Stéphane Ouellet; Sylvie Langlois; Mathieu Larivière; Régen Drouin; Daniel Sinnett (pp. 182-193).

Glypican 3 (GPC3) is an X-linked gene that has its peak expression during development and is down-regulated in all studied tissues after birth. We have shown that GPC3 was expressed in neuroblastoma and Wilms' tumor. To understand the mechanisms regulating the transcription of this gene in neuroblastoma cells, we have focused our study on the identification of putative transcription factors binding the promoter. In this report we performed in vivo dimethylsulfate, UV type C irradiation and DNaseI footprinting analyses coupled with ligation-mediated PCR on nearly 1000 bp of promoter in two neuroblastoma cell lines, SJNB-7 (expressing GPC3) and SK-N-FI (not expressing GPC3). Nucleosome signature footprints were observed in the most distal part of the studied region in both cell lines. We detected eight large differentially protected regions, suggesting the presence of binding proteins in both cell lines but more DNA–protein interactions in GPC3-expressing cells. Sp1 was previously shown to be able to bind some of these regions. Here by combining electromobility shift assays and chromatin immunoprecipitations we showed that the transcription factor NFY was part of the DNA–protein complex found in footprinted regions upstream of the described minimal promoter. These studies performed on chromatin in situ suggest that NFY and yet unknown cell type-specific factors may play an important role in the regulation of GPC3.

Keywords: GPC3; Neuroblastoma; In; vivo; footprinting; Transcription factor

The KH and S1 domains of Escherichia coli polynucleotide phosphorylase are necessary for autoregulation and growth at low temperature by Maura Epifanía Matus-Ortega; Maria Elena Regonesi; Alberto Piña-Escobedo; Paolo Tortora; Gianni Dehò; Jaime García-Mena (pp. 194-203).

PNPase is a phosphate-dependent exonuclease of Escherichia coli required for growth in the cold. In this work we explored the effect of specific mutations in its two RNA binding domains KH and S1 on RNA binding, enzymatic activities, autoregulation and ability to grow at low temperature. We removed critical motifs that stabilize the hydrophobic core of each domain, as well as made a complete deletion of both (ΔKHS1) that severely impaired PNPase binding to RNA. Nevertheless, a residual RNA binding activity, possibly imputable to catalytic binding, could be observed even in the ΔKHS1 PNPase. These mutations also resulted in significant changes in the kinetic behavior of both phosphorolysis and polymerization activities of the enzyme, in particular for the double mutant Pnp-ΔKHS1-H. Additionally, PNPases with mutations in these RNA binding domains did not autoregulate efficiently and were unable to complement the growth defect of a chromosomal Δ pnp mutation at 18 °C. Based on these results it appears that in E. coli the RNA binding domains of PNPase, in particular the KH domain, are vital at low temperature, when the stem–loop structures present in the target mRNAs are more stable and a machinery capable to degrade structured RNA may be essential.

Keywords: Abbreviations; aa; aminoacids; ADP; adenosin diphosphate; c.f.u.; colony forming units; DTT; dithiothreitol; EDTA; ethylenediaminetetraacetic acid; H; six-histidine tag; IPTG; isopropyl-beta-; d; -thiogalactopyranoside; K; _d; dissociation constant; kDa; kilo Daltons; K; _m; Michaelis–Menten constant; OD; optical density; PAGE; polyacrylamide gel electrophoresis; Pi; inorganic phosphate; PMSF; Phenylmethylsulphonylfluoride; PNPase; polynucleotide phosphorylase; R-EMSA; RNA-electrophoretic mobility shift assay; rpm; revolution per min; SDS; sodium lauryl sulfatePNPase; Protein aggregation; RNA-binding domains; Cold adaptation

Promoter analysis of human CC chemokine CCL23 gene in U937 monocytoid cells by Yong-Hyun Shin; Guy Wilhem Lee; Kyung-No Son; Sang Min Lee; Chang Joong Kang; Byoung S. Kwon; Jiyoung Kim (pp. 204-208).

Expression of CCL23 is induced by external stimuli including PMA in monocytes, but its transcriptional regulation has not been studied to date. Serial deletion analysis of its 5′ flanking region revealed that the region −293 to +31 was important for induction by PMA. Cis-acting elements at the −269/−264 (NFAT site), −167/−159 (NF-κB site), and −51/−43 (AP-1 site) positions were identified as the critical sites for the CCL23 expression in U937 cells. We demonstrated the binding of the transcription factors to the consensus sites. Specific inhibitors for signal pathways reduced PMA-induced expression of CCL23, confirming involvement of these transcription factors.

Keywords: Chemokine; CCL23; Gene expression; Transcription factor; PMA

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BBA - Gene Structure and Expression (v.1769, #3)