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BBA - Gene Structure and Expression (v.1732, #1-3)

Editorial Board (pp. ii).

Zebrafish z-otu, a novel Otu and Tudor domain-containing gene, is expressed in early stages of oogenesis and embryogenesis by Saijun Mo; Ping Song; Daoyuan Lv; Yungui Chen; Wei Zhou; Wuming Gong; Zuoyan Zhu (pp. 1-7).

Several studies have suggested that Otu domain had de-ubiquitinating activity and Tudor domain was important for the formation of germ cells. Here, we reported a novel zebrafish ovary-specific gene containing Otu and Tudor domain, z-otu, which was expressed at stages I–III oocytes and embryonic stages from zygotes to early blastula during embryonic cells maintained their totipotency. Therefore, z-otu might link the ubiquitin signaling pathway to early oogenesis and maintaining the totipotency of embryonic cell.

Keywords: Z-otu; Otu domain; Tudor domain; Oogenesis; Embryogenesis; Zebrafish

Identification and characterization of the variants of metastasis-associated protein 1 generated following alternative splicing by Masahiro Yaguchi; Yoriko Wada; Yasushi Toh; Haruo Iguchi; Akira Kono; Kimihiko Matsusue; Soichi Takiguchi (pp. 8-14).

The metastasis-associated gene 1 (mta1) was identified initially in rat highly metastatic cancer cell lines and found to be a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. The gene for mouse mta1 was screened and its genomic structure was determined. It consists of 21 exons spanning 40 kb of genomic DNA. The full-length mouse Mta1 cDNA contained a 2145 nucleotide open reading frame encoding 715 amino acids. In addition to the full-length cDNA, several alternative splicing variants were found. Some differences in the splicing variants found were observed among various mouse organs and cells examined by the semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cDNAs of the splicing variants were inserted into green fluorescent protein (GFP) expression vector and the subcellular localization of the GFP-Mta1 fusion proteins were analyzed. Knowledge of the Mta1 gene expression pattern will be useful in better understanding its functional diversity.

Keywords: Mta1 gene; NuRD complex; Alternative splicing; Subcellular localization

Lead-induced upregulation of the heme-regulated eukaryotic initiation factor 2α kinase is compromised by hemin in human K562 cells by Angshuman Sarkar; Abhijeet Kulkarni; Samit Chattopadhyay; Devraj Mogare; Kiran K. Sharma; Kamini Singh; Jayanta K. Pal (pp. 15-22).

Expression and kinase activity of the heme-regulated-eIF-2α kinase or -inhibitor (HRI) are induced during cytoplasmic stresses leading to inhibition of protein synthesis. Using a reporter construct with HRI promoter, we have determined the promoter activity during heat-shock and lead toxicity in human K562 cells. These two conditions induced HRI promoter activity by 2- to 3-fold. Contrary to this, hemin, a suppressor of HRI kinase activity, downregulated HRI promoter activity and stimulated hemoglobin synthesis. Interestingly, when hemin-treated cells were transfected and exposed to lead, hemin compromised lead-effect substantially by downregulating HRI promoter activity, HRI transcription and HRI kinase activity. These results together suggest that heme signaling in relation to translation regulation is not only restricted to the cytoplasm (modulating HRI kinase activity) alone but it also spans to the nucleus modulating HRI expression. Hemin may thus be useful for alleviation of stress-induced inhibition of protein synthesis.

Keywords: Heme-regulated inhibitor; Promoter activity; Expression; Kinase activity; Heat shock; Lead toxicity

Transcriptional regulation of the Drosophila orc2 gene by the DREF pathway by Koji Okudaira; Katsuhito Ohno; Hideki Yoshida; Maki Asano; Fumiko Hirose; Masamitsu Yamaguchi (pp. 23-30).

DNA replication-related element (DRE) and the DRE-binding factor (DREF) play an important role in regulating DNA replication-related genes such as PCNA and DNA polymerase α in Drosophila. We have previously reported that overexpression of DREF in developing eye imaginal discs induced ectopic DNA synthesis and apoptosis, which results in rough eyes. To identify genetic interactants with the DREF gene, we have carried out a screen for modifiers of the rough eye phenotype. One of the suppressor genes identified was the Drosophila orc2 gene. A search for known transcription factor recognition sites revealed that the orc2 gene contains three DREs, named DRE1 (+14 to +21), DRE2 (−205 to −198), and DRE3 (−709 to −702). Band mobility shift analysis using Kc cell nuclear extracts detected the specific complex formed between DREF and the DRE1 or DRE2. Specific binding of DREF to genomic region containing the DRE1 or DRE2 was further demonstrated by chromatin immunoprecipitation assays, suggesting that these are the genuine complexes formed in vivo. The luciferase assay in Kc cells indicated that the DRE sites in the orc2 promoter are involved in a transcriptional regulation of the orc2 gene. The results, taken together, demonstrate that the orc2 gene is under the control of DREF pathway.

Keywords: orc2; DREF; DNA replication-related gene; Transcription

FTF and LRH-1, two related but different transcription factors in human Caco-2 cells: Their different roles in the regulation of bile acid transport by Debra H. Pan; Frank Chen; Ezequiel Neimark; Xiaoping Li; Benjamin L. Shneider (pp. 31-37).

The apical sodium dependent bile acid transporter (ASBT) mediates ileal bile acid reabsorption. The transcription factors, liver receptor homologue-1 (LRH-1:mouse) and fetoprotein transcription factor (FTF:human), are presumably orthologues. Bile-acid induced negative feedback regulation of mouse (m) and human (h) ASBT occurs via LRH-1 and RAR/RXR, respectively. hASBT has a potential FTF cis-element, although its functional role is unknown. hASBT and mASBT promoter constructs and an FTF cis-element mutated hASBT (hASBT/FTFμ) were assessed in human Caco-2 cells treated with chenodeoxycholic acid (CDCA) and/or co-transfected with hFTF, mLRH-1, or specific small interfering FTF or LRH-1 RNA (siFTF or siLRH). Basal promoter activity was reduced in hASBT/FTFμ, although bile acid response persisted. hFTF activated hASBT but not mASBT, while mLRH-1 activated mASBT but not hASBT. siFTF reduced hASBT but not mASBT activity; siLRH reduced mASBT but not hASBT activity. siLRH but not siFTF abrogated bile acid responsiveness. Electrophoretic mobility shift assays demonstrated distinct and specific binding of the mLRH-1 or hFTF cis-elements. In conclusion, FTF and LRH-1 are two related but different transcription factors in human Caco-2 cells, suggesting that they may be homologues and not orthologues. FTF is not involved directly in bile acid mediated negative feedback regulation of the ASBT.

Keywords: Ileum; Liver; Nuclear receptor; Transcription; Enterohepatic

Transcriptional regulation of human CC chemokine CCL15 gene by NF-κB and AP-1 elements in PMA-stimulated U937 monocytoid cells by Yong-Hyun Shin; Kyung-No Son; Guy Wilhem Lee; Byoung S. Kwon; Jiyoung Kim (pp. 38-42).

CCL15 exerts biological effects on a variety of cells, including monocytes. NF-κB has been reported to be involved in the transcription of the CCL15 gene. In this study, we have identified an AP-1 element located at −76/−65, which appears to regulate the transcription of the CCL15 gene. We also confirmed that the AP-1 factor binds to the element. Specific inhibitors for MAPK pathways and expression of dominant negative MKK4 or JNK1 reduced PMA-induced transcriptional activation of CCL15. Our findings indicate that transcription of the CCL15 gene is regulated by AP-1 and NF-κB through MEK and JNK MAPK pathways in monocytoid cells.

Keywords: Chemokine; CCL15; Leukotactin-1; NF-κB; AP-1; Transcription

Alternative promoters and renal cell-specific regulation of the mouse type IIa sodium-dependent phosphate cotransporter gene by Hironori Yamamoto; Yoshiko Tani; Kumi Kobayashi; Yutaka Taketani; Tadatoshi Sato; Hidekazu Arai; Kyoko Morita; Ken-ichi Miyamoto; John Wesley Pike; Shigeaki Kato; Eiji Takeda (pp. 43-52).

The type IIa sodium-dependent phosphate cotransporter (NPT2a) expressed in renal proximal tubules represents an important determinant in maintaining inorganic phosphate (Pi) homeostasis. In the present study, we identified two variant transcripts of the mouse NPT2a gene, Npt2a-v1 and Npt2a-v2, characterized by the presence of alternative first exons (either exon 1A or exon 1B). The chromosomal structure analysis revealed that the Npt2a gene comprises of two promoters (promoters 1 and 2) and 14 exons, and spans approximately 17 kb. Quantitative PCR analysis showed that renal mRNA levels of both the variants markedly decreased in X-linked vitamin D-resistant hypophosphatemic rickets (Hyp) mice compared to normal littermates. Interestingly, transcriptional activity of a reporter gene, containing Npt2a promoters 1 and 2, was renal cell—specifically increased by 1α, 25(OH)₂D₃ and its analogs. The deletion analysis revealed that the CAAT box in the Npt2a promoter 2 is important for the 1α, 25(OH)₂D₃-dependent renal cell-specific activation of the reporter gene. These data suggested that two alternative promoters control the renal expression of Npt2a gene and both Npt2a variant transcripts are down regulated in Hyp mice.

Keywords: Phosphate cotransporter; Splicing variant; Gene promoter; Hypophosphatemia; Vitamin D

Inflammation-responsive transcription factors SAF-1 and c-Jun/c-Fos promote canine MMP-1 gene expression by Alpana Ray; Arvind Shakya; Bimal K. Ray (pp. 53-61).

Matrix metalloproteinase-1 (MMP-1) has been implicated in the pathogenesis of osteoarthritis (OA) due to its ability to degrade extracellular matrix component of the joint cartilage tissue that cushions the bone from frictional damage. Canine hip dysplasia, a developmental orthopedic disease which results in arthritic condition as is seen in human OA is an excellent system to study the involvement of MMP-1 in the pathogenesis of OA. To date, however, no report is available regarding canine MMP-1 promoter and the regulatory mechanism by which increased synthesis of MMP-1 protein might be regulated. To gain an insight, we have investigated the promoter region of canine MMP-1. MMP-1 synthesis in the resident cells of arthritic joints is regulated via two major cytokines, IL-1β and TNF-α. By using a series of progressively deleted reporter constructs, multiple cytokine-responsive elements were identified in the proximal promoter region of canine MMP-1. These include DNA-binding elements of AP-1 and SAF-1 transcription factors. Mutation of AP-1 or SAF-1 element resulted in marked reduction in the cytokine responsiveness of MMP-1 promoter. We show that AP-1 and SAF-1 DNA-binding activities are increased in cytokine-stimulated cells as well as in osteoarthritic cartilage tissues. In correlation, immunohistochemical analysis indicated higher levels of MMP-1, SAF-1 and AP-1 proteins in osteoarthritic but not in the normal cartilage tissue. These results show that induction and activation of AP-1 and SAF-1 transcription factors are involved in the regulation of MMP-1 expression in the chondrocytes which could be used as therapeutic targets to combat pathogenesis of OA.

Keywords: Transcription factor; Gene expression; Osteoarthritis; MMp-1; Chondrocyte

DNA macroarray and real-time PCR analysis of two nuclear photosystem I mutants from Chlamydomonas reinhardtii reveal downregulation of Lhcb genes but different regulation of Lhca genes by Carsten Balczun; Astrid Bunse; Minou Nowrousian; Alexandra Korbel; Stephanie Glanz; Ulrich K�ck (pp. 62-68).

In photoautotrophic organisms, the expression of nuclear genes encoding plastid proteins is known to be regulated at various levels. In this study, we present the analysis of two non-photosynthetic mutants (CC1051 and TR72) from the unicellular green alga Chlamydomonas reinhardtii. Both mutant strains show a defect in the processing of chloroplast psaA mRNA, and therefore they are assumed to be defective in photosystem I (PSI) assembly. We have performed macroarray experiments with trans-splicing mutants CC1051 and TR72 in order to analyse putative pleiotropic effects of nuclear-located mutations leading to a non-functional PSI. To the best of our knowledge, this is the first example of Chlamydomonas cDNA macroarray analysis comparing the transcriptional regulation of nuclear genes in wild-type and photosystem I mutants. The macroarray results demonstrated a transcriptional downregulation of members of the Lhcb gene family more than 2-fold in both mutant strains. In addition, real-time RT-PCR experiments found a 4- to 16-fold reduction in transcript levels of several Lhca genes in TR72; whereas in CC1051, no significant change in transcript levels was observed. Taken together, our data suggest that a signal is transmitted from the chloroplast to the nucleus that serves to regulate the level of light harvesting polypeptides in the organelle.

Keywords: Chlamydomonas reinhardtii; Photosystem I mutant; Transcriptome analysis; LHC; light-harvesting complex; Chloroplast-to-nucleus signaling

Cloning and characterization of a novel zinc finger protein (rZFP96) in the rat corpus luteum by Ricky R. Lareu; Markus D. Lacher; Robert R. Friis; Arun M. Dharmarajan (pp. 69-75).

The corpus luteum (CL) is a temporary organ involved in the maintenance of pregnancy. In the course of its life-cycle, the CL undergoes two distinct and consecutive processes for its inevitable removal through apoptosis: functional and structural luteolysis. We isolated a gene encoding for a novel rat zinc finger protein (ZFP), named rat ZFP96 (rZFP96) from an ovarian lambda cDNA library. Sequence analysis revealed close sequence and structural similarity to mouse ZFP96 and human zinc finger protein 305 (ZNF305). Quantitative reverse transcription-polymerase chain reaction analysis revealed a positive correlation with the end of pregnancy, that is, the onset of structural luteolysis of the CL. Messenger RNA levels increased 3-fold ( P<0.01) between days 13 and 22 of pregnancy and 8-fold ( P<0.01) between day 13 of pregnancy and day 1 post-partum. In addition, we detected rZFP96 expression in mammary, placenta, heart, kidney and skeletal muscle. Sequence analysis predicted that rZFP96 has a high probability of localizing to the nuclear compartment. The presence of both a perfect consensus TGEKP linker sequence between zinc fingers 2 and 3 as well as several similar sequences between the other zinc fingers suggests physical interaction with DNA. Speculatively, rZFP96 may therefore function as a transcription factor, switching-off pro-survival genes and/or upregulating pro-apoptotic genes and thereby contributing to the demise of the CL.

Keywords: Zinc finger protein; rZFP96; Apoptosis; Corpus luteum; Structural luteolysis

Functional analysis of two regulatory regions of the human Na⁺-dependent vitamin C transporter 2, SLC23A2, in human vascular smooth muscle cells by Stanley A. Rubin; Sanjit Dey; Jack C. Reidling (pp. 76-81).

Uptake of vitamin C occurs through the Na⁺-dependent vitamin C transporters (SVCT1 and 2), the products of two separate genes. In cultured human vascular smooth muscle cells (hVSMC), we found expression of only the hSVCT2 transcript and identified an additional 5′-UTR transcript variant that we termed exon 1b, in addition to the previously described exon 1a. We cloned and tested the promoter functionality of the two genomic regions of the hSVCT2 upstream of these alternative first exons in hVSMC. Both demonstrated activity, and deletion constructs demonstrated that the minimal promoter regions were within ∼100 bp relative to their adjacent exons.

Keywords: Ascorbic acid; Transcript variant; Gene promoter; Cardiovascular system

KLF6 is one transcription factor involved in regulating acid ceramidase gene expression by Jae-Ho Park; Efrat Eliyahu; Goutham Narla; Analisa DiFeo; John A. Martignetti; Edward H. Schuchman (pp. 82-87).

Acid ceramidase (AC; E.C.3.5.1.23) activity is required to hydrolyze ceramide into sphingosine. An inherited deficiency of this enzymatic activity leads to the lipid storage disorder, Farber Lipogranulomatosis. Aberrant AC activity also has been demonstrated in several human cancers. We have characterized a 1931-bp putative promoter region of the murine AC gene by Luciferase reporter assays, electrophoretic mobility shift assays, and mutational analysis. A 143-bp sequence essential for AC promoter activity was found, and mobility shift and super-shift experiments using nuclear extracts of NIH3T3 cells demonstrated that a 34-bp, GC-rich sub-region could bind the transcription factors KLF6, Sp1, and AP2. Transient over-expression of KLF6 in NIH3T3 cells significantly increased the activity of a co-transfected Luciferase reporter construct containing the wild-type AC promoter, and a positive correlation was observed between AC and KFL6 RNA and protein expression in two different human cancer cell lines in which KLF6 expression was either “knocked-down�? by RNAi or increased by retroviral-mediated gene transfer. Northern blot analysis also revealed a correlation of KLF6 and AC gene expression in various human tissues. These results provide the first characterization of the AC promoter from any species and demonstrate that KLF6 is one transcription factor involved in the regulation of AC gene expression.

Keywords: Abbreviations; AC; acid ceramidase; ASM; acid sphingomyelinase; Sph; sphingosine; Sph1P; sphingosine�?1-phosphate; G3PDH; glyceraldehydes-3-phosphate dehydrogenase; EMSA; electrophoretic mobility shift assays; NSCLC; non-small cell lung cancer; RPS11; ribosomal proteins S11Acid ceramidase; Ceramide; Promoter; Gel shift; KLF6; GC-rich

The phylogeny of teleost ZIP and ZnT zinc transporters and their tissue specific expression and response to zinc in zebrafish by Graham P. Feeney; Dongling Zheng; Peter Kille; Christer Hogstrand (pp. 88-95).

In contrast to mammals, zinc transporter genes remain largely uncharacterised in teleosts. Teleost zinc transporter genes were data-mined and phylogenetically assigned to mammalian orthologues. For the first time in animals, the tissue-distribution and mRNA expression response to zinc for most zinc transporter genes was tested in zebrafish.

Keywords: ZIP; ZnT; Zinc; SLC30A; SLC39A

Genomic structure of human omentin, a new adipocytokine expressed in omental adipose tissue by A. Sch�ffler; M. Neumeier; H. Herfarth; A. F�rst; J. Sch�lmerich; C. B�chler (pp. 96-102).

Genomic structure, promoter region, amino acid sequence and exon-specific primer combinations of the human omentin gene are presented. Omentin mRNA expression differs between omental adipose tissue probes from patients with chronic inflammatory bowel diseases such as Crohn's disease. Sequence comparisons revealed a 100% identity of omentin with human intelectin. Based on this, omentin might be a new adipocytokine playing a role in the defense against intestinal bacterial translocation in the context of Crohn's disease.

Keywords: Omentin; Intelectin; Adipocyte; Chronic inflammatory bowel disease; Crohn's disease

A stable, inducible, dose-responsive ODC overexpression system in human cell lines by Shannon M. Wilson; Leo Hawel III; Kirk E. Pastorian; Craig V. Byus (pp. 103-110).

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.

Keywords: Ornithine decarboxylase (ODC); Overexpression; Inducible; Putrescine; Polyamine; Spermidine/spermine N; ¹; -acetyltransferase (SSAT)

Gene Structure and Expression Cumulative Contents (pp. 111-111).

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BBA - Gene Structure and Expression (v.1732, #1-3)