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BBA - Gene Structure and Expression (v.1729, #1)

Editorial Board (pp. ii).

Identification of a novel Wee1 isoform by Yaping Yu; Biao Chen; Zheng Chen; Cathy Fan; Yifan Han; Jing Zhang; Lizhi He; Alistair Ingram; Anil Kapoor; Jerry H. Wang; Damu Tang (pp. 1-9).

We have identified a novel isoform of Wee1 kinase (Wee1i), which uses Met215 of Wee1 as its initiation codon. RT-PCR, Western blot, and in situ hybridization verified wee1i expression in mammalian cells, rat brain, and rat thymus. Recombinant and partially purified Wee1i from rat thymus displayed kinase activity comparable to or higher than Wee1. The N-terminal 214 residues of Wee1 facilitate its ubiquitin-dependent degradation to trigger mitotic entry. Since Wee1i, lack of these 214 residues, it may evade this degradation and thus provide constitutive Wee1-like kinase activity to inhibit mitotic cell proliferation. Thus, Wee1i may play an important role in differentiation and in tumor suppression.

Keywords: Wee1; Wee1 isoform; Cyclin-dependent kinase; Cell cycle regulation; Neuron; Differentiation

The influence of nucleotide sequences at and near ribosome-binding site on translational efficiency of the Bacillus subtilis rho gene by Gwo-Chyuan Shaw; Mei-Yi Wu; Tian-Ren Lee; Chun-Wei Hsu (pp. 10-13).

The Bacillus subtilis rho gene encodes the transcription termination factor Rho that is produced at a low level in B. subtilis cells. No typical Shine–Dalgarno (SD) sequence lies at an appropriate distance from the translational start site of rho. However, the nucleotide sequence GTGGTG present upstream of the rho translational start site is highly conserved among rho genes of Bacilli. Base substitutions at the central GG or its downstream T abolished expression of rho-lacZ translational fusion, suggesting their importance in rho expression. Mutations at the relatively conserved sequence AAAG located further upstream of GTGGTG could also affect translational efficiency. Moreover, insertion of two or three nucleotides between these two conserved regions abrogated rho-lacZ expression, suggesting that the spacing is important. The possibility that the rho gene may contain a split SD sequence is discussed.

Keywords: Transcription termination factor Rho; Ribosome-binding site; Shine–Dalgarno sequence; Translational efficiency; Bacillus subtilis

Characterization of the human zinc finger protein 267 promoter: Essential role of nuclear factor Y by Kanghong Hu; Marina Fink; Matthias Froh; Erwin G�bele; Claus Hellerbrand; Marcus M�hlbauer; Reiner Wiest; J�rgen Sch�lmerich; Bernd Schnabl (pp. 14-23).

Liver fibrosis results from an excessive deposition of extracellular matrix proteins secreted by activated hepatic stellate cells (HSCs). The activation process is accompanied by an increased activity of various transcription factors, including zinc finger protein 267 (ZNF267). Recently, ZNF267 has been shown to modulate gene expression and to function as a transcriptional repressor. MMP-10 was identified as a target gene; its gene expression and promoter activity are inhibited by ZNF267, which might promote liver fibrogenesis through diminished matrix degradation. However, the transcriptional regulation of the ZNF267 gene is unknown. In the present study, we have cloned and characterized the human ZNF267 promoter containing a 1.5 kb fragment of the 5′-flanking region (−1414/+173). The ZNF267 gene has a TATA-less promoter with multiple transcription initiation sites. Analysis of serial 5′-deletions of luciferase reporter constructs revealed a minimal promoter between −72 and +173 bp. Mutational analysis of putative regulatory elements indicated that a CCAAT box within this region was essential for ZNF267 promoter activity. Electrophoretic mobility shift assays demonstrated that transcription factor nuclear factor Y (NF-Y) bound to the CCAAT box. In co-transfection experiments, NF-YA increased the promoter activity of ZNF267. In conclusion, our results suggest that the binding site for NF-Y is critical for ZNF267 gene regulation and, herewith, the activation of this transcriptional factor may play an important role in the activation process of HSCs and in liver fibrosis.

Keywords: Abbreviations; HSCs; hepatic stellate cells; ZNF267; zinc finger protein 267; ECM; extracellular matrix; C/EBP; CCAAT/enhancer-binding protein; NF-Y; nuclear factor Y; KLF; Kruppel-like factor; KRAB; Kruppel associated box; MMP; matrix metalloproteinase; HEK; human embryonic kidney; FCS; fetal calf serum; EMSA; electrophoretic mobility shift assay; IL; interleukin; TGFβ; transforming growth factor β; TNFα; tumor necrosis factor α; INFγ; interferon γ; SNAP; S; -Nitroso-; N; -acetylpenicillamine; RACE; rapid amplification of cDNA ends; CTF; CCAAT transcription factorKruppel-like zinc finger proteins; Hepatic stellate cell; Promoter; Gene regulation; Nuclear factor Y; CCAAT box

Extensive expression studies revealed a complex alternative splicing pattern of the HMGA2 gene by Sven Hauke; Silke Leopold; Claudia Schlueter; Aljoscha M. Flohr; Hugo Murua Escobar; Piere Rogalla; J�rn Bullerdiek (pp. 24-31).

Chromosomal rearrangements of the HMGA2 locus belong to the most common aberrations in human benign tumors. HMGA2 rearrangements often result in chimeric genes expressing transcripts consisting of the first three exons of HMGA2 followed by ectopic sequences derived from intron 3 of that gene.RT-PCR-based expression studies of 4 of these HMGA2 transcripts revealed a co-expression with the “wild-type�? HMGA2a in tumor samples as well as in normal tissues. Northern blot hybridizations of the lipoma cell line Li-14 revealed the expression of five additional HMGA2 transcripts consisting of exons 1 to 3 but not exons 4 to 5 besides the full-length HMGA2a transcript. In silico analyses have been performed showing a high homology to well-established consensus sequences for the 3′ splice acceptor site, the branch site, and poly(A) signal. Thus, it is quite obvious that the HMGA2 transcripts described herein are alternative, not aberrant, splice-products of the HMGA2 gene.It is hypothesized that HMGA2-dependent tumorigenesis is caused by a disturbed equilibrium in the co-expression of the HMGA2 splice variants leading to aberrant cell proliferation and/or malignant transformation of cells.

Keywords: High mobility group protein gene HMGA2; Alternative splicing; Benign tumor; Chromosomal rearrangement

Cloning and expression of hypoxia-inducible factor 1α from the hibernating ground squirrel, Spermophilus tridecemlineatus by Pier Morin Jr.; Kenneth B. Storey (pp. 32-40).

Mammalian hibernation is associated with apnoic breathing patterns and a hypoxia–hypothermia connection has been suggested as part of the mechanism by which body temperature is reduced as animals enter torpor. Hence, we hypothesized that changes in the expression of the hypoxia inducible factor (HIF-1) may potentially be involved in regulating hibernation-responsive gene targets. The expression of the alpha subunit of HIF-1 was quantified at both gene and protein levels in four organs of the thirteen-lined ground squirrel, Spermophilus tridecemlineatus. Reverse transcription-PCR showed no change in hif-1α transcript levels in the liver, lung, skeletal muscle or brown adipose tissue of euthermic versus hibernating animals but HIF-1α protein levels were elevated by 60–70% in the two organs responsive for thermogenesis (brown adipose and skeletal muscle). Furthermore, assessment of DNA binding by HIF-1 in nuclear extracts from brown adipose revealed 6-fold higher levels in hibernator tissue than in euthermic controls suggesting increased expression of HIF-1 responsive genes during hibernation. The complete nucleotide sequence of hif-1α from ground squirrels, the first hif-1α sequence amplified from a hibernating mammal, was obtained using PCR amplification and 3′ and 5′ RACE. Amino acid sequence analysis revealed 90–95% identity with the HIF-1α protein from other mammals. Several unique amino acid sequence substitutions were identified that may affect protein conformation and could possibly function to counteract low temperature effects on HIF-1α conformation at near 0 °C body temperatures during torpor.

Keywords: Mammalian hibernation; Hypoxia-inducible factor 1; 13-Lined ground squirrel; Brown adipose tissue; Muscle; Thermogenesis

MAPK pathway contributes to density- and hypoxia-induced expression of the tumor-associated carbonic anhydrase IX by Juraj Kopacek; Monika Barathova; Franck Dequiedt; Janka Sepelakova; Richard Kettmann; Jaromir Pastorek; Silvia Pastorekova (pp. 41-49).

Transcription of the CA9 gene coding for a tumor-associated carbonic anhydrase IX (CA IX) isoform is regulated by hypoxia via the hypoxia-inducible factor 1 (HIF-1) and by high cell density via the phosphatidylinositol-3-kinase (PI3K) pathway. We examined the role of the mitogen-activated protein kinase (MAPK) pathway in the control of CA9 gene expression. Inhibition of MAPK signaling by U0126 in HeLa cells led to reduced activity of the PR1-HRE-luc CA9 promoter construct and decreased CA IX protein levels in dense culture as well as in hypoxia. Similar reduction was obtained by expression of a dominant-negative ERK1 mutant and was also observed in U0126-treated HIF-1α-deficient Ka13 cells. Simultaneous treatment with the MAPK and PI3K inhibitors U0126 and LY 294002 had stronger effect than individual inhibition of these pathways. Taken together, our results suggest that besides the PI3K pathway, the MAPK cascade is involved in the regulation of CA9 gene expression under both hypoxia and high cell density.

Keywords: Abbreviations; CA IX; carbonic anhydrase protein; CA9; carbonic anhydrase gene; HIF; hypoxia inducible factor; PI3K; phosphatidylinositol-3-kinase; MAPK; mitogen-activated kinaseCarbonic anhydrase IX; Hypoxia; Cell density; MAPK pathway; Promoter activity; Transcriptional regulation

The C terminus of MINT forms homodimers and abrogates MINT-mediated transcriptional repression by Junlin Li; Junfeng Li; Xi Yang; Hongyan Qin; Peng Zhou; Yingmin Liang; Hua Han (pp. 50-56).

Notch signaling plays a pivotal role in numerous cell fate determination events during development, and therefore its regulation has been studied intensively. MSX2-interacting nuclear target protein (MINT) modifies the Notch signaling by interacting with and inhibiting the downstream transcription factor RBP-J/CBF-1 of Notch. In this study, by a yeast two hybrid screening, we found that the C terminal fragment of MINT interacted with each other. We confirmed the interaction between two MINT C terminal fragments both in vitro and in vivo. We further demonstrated that the overexpression of the C terminal fragment of MINT cancelled its inhibitory effect on the transactivation of an RBP-J-dependent promoter by Notch. These results suggest that MINT may form a dimer or multimer in cells through its C terminus, and that the C terminal fragment of MINT may work as its dominant-negative version.

Keywords: MSX2-interacting nuclear target protein; Notch; RBP-J; Protein interaction; Transcription

Biochemical observation of the rapid mobility of nuclear HMGB1 by Nelly Sapojnikova; Joseph Maman; Fiona A. Myers; Alan W. Thorne; Vladimir I. Vorobyev; Colyn Crane-Robinson (pp. 57-63).

Formaldehyde-crosslinked and sonicated chromatin fragments were obtained from 15-day chicken embryo erythrocytes and purified on caesium chloride gradients. Polyclonal antibodies raised against chicken HMGB1 were used to immuno-precipitate fragments carrying HMGB1 in two protocols: (1) affinity purified antibodies covalently coupled to agarose beads and (2) diluted antiserum. The DNA of the antibody-bound chromatin was quantified and its sequence content assessed by quantitative real-time PCR to give values of the absolute enrichments generated. Amplicons were monitored within the active β-globin locus, in the adjacent heterochromatin, in the lysozyme locus (containing an active housekeeping gene and the inactive lysozyme gene) and at the promoter of the inactive ovalbumin gene. For all amplicons the Bound/Input ratio was close to unity, implying no preferential location of HMGB1 on the chromatin. This initially unexpected result can now be understood in the light of the exceptional mobility of HMGB1 revealed by FLIP experiments showing that only 1–2 s are needed for HMGB1 to cross the nucleus: crosslinking times of 1 min were used in the present experiments.

Keywords: HMGB1; Chromatin immunoprecipitation; Chicken genome

Identification and expression profile of the ID gene family in the rainbow trout ( Oncorhynchus mykiss) by Scott A. Gahr; M. Fernanda Rodriguez; Caird E. Rexroad III (pp. 64-73).

ID proteins are negative regulators of basic helix–loop–helix transcription factors governing growth and development in mammals. However, little is known about the ID gene function and expression in fish. We report the identification and characterization of two new rainbow trout ID genes (ID1D and ID2B) and extend our expression analyses of two previously identified ID genes (ID1A and ID2A). Phylogenetic analyses indicate an evolutionary relationship between ID1A and ID1D and between ID1B and ID1C, suggesting a mechanism of divergence throughout salmonid evolution. To access the expression of these genes in adult and developing fish, we measured the relative transcript abundance of four ID1 and two ID2 genes by real-time PCR. ID1 transcripts were expressed in a variety of tissues and the ID1 paralogues showed similar patterns of expression, whereas the ID2 paralogues were differentially expressed. To access the role of the ID genes during embryonic development, gene expression was measured at early (day 0 and day 2), mid (day 9 and day 18) and late (day 30 and day 50) embryonic development. ID1A and ID1D expression remained unchanged throughout embryonic development, while ID1B and ID1C were lowest during early, highest at mid, and decreased during late embryonic development. The ID2 transcripts revealed the highest expression in unfertilized eggs and day 2 embryos, and remained low throughout the remainder of embryonic development. The sequence analyses and gene expression patterns implicate gene and genome duplication in rainbow trout ID gene evolution and suggest an extensive role for the IDs in rainbow trout growth and development.

Keywords: Inhibitor of differentiation/DNA binding; Embryonic expression; Gene structure; Tissue distribution; Real-time PCR

C/EBPδ and C/EBPγ bind the CCAAT-box in the human β-globin promoter and modulate the activity of the CACC-box binding protein, EKLF by Christopher T. Gordon; Vanessa J. Fox; Suzana Najdovska; Andrew C. Perkins (pp. 74-80).

Developmental- and tissue-specific expression of globin genes is mediated by a few key elements within the proximal promoter of each gene. DNA-binding assays previously identified NF-Y, GATA-1, C/EBPβ and C/EBPγ as candidate regulators of β-globin transcription via the CCAAT-box, a promoter element situated between CACC- and TATA-boxes. We have identified C/EBPδ as an additional β-globin CCAAT-box binding protein. In reporter assays, we show that C/EBPδ can co-operate with EKLF, a CACC-box binding protein, to activate the β-globin promoter, whereas C/EBPγ inhibits the transcriptional activity of EKLF in this assay.

Keywords: CCAAT-box; C/EBPδ; C/EBPγ; EKLF; β-globin promoter; Luciferase assay

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BBA - Gene Structure and Expression (v.1729, #1)