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BBA - Gene Regulatory Mechanisms (v.1779, #1)

Editorial Board (pp. ii).

Acknowledgement (pp. i-iii).

Editorial by Steven J. Triezenberg Executive Editor, BBA_GRM; Craig L. Peterson Executive Editor, BBA_GRM (pp. 1-2).

Human chromosome fragility by T. Lukusa; J.P. Fryns (pp. 3-16).

Fragile sites are heritable specific chromosome loci that exhibit an increased frequency of gaps, poor staining, constrictions or breaks when chromosomes are exposed to partial DNA replication inhibition. They constitute areas of chromatin that fail to compact during mitosis.They are classified as rare or common depending on their frequency within the population and are further subdivided on the basis of their specific induction chemistry into different groups differentiated as folate sensitive or non-folate sensitive rare fragile sites, and as aphidicolin, bromodeoxyuridine (BrdU) or 5-azacytidine inducible common fragile sites. Most of the known inducers of fragility share in common their potentiality to inhibit the elongation of DNA replication, particularly at fragile site loci.Seven folate sensitive (FRA10A, FRA11B, FRA12A, FRA16A, FRAXA, FRAXE and FRAXF) and two non-folate sensitive (FRA10B and FRA16B) fragile sites have been molecularly characterized. All have been found to represent expanded DNA repeat sequences resulting from a dynamic mutation involving the normally occurring polymorphic CCG/CGG trinucleotide repeats at the folate sensitive and AT-rich minisatellite repeats at the non-folate sensitive fragile sites. These expanded repeats were demonstrated, first, to have the potential, under certain conditions, to form stable secondary non-B DNA structures (intra-strand hairpins, slipped strand DNA or tetrahelical structures) and to present highly flexible repeat sequences, both conditions which are expected to affect the replication dynamics, and second, to decrease the efficiency of nucleosome assembly, resulting in decondensation defects seen as fragile sites. Thirteen aphidicolin inducible common fragile sites (FRA2G, FRA3B, FRA4F, FRA6E, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA8C, FRA9E, FRA16D and FRAXB) have been characterized at a molecular level and found to represent relatively AT-rich DNA areas, but without any expanded repeat motifs. Analysis of structural characteristics of the DNA at some of these sites (FRA2G, FRA3B, FRA6F, FRA7E, FRA7G, FRA7H, FRA7I, FRA16D and FRAXB) showed that they contained more areas of high DNA torsional flexibility with more highly AT-dinucleotide-rich islands than neighbouring non-fragile regions. These islands were shown to have the potential to form secondary non-B DNA structures and to interfere with higher-order chromatin folding. Therefore, a common fragility mechanism, characterized by high flexibility and the potential to form secondary structures and interfere with nucleosome assembly, is shared by all the cloned classes of fragile sites.From the clinical point of view, the folate sensitive rare fragile site FRAXA is the most important fragile site as it is associated with the fragile X syndrome, the most common form of familial mental retardation, affecting about 1/4000 males and 1/6000 females. Mental retardation in this syndrome is considered as resulting from the abolition of the FMR1 gene expression due to hypermethylation of the gene CpG islands adjacent to the expanded methylated trinucleotide repeat. FRAXE is associated with X-linked non-specific mental retardation, and FRA11B with Jacobsen syndrome. There is also some evidence that fragile sites, especially common fragile sites, are consistently involved in the in vivo chromosomal rearrangements related to cancer, whereas the possible implication of common fragile sites in neuropsychiatric and developmental disorders is still poorly documented.

Keywords: Rare fragile site; Common fragile site; CCG/CGG trinucleotide repeat; AT-rich minisatellite repeat; Dynamic mutation; DNA torsional flexibility

Transcriptional regulation of the human soluble epoxide hydrolase gene EPHX2 by Hiromasa Tanaka; Shizuo G. Kamita; Nicola M. Wolf; Todd R. Harris; Zhaoju Wu; Christophe Morisseau; Bruce D. Hammock (pp. 17-27).

Soluble epoxide hydrolase (sEH) is a multifunctional protein encoded by the EPHX2 gene. The biological functions and enzyme kinetics of sEH have been extensively investigated, however, little is known about its transcriptional regulation and mechanisms of tissue specific expression. Here, a luciferase gene based reporter assay was used to identify the minimal promoter and cis regulatory elements of EPHX2. The minimal promoter was identified as a GC-rich region between nts −374 and +28 with respect to the putative transcriptional start site. A reporter plasmid carrying this minimal promoter showed higher or similar activities in comparison to a reporter plasmid carrying nts −5,974 to +28 of EPHX2 in 9 human cell lines that were tested. Sp1 binding sites that are involved in augmenting the minimal promoter activity of EPHX2 were identified by nested deletion analysis, site-specific mutation, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay.

Keywords: Epoxide hydrolase; EPHX2; Promoter; Transcription

Retinoic acid is a negative regulator of matrix Gla protein gene expression in teleost fish Sparus aurata by Natércia Conceição; Vincent Laizé; Brigite Simões; António R. Pombinho; M. Leonor Cancela (pp. 28-39).

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues while accumulated only in bone and cartilage under physiological conditions. Although the precise molecular mechanism of action of MGP remains unknown, all available evidence indicates that it acts as a physiological inhibitor of mineralization. This work presents the cloning of gilthead seabream MGP gene ( SaMGP) and the functional analysis of its promoter. SaMGP gene was found to be organized in five exons and to be under control of a distal and a proximal promoter, both, capable of activating SaMGP transcription in transient transfections. Furthermore, we present strong evidence that retinoic acid down-regulates SaMGP gene transcription by interacting, through binding of its receptor, with a specific region within distal promoter. Interestingly, the presence of repetitive motifs in the proximity of SaMGP gene regulatory regions suggests that they may modulate promoter accessibility to transcription machinery, as already seen for other genes. This work provides additional evidence of the usefulness of non-mammalian model systems to elucidate the complex regulation of MGP gene transcription.

Keywords: Matrix Gla protein; Gilthead seabream; Sparus aurata; Retinoic acid; Regulation of gene expression; Functional analysis of promoter

Analysis of the promoter region of human placenta-specific DSCR4 gene by Satoko Asai; Akiko Yamaki; Jun Kudoh; Nobuyoshi Shimizu; Yoshiko Shimizu (pp. 40-50).

The gene DSCR4 locates in the band q22.2 of human chromosome 21 and encodes a protein of 118 amino acids. Expression of DSCR4 is restricted to human placenta and placental choriocarcinoma cell lines BeWo and JEG3. The 5′-RACE method using RNA from human placenta indicated the major transcription start site at 93 nt upstream (nt −93) of the initiation codon. Transfection assay using a series of deletion constructs of the 5′-flanking region fused to the luciferase reporter gene identified three positive regions nt −2200 to −2088, nt −2064 to −1924, nt −810 to −632 and two negative regions nt −1923 to −1740, nt −631 to −425. The computer analysis predicted the presence of several cis-elements in these regions and the promoter assay using various mutants of consensus sequence identified two distinct cis-elements for OLF-1 and E47. The electrophoretic mobility shift assay (EMSA) using the extracts of DSCR4-expressing cells confirmed the binding of certain protein factors to these cis-elements. In fact, OLF-1-like transcription factor, EBF-3 and EBF-4 were detected in the DSCR4-expressing cell lines and human placenta. Based on these data, we postulated that transcription of DSCR4 gene is regulated positively by binding of OLF-1-like transcription factor and negatively by binding of E47-like transcription factor.

Keywords: Abbreviations; DSCR; Down syndrome chromosomal region; DSCR4; Down syndrome critical region 4; RACE; rapid amplification of cDNA ends; OLF-1; olfactory neuron-specific transcription factor; EMSA; electrophoretic mobility shift assay; EBF; early B-cell factor; ERV; endogenous retrovirus; LTR; long terminal repeatsDSCR4; Choriocarcinoma; Luciferase; OLF-1; E47; EMSA

A lasp family protein of Ciona intestinalis by Asako G. Terasaki; Jin Hiruta; Junko Suzuki; Sachiko Sakamoto; Tatsuji Nishioka; Hiroshi Suzuki; Kazuyo Ohashi; Kaoru Azumi; Michio Ogasawara (pp. 51-59).

Lasp-1 and lasp-2 are actin-binding proteins that contain a LIM domain, nebulin repeats, and an SH3 domain and they are significantly conserved in mammalian and avian. Lasp-1 is widely expressed in nonmuscle tissues and lasp-2 is specifically expressed in the brain. Genes encoding proteins homologous to lasp-1 and lasp-2 were deposited in the genome/cDNA database of invertebrates such as sea urchins, nematodes, and insects; however, function of their proteins have not been studied in detail.In this study, we analyzed the gene structure, actin-binding activity, and expression of the lasp protein of the ascidian Ciona intestinalis ( Ci lasp). A single gene encoding lasp protein was found in the ascidian, and the amino acid sequences of Ci lasp and other invertebrate lasp proteins exhibited similarity to vertebrate lasp-1 and lasp-2 to the same extent. A part of the exon–intron boundaries was conserved between the vertebrate lasp-1, the vertebrate lasp-2 and the invertebrate lasp genes. Ci lasp exhibited actin-binding activity in a co-sedimentation assay. In situ hybridization revealed that the expression of Ci lasp mRNA was apparent in nervous system of early embryos and was detected in various tissues in young adults. This suggests that the functions of invertebrate lasp proteins might include the functions of vertebrate lasp-1 and lasp-2.

Keywords: Ascidian; Lasp; Actin-binding; LIM domain; Nebulin repeat; SH3 domain

The 3′ UTR of the human CTLA4 mRNA can regulate mRNA stability and translational efficiency by Lorenzo Malquori; Laura Carsetti; Giovina Ruberti (pp. 60-65).

T cell activation results from the integration of signals generated through the T cell antigen receptor–CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by the engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. CTLA-4 plays a critical role in downregulating T cell responses. Autoimmune diseases have shown genetic linkage to the CTLA4 locus. In this report we demonstrate that the 3′ UTR of CTLA4 regulates firefly luciferase reporter gene expression, can confer instability to CTLA4 mRNA and can influence its translation efficiency in vitro.

Keywords: CTLA4; 3′ UTR; Gene regulation; (AT)n polymorphism; Autoimmune disease

Human CpG binding protein interacts with MLL1, MLL2 and hSet1 and regulates Hox gene expression by Khairul I. Ansari; Bibhu P. Mishra; Subhrangsu S. Mandal (pp. 66-73).

Human encodes several histone H3-Lysine 4 (H3K4) specific methyl-transferases (HMTs) such as MLL1 (mixed lineage leukemia 1), MLL2, MLL3, hSet1 etc, that play critical roles in gene expression. These HMTs are present as distinct multi-protein complexes with several proteins in common. Herein, we have affinity purified and characterized human CpG binding protein (CGBP) and its interacting proteins from human cells. We demonstrated that CGBP is co-purified with three H3K4 specific HMTs MLL1, MLL2, and hSet1. We also performed independent immuno-precipitation of MLL1, MLL2 and hSet1 complexes from human cell and demonstrated that each of these complexes contains CGBP. In addition, CGBP is co-localized with MLL1, MLL2 and hSet1 in vivo and binds to the promoter of MLL target gene HoxA7. Antisense mediated knock down of CGBP diminished the recruitment of MLL1 and down regulated levels of H3K4 trimethylation in HoxA7 promoter affecting its expression. These results demonstrated that CGBP interacts with MLL1, MLL2 as well as hSet1 HMTs and plays critical roles in regulations of MLL target genes.

Keywords: Histone methyl-transferase; CpG binding protein; MLL1; MLL2; Set1; Gene expression

Identification of two E-boxes that negatively modulate the activity of MyoD on the alpha-sarcoglycan core promoter by Paul Delgado-Olguín; J. Manuel Hernández-Hernández; Fabio Salamanca; Félix Recillas-Targa; Ramón M. Coral-Vázquez (pp. 74-80).

The α-SG promoter is composed of a plethora of cis-regulatory elements, whose individual contribution to α-SG gene expression modulation remains unknown. We have identified a negative regulatory element in the α-SG distal promoter including two conserved E-boxes (E1 and E2), which interact with MyoD. We found that E1 and E2 negatively modulate the transactivation potential of MyoD on the α-SG core promoter. Moreover, such negative effect is mainly mediated by E2, which is surrounded by conserved nucleotides conferring MyoD binding capacity. Our results suggest that modulation of MyoD activity by E1, and particularly E2, contributes to the negative regulation of α-SG gene expression during myogenic differentiation.

Keywords: α-sarcoglycan promoter; MyoD transactivation; Core promoter

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BBA - Gene Regulatory Mechanisms (v.1779, #1)