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BBA - Gene Structure and Expression (v.1731, #2)

Editorial Board (pp. ii).

Commentary on “Stable incorporation of sequence-specific repressors Ash1 and Ume6 into the Rpd3L complex�? by Caroline S. Dacwag; Anthony N. Imbalzano (pp. 75-76).

Rpd3; Histone deacetylase; Gene regulation

Stable incorporation of sequence specific repressors Ash1 and Ume6 into the Rpd3L complex by Michael J. Carrozza; Laurence Florens; Selene K. Swanson; Wei-Jong Shia; Scott Anderson; John Yates; Michael P. Washburn; Jerry L. Workman (pp. 77-87).

Histone deacetylation by Saccharomyces cerevisiae Rpd3 represses genes regulated by the Ash1 and Ume6 DNA-binding proteins. Rpd3 exists in a small 0.6 MDa (Rpd3S) and large 1.2 MDa (Rpd3L) corepressor complex. In this report, we identify by mass spectrometry and MudPIT the subunits of the Rpd3L complex. These included Rpd3, Sds3, Pho23, Dep1, Rxt2, Sin3, Ash1, Ume1, Sap30, Cti6, Rxt3 and Ume6. Dep1 and Sds3, unique components of Rpd3L, were required for Rpd3L integrity and HDAC activity. Similar to RPD3, deletion of DEP1 enhanced telomeric silencing and derepressed INO1. Two sequence-specific repressors, Ash1 and Ume6, were stably associated with Rpd3L. While both of these proteins localized to the INO1 and HO promoters, the repression of these genes were dependent only on Ume6 and Ash1, respectively. Thus, the Rpd3L complex is directly recruited to specific promoters through multiple integral DNA-binding proteins.

Keywords: Yeast; Chromatin; Histone; Acetylation; Deacetylase; Repressor

Identification and organ expression of peroxisome proliferator activated receptors in brown trout ( Salmo trutta f. fario) by Carla Batista-Pinto; Pedro Rodrigues; Eduardo Rocha; Alexandre Lobo-da-Cunha (pp. 88-94).

Although widely studied in mammals, little information about fish peroxisome proliferator activated receptors (PPARs) is yet available. As a baseline for future studies, the three PPAR isotypes were identified in brown trout ( Salmo trutta f. fario) and their organ distribution pattern was established. The cDNA fragments encoding PPARs α, β and γ were amplified by PCR, and the deduced sequences of the correspondent peptides were compared with other species sequences. Both the 183 amino acid sequence from PPARα and the 103 amino acid sequence from PPARβ shared high levels of homology with the correspondent peptides of other fishes and terrestrial vertebrates, whereas PPARγ 108 amino acid sequence showed much less similarity with non-fish PPARγ. According to both semi-quantitative RT-PCR and real-time RT-PCR, PPARα mRNA predominates in white muscle, heart and liver and PPARβ is more expressed in testis, heart, liver, white muscle and trunk kidney. PPARγ was only detected in trunk kidney and liver by real-time RT-PCR and also in spleen by semi-quantitative RT-PCR. PPARβ seems to be the most strongly expressed isotype, whereas PPARγ shows a much weaker global expression.

Keywords: Peroxisome; PPAR; Expression; Teleost

Determination of osteoprogenitor-specific promoter activity in mouse mesenchymal stem cells by recombinant adeno-associated virus transduction by Sanjay Kumar; Gandham Mahendra; Selvarangan Ponnazhagan (pp. 95-103).

Towards utilizing gene-targeted, repopulating mesenchymal stem cells (MSC) to increase osteogenesis, we evaluated the expression of bone-specific promoters during MSC differentiation. Multi-lineage potential of cultured MSC was confirmed by osteogenic, adipogenic and chondrogenic differentiation under controlled conditions. Recombinant adeno-associated virus (rAAV) encoding luciferase under the human cytomegalovirus (CMV), mouse alkaline phosphatase (ALP), Runx-2/cbfa1 (RUNX), osteopontin (OPN), collagen type 1a (COL), and osteocalcin (OCN) promoters was used to transduce mouse MSC. Replicate cultures were maintained undifferentiated or differentiated to osteoblast lineage. Luciferase expression was determined on days 1, 2, 3, 7, 14, or 21 as a measure of promoter activity. Expression of osteogenic markers and mineralization was determined as correlates of osteopoiesis. Results indicated expression from CMV promoter in undifferentiated and differentiated cultures at early stage. However, expression from COL and RUNX promoters was abundant only in differentiating cultures as early as 24 h but declined gradually. Expression from OPN and ALP promoters was evident 24 h following osteogenic differentiation and peaked gradually until 2 weeks before declining. Expression from OC promoter was evident only after 7 days of differentiation but remained until final analysis on day 21. That rAAV transduction of MSC does not induce differentiation was also confirmed by quantitative reverse-transcription polymerase chain reaction (QRT-PCR). The observed stage-specific expression of analyzed promoters was not significant when the MSC were differentiated to adipocytes. Thus, the use of RUNX2 or COL promoter to stably express osteoinductive factors in MSC may allow both self-renewal of modified MSC and enrichment of osteoblast commitment.

Keywords: Adeno-associated virus; Mesenchymal stem cell; Osteoprogenitor; Promoter activity

Regulatory studies of murine methylenetetrahydrofolate reductase reveal two major promoters and NF-κB sensitivity by Laura Pickell; Pamela Tran; Daniel Leclerc; John Hiscott; Rima Rozen (pp. 104-114).

Two promoters of the murine methylenetetrahydrofolate reductase gene ( Mthfr), a key enzyme in folate metabolism, were characterized in Neuro-2a, NIH/3T3 and RAW 264.7 cells. Sequences of 189 bp and 273 bp were sufficient to achieve maximal activity of the upstream and downstream promoter, respectively. However, subtle differences in minimal promoter lengths and in promoter activities were observed between the cell lines. Both promoters demonstrated comparable activity in NIH/3T3 and RAW 264.7 cells, while in Neuro-2a cells, the upstream promoter was 15-fold more active than the downstream promoter. Alignment and data mining tools identified a candidate nuclear factor kappa B (NF-κB) binding site at the 3′end of the downstream promoter that is conserved throughout several species. NF-κB activation experiments in cultured cells were associated with increased Mthfr mRNA. Co-transfection of NF-κB and promoter constructs demonstrated Mthfr up-regulation by at least 2-fold through its downstream promoter in Neuro-2a cells; this increase was significantly reduced when the putative binding site was mutated. EMSA analysis demonstrated direct binding of NF-κB to this non-mutated site. This study, a first step into the elucidation of Mthfr regulation, demonstrates that two TATA-less, GC-rich promoters differentially drive transcription of Mthfr in a cell-specific manner, and provides a novel link of Mthfr to possible roles in the immune response and cell survival.

Keywords: Methylenetetrahydrofolate reductase; Folic acid; Nuclear factor kappa B; Promoter; Expression; Regulation

Transcriptional regulation of the human reduced folate carrier A1/A2 promoter: Identification of critical roles for the USF and GATA families of transcription factors by Scott G. Payton; Mingjun Liu; Yubin Ge; Larry H. Matherly (pp. 115-124).

The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5′ and 3′ deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation.

Keywords: Reduced folate carrier; Methotrexate; Transcription; USF; GATA

Structure and expression of the zebrafish mest gene, an ortholog of mammalian imprinted gene PEG1/ MEST by Yoonsoo Hahn; Seung Kyoung Yang; Jae Hoon Chung (pp. 125-132).

PEG1/MEST is a paternally expressed gene in placental mammals. Here, we report identification of zebrafish ( Danio rerio) gene mest, an ortholog of mammalian PEG1/MEST. Zebrafish mest encodes a polypeptide of 344 amino acids and shows a significant similarity to mammalian orthologs. Zebrafish mest is present as a single copy in the zebrafish genome and is closely linked to copg2 as in mammals. It is notable that 10 of 11 intron positions in mest are conserved among mammalian PEG1/MEST genes, indicating that the genomic organization and linkage between mest and copg2 loci was established in ancient vertebrates. Zebrafish mest is expressed in blastula, segmentation, and larval stages, exhibiting gradually increased expression as the development proceeds. Allelic expression analysis in hybrid larvae shows that both parental alleles are transcribed. We also observed one-codon alternative splicing involving an alternative usage of the two consecutive splice acceptors of intron 1, generating two protein isoforms with different lengths of a single amino acid.

Keywords: Mest; Zebrafish; Developmental expression; Allelic expression; One-codon alternative splicing

Cloning and analysis of the murine Foxi2 transcription factor by Patrick J.E.C. Wijchers; Marco F.M. Hoekman; J. Peter H. Burbach; Marten P. Smidt (pp. 133-138).

Forkhead transcription factors comprise a large family of key regulators of embryonic development. Here, we describe the cloning and analysis of the murine Foxi2 gene, coding for a putative 311 amino acid protein resembling Foxi subfamily members in mice and other species. Expression analysis during the final stages of embryonic development revealed that Foxi2 expression is mainly confined to subsets of cells in epithelial structures and particular ducts, in addition to the developing forebrain and neural retina. Since FoxI factors are thought to be implicated in the regulation of cell fate, the highly restricted expression pattern of Foxi2 suggestive of a possible role in controlling cellular identity.

Keywords: Forkhead; Transcription factor; Foxi2; Development; Cell identity; Differentiation

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BBA - Gene Structure and Expression (v.1731, #2)