| 1. |
- Grote, Jens, et al.
(författare)
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Identification of poly(ADP-ribose) polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression
- 2006
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Background: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. ( 2002) J. Biol. Chem. 277, 41879-41887). Results: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose) polymerase-I (PARP-I) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-l with 1,5- isoquinolinediol (DiQ). Conclusion: The candidates, poly(ADP-ribose) polymerase-l (PARP-l) and the heterodimeric complex Ku70/ Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.
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| 2. |
- Ajore, Ram, et al.
(författare)
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The leukemia associated ETO nuclear repressor gene is regulated by the GATA-1 transcription factor in erythroid/megakaryocytic cells
- 2010
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The Eight-Twenty-One (ETO) nuclear co-repressor gene belongs to the ETO homologue family also containing Myeloid Translocation Gene on chromosome 16 (MTG16) and myeloid translocation Gene-Related protein 1 (MTGR1). By chromosomal translocations ETO and MTG16 become parts of fusion proteins characteristic of morphological variants of acute myeloid leukemia. Normal functions of ETO homologues have as yet not been examined. The goal of this work was to identify structural and functional promoter elements upstream of the coding sequence of the ETO gene in order to explore lineage-specific hematopoietic expression and get hints to function.RESULTS: A putative proximal ETO promoter was identified within 411 bp upstream of the transcription start site. Strong ETO promoter activity was specifically observed upon transfection of a promoter reporter construct into erythroid/megakaryocytic cells, which have endogeneous ETO gene activity. An evolutionary conserved region of 228 bp revealed potential cis-elements involved in transcription of ETO. Disruption of the evolutionary conserved GATA -636 consensus binding site repressed transactivation and disruption of the ETS1 -705 consensus binding site enhanced activity of the ETO promoter. The promoter was stimulated by overexpression of GATA-1 into erythroid/megakaryocytic cells. Electrophoretic mobility shift assay with erythroid/megakaryocytic cells showed specific binding of GATA-1 to the GATA -636 site. Furthermore, results from chromatin immunoprecipitation showed GATA-1 binding in vivo to the conserved region of the ETO promoter containing the -636 site. The results suggest that the GATA -636 site may have a role in activation of the ETO gene activity in cells with erythroid/megakaryocytic potential. Leukemia associated AML1-ETO strongly suppressed an ETO promoter reporter in erythroid/megakaryocytic cells.CONCLUSIONS: We demonstrate that the GATA-1 transcription factor binds and transactivates the ETO proximal promoter in an erythroid/megakaryocytic-specific manner. Thus, trans-acting factors that are essential in erythroid/megakaryocytic differentiation govern ETO expression.
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| 3. |
- Ajore, Ram, et al.
(författare)
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The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells
- 2012
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 13:11
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Tidskriftsartikel (refereegranskat)abstract
- Background: MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis. Results: 5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA-and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16/MTGR1 promoters by the leukemia associated AML1-ETO fusion gene may have a role in hematopoietic dysfunction of leukemia. Conclusions: An evolutionary conserved GATA binding site is critical in transcriptional regulation of the MTG16 promoter. In contrast, the MTGR1 gene depends on a GC-box-rich sequence for transcriptional regulation and possible ubiquitous expression. Our results demonstrate that the ETO homologue promoters are regulated differently consistent with hematopoietic cell-type-specific expression and function.
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| 4. |
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| 5. |
- Dhanda, R. S., et al.
(författare)
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The human SIN3B corepressor forms a nucleolar complex with leukemia-associated ETO homologues
- 2008
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 9, s. 1-17
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Tidskriftsartikel (refereegranskat)abstract
- Background: SIN3 ( SWI-Independent) is part of a transcriptional deacetylase complex, which generally mediates the formation of repressive chromatin. The purpose of this work was to study possible interactions between corepressors human SIN3B (hSIN3B) and the ETO homologues ETO ( eight twenty-one), MTG16 ( myeloid-transforming gene 16) and MTGR1 ( MTG-related protein 1). In addition, the subnuclear localization of the hSIN3B and the ETO homologues was also examined. Results: A ubiquitous expression of hSIN3B was observed in adult and fetal tissues. Results with both ectopically expressed proteins in COS-7 cells and endogeneous proteins in the K562 human erytholeukemia cell line demonstrated interactions between hSIN3B and ETO or MTG16 but not MTGR1. Furthermore, nuclear extract of primary placental cells showed complexes between hSIN3B and ETO. The interaction between hSIN3B and ETO required an intact amino-terminus of ETO and the NHR2 domain. A nucleolar localization of hSIN3B and all the ETO homologues was demonstrated upon overexpression in COS-7 cells, and confirmed for the endogeneously expressed proteins in K562 cells. However, hSIN3B did not colocalize or interact with the leukemia-associated AML1 -ETO. Conclusion: Our data from protein-protein interactions and immunolocalization experiments support that hSIN3B is a potential member of a corepressor complex involving selective ETO homologues.
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| 6. |
- Forsare, Carina, et al.
(författare)
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RNA quality in frozen breast cancer samples and the influence on gene expression analysis - a comparison of three evaluation methods using microcapillary electrophoresis traces
- 2007
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Background: Assessing RNA quality is essential for gene expression analysis, as the inclusion of degraded samples may influence the interpretation of expression levels in relation to biological and/ or clinical parameters. RNA quality can be analyzed by agarose gel electrophoresis, UV spectrophotometer, or microcapillary electrophoresis traces, and can furthermore be evaluated using different methods. No generally accepted recommendations exist for which technique or evaluation method is the best choice. The aim of the present study was to use microcapillary electrophoresis traces from the Bioanalyzer to compare three methods for evaluating RNA quality in 24 fresh frozen invasive breast cancer tissues: 1) Manual method = subjective evaluation of the electropherogram, 2) Ratio Method = the ratio between the 28S and 18S peaks, and 3) RNA integrity number (RIN) method = objective evaluation of the electropherogram. The results were also related to gene expression profiling analyses using 27K oligonucleotide microarrays, unsupervised hierarchical clustering analysis and ontological mapping. Results: Comparing the methods pair-wise, Manual vs. Ratio showed concordance (good vs. degraded RNA) in 20/ 24, Manual vs. RIN in 23/ 24, and Ratio vs. RIN in 21/ 24 samples. All three methods were concordant in 20/ 24 samples. The comparison between RNA quality and gene expression analysis showed that pieces from the same tumor and with good RNA quality clustered together in most cases, whereas those with poor quality often clustered apart. The number of samples clustering in an unexpected manner was lower for the Manual (n = 1) and RIN methods (n = 2) as compared to the Ratio method (n = 5). Assigning the data into two groups, RIN = 6 or RIN < 6, all but one of the top ten differentially expressed genes showed decreased expression in the latter group; i. e. when the RNA became degraded. Ontological mapping using GoMiner (p = 0.05; = 3 genes changed) revealed deoxyribonuclease activity, collagen, regulation of cell adhesion, cytosolic ribosome, and NADH dehydrogenase activity, to be the five categories most affected by RNA quality. Conclusion: The results indicate that the Manual and RIN methods are superior to the Ratio method for evaluating RNA quality in fresh frozen breast cancer tissues. The objective measurement when using the RIN method is an advantage. Furthermore, the inclusion of samples with degraded RNA may profoundly affect gene expression levels.
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| 7. |
- Lamba, Pankaj, et al.
(författare)
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Novel forms of Paired-like homeodomain transcription factor 2 (PITX2): Generation by alternative translation initiation and mRNA splicing
- 2008
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 9:31
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Tidskriftsartikel (refereegranskat)abstract
- Background: Members of the Paired-like homeodomain transcription factor ( PITX) gene family, particularly PITX1 and PITX2, play important roles in normal development and in differentiated cell functions. Three major isoforms of PITX2 were previously reported to be produced through both alternative mRNA splicing (PITX2A and PITX2B) and alternative promoter usage (PITX2C). The proteins derived from these mRNAs contain identical homeodomain and carboxyl termini. Differences in the amino-termini of the proteins may confer functional differences in some contexts. Results: Here, we report the identification of two novel PITX2 isoforms. First, we demonstrate that the Pitx2c mRNA generates two protein products, PITX2C alpha and PITX2C beta, via alternative translation initiation. Second, we identified a novel mRNA splice variant, Pitx2b2, which uses the same 5' splice donor in intron 2 as Pitx2b (hereafter referred to as Pitx2b1), but employs an alternative 3' splice acceptor, leading to an in-frame deletion of 39 base pairs relative to Pitx2b1. Pitx2b2 mRNA is expressed in both murine and human pituitary. The data show that in a murine gonadotrope cell line and adult murine pituitary what was previously thought to be PITX2B1 is actually PITX2C beta, or perhaps PITX2B2. PITX2B1 is expressed at lower levels than previously thought. PITX2C beta and PITX2B2 activate gonadotrope-specific gene promoter-reporters similarly to known PITX2 isoforms. Conclusion: We have identified and characterized two novel isoforms of PITX2, generated by alternative translation initiation (PITX2C beta) and alternative mRNA splicing (PITX2B2). These proteins show similar DNA binding and trans-activation functions as other PITX2 isoforms in vitro, though their conservation across species suggests that they may play distinct, as yet unidentified, roles in vivo.
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| 8. |
- Andersen, Malin, 1977-, et al.
(författare)
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Alternative promoter usage of the membrane glycoprotein CD36
- 2006
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 7, s. 8-
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Tidskriftsartikel (refereegranskat)abstract
- Background: CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation.Results: We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters.Conclusion: Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.
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| 9. |
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| 10. |
- Cheung, Louisa, et al.
(författare)
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Hormonal and nutritional regulation of alternative CD36 transcripts in rat liver : a role for growth hormone in alternative exon usage
- 2007
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Ingår i: BMC Molecular Biology. - : Springer Science and Business Media LLC. - 1471-2199. ; 8:60, s. 12-
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Tidskriftsartikel (refereegranskat)abstract
- Background: CD36 is a multiligand receptor involved in various metabolic pathways, including cellular uptake of long-chain fatty acids. Defect function or expression of CD36 can result in dyslipidemia or insulin resistance. We have previously shown that CD36 expression is female-predominant in rat liver. In the present study, hormonal and nutritional regulation of hepatic CD36 expression was examined in male and female rats. Since alternative transcription start sites have been described in murine and human Cd36, we investigated whether alternative CD36 transcripts are differentially regulated in rat liver during these conditions.Results: Sequence information of the rat Cd36 5'-UTR was extended, showing that the gene structure of Cd36 in rat is similar to that previously described in mouse with at least two alternative first exons. The rat Cd36 exon 1a promoter was sequenced and found to be highly similar to murine and human Cd36. We show that alternative first exon usage is involved in the female-predominant expression of CD36 in rat liver and during certain hormonal states that induce CD36 mRNA abundance. Estrogen treatment or continuous infusion of growth hormone (GH) in male rats induced CD36 expression preferentially through the exon 1a promoter. Old age was associated with increased CD36 expression in male rats, albeit without any preferential first exon usage. Intermittent GH treatment in old male rats reversed this effect. Mild starvation (12 hours without food) reduced CD36 expression in female liver, whereas its expression was increased in skeletal muscle.Conclusion: The results obtained in this study confirm and extend our previous observation that GH is an important regulator of hepatic CD36, and depending on the mode of treatment (continuous or intermittent) the gene might be either induced or repressed. We suggest that the effects of continuous GH secretion in females (which is stimulatory) and intermittent GH secretion in males (which is inhibitory) explains the sex-different expression of this gene. Furthermore, a female-specific repression of hepatic CD36 in response to food deprivation was found, which was in contrast to a stimulatory effect in skeletal muscle. This demonstrates a tissue-specific regulation of Cd36.
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