Untersuchungen zur Regulation der 5-Lipoxygenase-Gen-Expression durch DNA-Methylierung und Histon-Deacetylierung/Acetylierung

5-LO is the key enzyme in the biosynthesis of proinflammatory leukotrienes, converting arachidonic acid to 5-HPETE, and in a second step 5-HPETE to leukotriene A4. Although the 5-LO promoter possesses characteristics of so called housekeeping genes, such as lack of TATA/CCAAT boxes and existence of several Sp1 binding sites, the 5 -LO gene is tissue specifically expressed in primarily immune competent cells of myeloid origin including granulocytes, monocytes, macrophages, mast cells and B-lymphocytes. 5-LO gene expression in MM6 and HL-60 cells is strongly induced after differentiation of the cells with TGF-beta and 1,25(OH)2D3. In some monocytic cancer cell lines, such as HL-60 TB and U937, TGF-beta and 1,25(OH)2D3 treatment are not able to activate 5-LO gene transcription. It was demonstrated, that in these cell lines the 5-LO core promoter is heavily methylated and that only demethylation by the DNA methyltransferase inhibitor 5-aza-2 deoxycytidine (Adc) upregulated the 5-LO mRNA levels. It was also shown that the histone deacetylase inhibitor TsA could induce 5-LO mRNA levels, but only in 1,25(OH)2D3/TGF-beta inducible MM6 cells. Interestingly the 1,25(OH)2D3/TGF-beta effect on 5-LO expression is reduced, when combined with TsA. Reporter gene assays revealed that 5-LO promoter activity is strongly induced after 24 h treatment with 330 nM TsA (construct N10 up to 35 fold in HeLa cells). The effect is dependent on the presence of the proximal Sp1 binding site GC4 (-53 bp to –48 bp in relation to the major TIS) in both HeLa and MM6 cells. In vitro binding of the transcription factor Sp1 to this site has been demonstrated in gel shift assays and DNase I footprints. Mutation of the binding site resulted in a loss of basal promoter activity in both 5-LO negative HeLa cells and in 5-LO positive MM6 cells, as well as in the loss of TsA inducibility. The mutational study of different Sp1 binding sites in a larger promoter context revealed the interaction or respectively the additive effect of the multiple Sp1 binding sites of the 5-LO promoter on basal as well as on TsA upregulated promoter activity. However, GC4 seems to be of special relevance for both the basal promoter activity, possibly recruiting the basal transcription machinery, as well as for the TsA induced upregulation of 5-LO promoter activity. TsA does not alter the protein expression levels of Sp1 and Sp3 as investigated in Western blot analysis, neither in HeLa nor in MM6 cells. DNA affinity purification assays revealed that TsA had no effect on the DNA affinity of Sp1 or Sp3. In vitro binding of both Sp1 and Sp3 to the 5-fold GC box, GC4 and GC5 was demonstrated by DAPA analysis, but histone deacetylase inhibition did not change the associated protein amounts. Finally, in vivo binding of Sp1 and Sp3 was investigated in chromatin immunoprecipitation assay (ChIP) in MM6 cells. TsA clearly induced the association of both proteins to the promoter area surrounding the TIS. Upon TsA treatment also RNA polymerase II binding to the area surrounding the TIS (-318 to +52 bp) was increased and even initiated in the more distal promoter parts –1049 to –292 bp, which are negatively regulated in reporter gene assays. Interestingly histone H4 is already highly acetylated without TsA treatment and the acetylation status of H4 remains unchanged after histone deacetylase inhibition, indicating an open chromatin structure of the 5-LO gene in MM6 cells. In a cotransfection study with Sp1 and Sp3, the transactivating potential of factors was investigated and in accordance with the ChIP data, Sp1 and Sp3 increased the promoter activity, but only after TsA treatment. In gel shift assays, the influence of DNA methylation on Sp1 binding was investigated. The results indicate different roles for the three proximal promoter sites. Whereas Sp1 binding to the 5-fold GC box and GC4 is impaired by DNA methylation, binding to GC5 is even increased. A cotransfection study with methylated 5-LO promoter constructs and the murine methyl-CpG binding proteins suggest MBD1 involvement in the regulation of the 5-LO promoter. Since in gel shifts Sp1 binding is inhibited by DNA methylation, at least to the 5-fold GC box and the activating element GC4, and similarly the mutation/deletion of the same sites strongly reduces or inhibits promoter activity, it is likely to assume, that the loss of promoter activity after in vitro methylation is in the first place due to impaired Sp1/Sp3 binding. Together the data underline the importance and complexity of Sp1/Sp3 binding to the GC rich sites in the regulation of 5-LO promoter activity in response to the histone deacetylase inhibitor TsA as well as in respect to DNA methylation.Die 5-Lipoxygenase ist das Schlüsselenzym in der Leukotrienbiosynthese. Neben den vielfältigen Regulationswegen auf Enzymebene, gestaltet sich auch die Regulation der 5-LO-Genexpression weit komplexer als zunächst erwartet. Obwohl der 5-LO-Promotor einige Eigenschaften von sogenannten Haushaltsgenen, wie das Fehlen einer TATA- oder CCAAT-Box und das Vorhandensein zahlreicher Sp1-Bindungsstellen aufweist, wird die 5-Lipoxygenase Zelltyp-spezifisch exprimiert und zwar vorwiegend in immunkompetenten Zellen wie Granulozyten, Monozyten, Makrophagen, Mastzellen und B-Lymphozyten. Ziel der vorgelegten Arbeit war es, die Regulation des 5-LO-Promotors durch epigenetische Mechanismen, wie der DNA-Methylierung genauer zu charakterisieren und zu untersuchen, wie der Histondeacetylase-Inhibitor Trichostatin A die Transkription der 5-LO steigert. Im Reportergen-Assay zeigte sich, dass die durch TsA vermittelte Steigerung der Aktivität des 5-LO-Promotors insbesondere von der Präsenz der proximalen Sp1-Bindungsstellen GC4, GC5 und einer 5-fach GC-Box abhängt, und dass diese Bindungsstellen für die basale 5-LO-Promotoraktivität essentiell sind. TsA steigert nicht die Proteinexpression der beiden GC Box bindenden Transkriptionsfaktoren Sp1 und Sp3. Im DAPA (DNA Affinity and Purification Assay) konnte auch keine verbesserte in vitro Bindung der beiden Faktoren an eine der proximalen Bindungsstellen gezeigt werden. Erst in ChIP Versuchen (Chromatin Immunpräzipitation) wurde eine Steigerung der Bindung von Sp1 und Sp3 im proximalen Promotor gefunden. Hand in Hand geht eine vermehrte Rekrutierung der RNA Polymerase II. Interessanterweise hatte TsA keinen Einfluss auf den Histonacetylierungsstatus von Histon H4. In der untersuchten Zelllinie MM6 liegt H4 bereits ohne TsA Behandlung stark acetyliert vor. TsA zeigte keinen Einfluss auf die Acetylierung von Sp1/Sp3 und es konnte keine Rekrutierung von Histondeacetylasen oder Histonacetyltransferasen nachgewiesen werden. Die in–vitro DNA-Methylierung hemmt die Aktivität von 5-LO-Promotorkonstrukten im Reportergen-Assay. Das Ausmaß der Hemmung korreliert dabei mit der Anzahl der methylierten CpG-Stellen. In Kotransfektionsstudien mit den murinen Methyl-CpG-bindenden Proteinen zeigte sich eine weitere Reduktion der Promotoraktivität durch Mbd1. Allerdings konnte bisher keine direkte Bindung von Mbd1 an den Promotor nachgewiesen werden und durch Mutationen auch keine diskrete Bindungsstelle lokalisiert werden. Im EMSA führt eine DNA-Methylierung der proximalen Sp1 Bindungsstelle GC4 und der 5-fach GC-Box zu einer verschlechterten Bindung von Sp1, was die erniedrigte Promotoraktivität im Reportergen Assay erklärt