Inverse PPARβ/δ Agonisten: Rekrutierung von Co-Repressoren und Mechanismen der transkriptionellen Repression

Der Peroxisomen-Proliferator-aktivierte Rezeptor β/δ (PPARβ/δ) bindet als Klasse II Kernrezeptor mit seinem obligatorischen Heterodimerisierungspartner RXR an PPAR-responsive Elemente (PPREs) der DNA. Im unligierten Zustand übt PPARβ/δ durch die Rekrutierung von NCoR/SMRT-Komplexen eine basale Repre...

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Bibliographic Details
Main Author: Legrand, Nathalie
Contributors: Adhikary, Till (PD Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
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The class II nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ) binds with its obligate heterodimerization partner RXR to PPAR responsive elements (PPREs) of the DNA. In the unliganded state, PPARβ/δ exerts basal repression on the transcription of its canonical target genes by recruiting NCoR/SMRT complexes. Binding of an agonist leads to the dissociation of these co-repressors and allows for co-activator recruitment, which results in activation of transcription. On the other hand, synthetic inverse agonists provoke reinforced repression, which prevents transcriptional induction of the target gene ANGPTL4 by different stimuli in a dominant manner. TGFβ mediated induction of ANGPTL4 transcription is a central promoting step in metastasis of breast cancer cells. Based on their ability to suppress this transcriptional activation of ANGPTL4, PPARβ/δ inverse agonists are promising candidates for the development of antineoplastic agents. The underlying mechanism of repression was largely unknown, and experiments of our group initially indicated, that the dominant repressive effect is not mediated by NCoR/SMRT complexes and the deacetylase activity of its HDAC3 subunit. The present thesis addresses the elucidation of the mechanism of transcriptional repression by PPARβ/δ inverse agonists. ANGPTL4 regulation by PPARβ/δ ligands is particularly strong, and this gene was therefore used as model locus. ChIP-qPCR experiments showed that the PPARβ/δ inverse agonist PT-S264 impairs transcription at the initiation or reinitiation step, respectively. Diminished recruitment of activating subunits of the Mediator complex (MED1, MED26 and MED13L) as well as the general transcription factor (GTF) TFIIB and RNA polymerase II to the ANGPTL4 promotor was detected. In contrast, recruitment of other GTFs such as TFIIA was not affected. It is known from literature, that the Mediator complex supports recruitment of TFIIB and that both, Mediator and TFIIB, are necessary for the recruitment of RNA polymerase II to the preinitiation complex. Therefore, the impaired recruitment of activating Mediator subunits (or the entire Mediator complex) could be causative for the impairment of TFIIB and RNA polymerase II recruitment. To identify co-repressors, which are recruited to PPARβ/δ in the presence of inverse agonists, ChIP mass spectrometry analyses were perfomed. Components of NCoR/SMRT complexes were detected as specific interactors upon binding of PT-S264. Among the PT-S264 dependent interactors no other known co-repressors were identified. The increased recruitment of NCoR/ SMRT complex components by PPARβ/δ upon binding of the inverse agonist was validated in ChIP-qPCR experiments. To obtain further insights into transcriptional regulation by PPARβ/δ and particularly the binding of co-repressors recruited in the presence of PT-S264, a functional screen of 80 PPARβ/δ mutants was performed. MDA-MB-231-luc2 PPARD KO cells were retrovirally transduced with mutant PPARD cDNAs and tested for an impairment of transcriptional regulation of the target gene ANGPTL4. Besides reinforced repression by the PPARβ/δ inverse agonist PT-S264, basal repression in the absence of synthetic ligands and activation by the PPARβ/δ agonist L165,041 were analysed. Several mutants identified in the functional screen showed a loss of basal repression and were of value for further investigation of the mechanisms. Compared to cells transduced with wildtype PPARβ/δ, reduced presence of NCoR/SMRT complex components at the PPREs of PPARβ/δ target genes was detected in the basal state, which was accompanied by increased recruitment of RNA polymerase II at their promoters. In cells expressing the mutants, recruitment of NCoR/SMRT complex components and the resulting diminished recruitment of RNA polymerase II to the promoter was restored by the PPARβ/δ inverse agonist PT-S264. These findings suggest that both basal repression by unliganded PPARβ/δ and repression by the PPARβ/δ inverse agonist are mediated by NCoR/SMRT complexes. The role of the HDAC3 subunit of NCoR/SMRT complexes in repression was tested by the use of deacetylase inhibitors. Basal repression as well as repression by the inverse agonist were largely insensitive to inhibition of deacetylase activity. Taken together, the results of the present thesis indicate that NCoR/SMRT complexes mediate the transcriptional repression by PPARβ/δ inverse agonists by both deacetylase-dependent and deacetylase-independent mechanisms. Prevention of recruitment of activating Mediator sub-units is probably causative for diminished TFIIB and RNA polymerase II recruitment to the transcription start site. Consequently, transcription is impaired at the initiation or reinitiation step. In conclusion, two hypotheses were proposed, which raise interesting questions for future approaches: I. NCoR and/ or SMRT prevent the formation of an active initiation complex by direct inhibition of acetyltransferase activity or through other mechanisms which do not rely on deacetylase activity. II. NCoR and/ or SMRT prevent contacts between regulatory elements and the promotor by regulating chromatin interactions. Given the stabilization of cohesin complexes by acetylation, this could be directly linked to hypothesis I.