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ATP-dependent chromatin remodelers - Analysis of expression patterns and impact on gene regulation
ATP-dependent chromatin remodelers are enzymes which use the energy from ATP hydrolysis to alter the chromatin structure. Thus, they play a key role in the transcriptional control of many important cellular processes such as proliferation,senescence and differentiation.The first part of this study f...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2013
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| Zusammenfassung: | ATP-dependent chromatin remodelers are enzymes which use the energy from ATP hydrolysis to alter the chromatin structure. Thus, they play a key role in the transcriptional control of many important cellular processes such as proliferation,senescence and differentiation.The first part of this study focused on CHD5, a novel chromatin remodeling enzyme. To gain further insight into its expression profile and biological function, polyclonal peptide antisera were, together with a commercially available antiserum, successfully established for use in western blot, immunoprecipitation and immunofluorescence staining. ATP-dependent chromatin remodelers are expressed by very different patterns ranging from ubiquitous expression to an expression restricted to specific cell populations. So far, CHD5 expression was thought to be restricted to neural-related tissues. Here, it was shown for the first time that CHD5 expression is not restricted to neural tissues but additionally expressed in rodent testes. Furthermore, several cell lines were tested for CHD5 expression on transcript and protein level. CHD5 expression was not detected in any of the tested neuroblastoma and glioblastoma cell lines. In neuroblastoma cell lines this was expected due to the suggested function as tumor suppressor. Furthermore, a primary astrocyte culture was established. No CHD5 could be detected in protein lysates from astrocytes but was found in those from murine neural stem cells. This suggests that CHD5 expression in brain is restricted to neural stem cells and probably also to neurons.
In the second part of this thesis, the impact of the chromatin remodelers CHD4 and BRG1 on a TNFα-induced inflammatory response was investigated. BRG1 and CHD4 expression was disrupted by a siRNA-mediated knockdown in HEK293 cells. After stimulations with TNFα for one to four hours, gene induction of typical inflammatory NF-κB target genes was determined by RT-qPCR. BRG1 and CHD4 were both required for the efficient induction of all tested target genes exclusively after one hour but not after four hours of TNFα treatment. Expression of the housekeeping gene ß-actin was unaffected. Surprisingly, although CHD4 is mainly known to be involved in transcriptional repression, a requirement for CHD4 in active transcription during this inflammatory response was revealed. To gain further insight into the mechanism of involvement, co-immunoprecipitations of the NF-κB subunit p65 with BRG1 and CHD4,respectively, were performed upon TNFα stimulation. No robust interactions between these proteins could be observed. Furthermore, ChIP experiments with the NF-κB subunit p50, histone H3 and CHD4 were carried out on the model target gene CXCL2. p50 was recruited to the promoter of CXCL2 rapidly after TNFα was added. Histone H3 binding to the promoter and the open reading frame of the gene strongly decreased within the first hour of TNFα stimulation, indicating that chromatin remodeling took place during the early period of gene induction. Recruitment of CHD4 was nonconclusive. Thus, the mechanism of how BRG1 and CHD4 act during TNFα response needs to be investigated further.
In summary, a major influence of the chromatin remodelers BRG1 and CHD4 on the TNFα-induced gene activation during its early phase in HEK293 cells was demonstrated. |
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| DOI: | 10.17192/z2013.0131 |