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PIH1 mediated SNF5 activate ribosomal RNA gene transcription regulation mechanism

Author: DiZuo
Tutor: ZhangYe；Shen
School: Peking Union Medical College , China
Course: Biochemistry and Molecular Biology
Keywords: RNA Histone Ribosomal PIH1 SNF5 Reporter gene Co-immunoprecipitation rDNA Chromatin remodeling Luciferase Promoter region Protein interactions Chromatin conformation Transcriptional activation Subunit Beijing Union Medical College Plasmid Transcriptional repressor Domain Regulatory mechanism
CLC: Q75
Type: PhD thesis
Year: 2010
Downloads: 105
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Abstract

Modification of its structure of chromatin remodeling is more than one molecule involved in multi-channel synergy, highly complex and orderly completion of the process. They are also eukaryotic the nucleus gene initiator in the internal and external environment under the influence of the most important events and gene transcription initiation the premise. Chromatin remodeling complexes depends on the ATP provide energy thus changing the state of the protein and DNA binding, according ATP enzyme subunit can be divided into four categories: SWI2/SNF2, ISWI, Mi-2/CHD INO80/SWR1 . Human SWI / SNF complex associated with RNA polymerase II, in addition to the ATP enzyme subunit BRG1 or BRM, 1N11/SNF5 is also one of the core subunits one. Nucleolar RNA polymerase I transcription to suppress the NoRC complex is dependent on the end of the ATP and histone H4 ISWI chromatin remodeling complex. In mammalian cells, the formation of the ribosome is a process of biosynthesis and energy consumption. Ribosome biosynthesis and cell in which energy environment and sugar is closely related to, ribosome synthesis and rRNA transcription inseparable, the rRNA transcription synthesis and energy metabolism and environmental changes. The SNF5 ATP-dependent chromatin remodeling complex SWI / SNF core subunits, one. SNF5 is considered to be a tumor suppressor gene, SNF5, mutations or deletions, can lead to early embryonic lethality induced tumors, and have found that in some tumor SNF5 not expressed, or mutations, such as MRT, AT / RT [1-7 Nobody SNF5 rDNA transcription related. The findings of our group PIH1 SNF5, binding protein, in yeast PIH1 congeners NOP17 involved in the pre-rRNA of shear [8,9]. Chromatin localized, reversible changes in gene selective expression plays a key role, and is one of today epigenetic regulation mechanism of the core content. The thesis of ribosomal RNA (rDNA) gene as a model to study the SNF5 induced by high glucose conditions, the rDNA transcriptional activation mechanism, which is mainly involved in histone acetylation and ATP-dependent chromatin remodeling complexes the regulation. By in vitro GST-pulldown experiments in vivo immune coprecipitation experiments and immunofluorescence (CoIP) interaction experiments confirmed SNF5 and PIH1, and find both the specific binding site. Through the the Northern organizations membrane hybridization to determine the wide distribution PIH1 organization. Luciferase dual-reporter gene and real-time RT-PCR detection SNF5 rDNA transcriptional activation; chromatin co-immunoprecipitation (ChIP) detection SNF5 the rDNA promoter District histone modification impact. Overexpression of SNF5 enhanced the rDNA promoter reporter gene activity and expression levels of pre-rRNA, also increased the level of histone acetylation, reduce histone methylation levels; contrary, knockdown the SNF5 reduce rDNA promoter reporter gene activity pre-rRNA expression, and also reduce the level of histone acetylation, increase the level of histone methylation. Luciferase dual-reporter gene and real-time RT-PCR detection system and chromatin immune co-precipitation (ChIP) technology found PIH1 and SNF5 have the same functionality. Further found that, to delete the binding site with the PIH1 the SNF5 deleted cut body did not affect expression of rDNA transcription;, SNF5 raised rDNA promoter region was significantly reduced under the conditions of low PIH1 knock; Furthermore, in the high glucose-induced the rDNA transcription process, SNF5 necessary raised by PIH1 rDNA promoter region, play a bridge role. The rDNA promoter area isobutyl chromatin state maintained, dependent on the binding of the subunit of Tip5 NoRC bromo domain and acetylated histone H4K16. By ChIP experiments found, PIH1 Tip5 competition is bound to histone H4K16, thereby activating rDNA transcription. Mass and in vitro binding experiments show, PIH1 and histone H4-specific binding, and H4K16 is a combination of critical parts. At the same time, we also found PIH1 pol I transcription activation factor UBF and influence in the rDNA promoter region raise, so SNF5 may activate transcription by UBF. Induced by high glucose can promote cell proliferation, the rDNA transcription significantly increased in this process. By real-time RT-PCR and ChIP experiments show SNF5 induced by high glucose to raise the amount of the rDNA promoter region increased significantly; SNF5 knockdown weaken rDNA transcription; the SNF5 role relies on PIH1 histone H4 combination. This thesis, SNF5 activate the PIH1 dependent rDNA the transcription, PIH1 may play a key role in cell proliferation competitive binding with Tip5 histone H4K16 suppression NoRC raising, thereby activating rDNA transcription. About SNF5 a lot of research, but its mechanism of action is not clear. Our research found that the SNF5 you can activate rDNA transcription and activation mechanisms rely on the PIH1 with Tip5 competitive binding histone H4K16.

PIH1 mediated SNF5 activate ribosomal RNA gene transcription regulation mechanism

Abstract

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