The. Pih1 Mediated Snf5 Activate The Transcription Of Ribosomal Rna Gene Regulation Mechanism

In the past decade, multiple lines of evidence have shown that the functions of the histone modification enzymes and that of ATP-dependent chromatin remodeling complexes are of critical importance in the derepression of genes in a chromatin environment. The effects of the two classes of enzymes on chromatin are deviating that the former modification enzymes serve to attach or remove key chemical groups of the core histones, while the latter utilize energy of ATP to repost nucleosome on the genomic DNA. There are four groups of chromatin remodeling complexes, SWI2/SNF2,ISWI, Mi-2/CHD,INO80/SWR1. SNF5 is a component of the ATP-dependent chromatin remodeling SWI/SNF complex that changes chromatin state of genes transcribed by Polâ…¡. But NoRC (nucleolar remodeling complex) which is an ISWI/SNF2-containing remodeling complex, maintains rDAN repeats in a closed chromatin state.Ribosome biogenesis plays an important role in growth, and transcription of 45s pre-rRNA by RNA polymeraseâ… (Polâ… ) is the key step for proliferation in most mammalian cells[10,11]. As a tumor suppressor, deletion and mutation analyses of CNS, renal and extrarenal rhabdoid tumors have demonstrated bi-allelic alterations of the SNF5 gene. But the real mechanism is still unknown, and yet nobody noticed that SNF5 related with rDNA transcription.Our previous study discovered that, PIH1, a novel binding protein of SNF5, whose homology protein named NOP17 in saccharomyces cerevisiae is involved in pre-rRNA process. In this study, we discovered that SNF5-mediated transcription activation was not restricted to Polâ…¡but had been shown to play a role in transcription of rRNA genes (rDNA) by Pol I. PIH1, a novel binding protein of SNF5, could be in competition with TIP5 for binding to the N terminal tail of histone H4. So we demonstrate that SNF5 participates in rDNA transcription through its interaction with PIH1. PIH1 was generated by yeast two-hybrid assay, using SNF5 as bait. To demonstrate the direct interaction between PIH1 with SNF5, the in vitro and in vivo binding assays were done. And the binding domain required for the interaction between SNF5 and PIH1 was determined.We next tested the subcellular localization of PIH1 and SNF5 in cells. Tissue distribution of PIH1 transcripts was also examined.We examined pre-rRNA synthesis and the activity of human rDNA promoter in HEK293T cells infected with SNF5 expression vectors or control vectors. Infection of Myc-SNF5 results in an obvious increase of pre-rRNA in cells tested, so does the reporter activity. Conversely, the level of pre-rRNA synthesis and the reporter activity were decreased in cells transfected with small-interfering RNAs against SNF5 (SNF5-siRNA). ChIP assays were carried out with one primer pairs to amplify genome fragments within the rDNA promoter (-47 to +29) (core element of rDNA promoter).ChIP assays with antibody against SNF5 indicated that the occupation of SNF5 in rDNA promoter decreases obviously in PIH1-knockdown cells, and the pre-rRNA level were almost the same in SNF5-knockdown or PIH1-knockdown or SNF5 and PIH1-knockdown cells. These results confirm that PIH1 is required for SNF5 activating poll transcription and SNF5 and PIH1 function in the same way.PIH1 interacts with histone H4 directly, and the acetylation of histone H4K16 increases in PIH1-knockdown cells, and stable in SNF5-knockdown cells. So PIH1 may interact with the N terminus of histone H4 competition with Tip5, especially at the acetylation site of histone H4K16, when knockdown of PIH1, Tip5 occupies more acetylated H4K16 sites, more NoRC were recruited to rDNA promoter to repress rDNA transcription. Indeed, ChIP assays with antibody against SNF2h revealed that the occupancy of SNF2h in rDNA promoter increased apparently in PIH1-knockdown or SNF5-knockdown cells. SNF5 functins through its binding protein PIH1. In addition, our study also indicates that PIH1 interacts with hUBF and influences the occupancy of hUBF in rDNA promoter. So PIH1 or SNF5 may activate rDNA transcription via hUBF.Condition of high glucose results in rDNA transcription activation, but the pre-rRNA levels decreased in either SNF5-knockdown or PIH1-knockdown cells when high glucose induced. High glucose inducing recruited more SNF5 to rDNA promoter, but the occupancy of PIH1 had no variation. Therefore we predicted that PIH1 recruited SNF5 to rDNA promoter. ChIP assays with antibody against SNF5 demonstrated that the occupancy of SNF5 in rDNA promoter decreased apparently in PIH1-knockdown cells, although high glucose induced.In this study, we discovered that SNF5-mediated transcription activation was not restricted to polâ…¡but had been shown to play a role in transcription of rRNA genes (rDNA) by polâ… . PIH1, a novel binding protein of SNF5, could be in competition with TIP5 for binding to the N terminal tail of histone H4. So we demonstrate that SNF5 participates in rDNA transcription through its interaction with PIH1 for the first time.