Ribosomal Rna Gene Transcription Mechanisms In Pih1 Regulation

Ribosome biogenesis is critical for the growth and proliferation of higher eukaryotic cells4,5. Direct transcription of the 45S precursor ribosomal RNA (pre-rRNA) from the activated rRNA genes is the first and rate-limiting step6; and is tightly regulated by metabolic or environmental changes'. It has been reported that the nucleolar chromatin repression complex NoRC plays a silencing role on the rRNA genes2. NoRC is composed of two subunits, the larger subunit of a TTF-I interacting protein2 (TIP5) that recognizes acetylated lysine 16 in histone H4 tail, and the SNF2h, an ATPase subunit of the ISWI chromatin remodeling complex family that conducts an ATP dependent nucleosome repositioning at the rRNA promoters8. Recent data have shown that the pRNA (Promoter-associated RNA) transcribed from the intergenic sequences of rRNA genes by RNA polymerase I (Pol I)9 and processed into<2000 nts is stabilized via binding to TIP5 and is a determinant of NoRC function in heterochromatin formation and rRNA silencing6. In contrast to the above, the findings of that TTF-I binds a mammalian TO element in the upstream of the UCE (upstream control element) of rRNA promoter10 that stimulates Pol I transcription in vivo 11,12 and in vitro 13,14; and those of the upstream binding factor (UBF) at the upstream control element (UCE), and the Cockayne Syndrome protein B (CSB) and nuclear myosine 1 (NM1) that are all known as activators for RNA polymerase I transcription15,16, however, the chromatin remodeling mechanisms involved in the activation of rRNA genes are still obscure.Human SNF5, a homolog of yeast SNF5, was first identified as an interacting protein of the ATPase subunits (hBrm and Brgl) of the SWI/SNF chromatin remodeling complex17-19. Using SNF5 as bait, we demonstrated earlier that SNF5 interacts with a 290-amino-acid hypothetical human protein in a yeast two-hybrid system20. This SNF5-interacting protein was later identified as the human ortholog of yeast nucleolar protein NOP17 and designated as PIH1-domain-containing protein 1, or PIH1D1 (abbreviated here as PIH1)21. As the protein identity between yeast PIH1 and its putative human orthologue was low (<30%) 21, it is thus intriguing to explore how human PIH1 functions, along with SNF5 in the ribosome biogenesis in human cells.Here, we demonstrate that PIH1 not only directly interacts with SNF5, a core subunit of the ATP-dependent SWI/SNF chromatin remodeling complexes17,19,but also specifically binds the N-terminal tail of histone H4 likely via. K16. The PIH1/H4 binding leads to the recruitment of SNF5 and then the ATPase subunit Brgl to release energy from ATP and remodel chromatin conformation associated with the rRNA promoter and facilitates pre-rRNA synthesis. This event also occurred under increased glucose concentration, in which, PIH1 may help relieving the cells from silencing via competing the TIP5 subunit of the NoRC at the acetylated K16 of H422.23. This is the first example regarding the BAF-SWI/SNF chromatin remodeling complex that functions in complex with PIH1 in the activation of pre-rRNA synthesis in human cells.Our results indicate that SNF5, a core subunit of the ATP-dependent SWI/SNF chromatin remodeling complex specifically binds a PIH1 domain protein (abbreviated as PIH1) in human cells. PIH1 preferentially recognizes Lys 16 of histone H4 (H4K16) in its native form at the promoter of rDNA. SNF5 further recruits an ATPase subunit Brgl to the site to generate an open chromatin conformation that is PIH1/H4K16 dependent. Additionally PIH1 also competes with the nucleolar silencing complex NoRC at the acetylated H4K16 to exclude NoRC from the site and facilitates Brgl-induced activation of the rRNA genes. A point mutation in the N-terminal domain of PIH1 abolished its H4 binding and prohibited the pre-rRNA synthesis. The key role of PIH1 is demonstrated here to be the H4K16 "reader" that determines the recruitment of SNF5 and Brgl to elicit a chromatin-remodeling environment in the rDNA promoter and activate pre-rRNA transcription. This is the first example of the requirement for PIH1 to act in concert with SNF5 and Brg1 in their critical role to initiate ribosome biogenesis in a histone-dependent manner. As the environmental nutrition conditions tightly regulate the pre-rRNA transcription, the findings here are of great importance in the recovery of human cells from nutrient starvation to glucose induced exponential growth in vivo. In eukaryotes, chromatin is the natural form of DNA in the nucleus. In the eukaryotic nucleus. DNA is packaged into chromatin.The basic unit of chromatin, the nucleosome core particle, consists of 146 bp of DNA wrapped 1.7 times around a core histone octamer that contains two copies each of histones H2A,H2B, H3, and H447. Chromatin is intimately involved in many cellular processes such as transcription, replication, repair,and recombination48-50.Hence, for these and other chromatin-utilizing processes, it is important to investigate how the structure of chromatin affects its function. To this end, it is necessary to reconstitute DNA and histones into chromatin in vitro.The recombinant ACF system developed by Kadonaga and co-workers51 employs purified histones, recombinant assembly factors (ATP-dependent chromatin remodeling factors and histone chaperones-Acfl, ISWI, and NAP1) and DNA.Here we purified these factors, assembled of chromatin templates in vitro, transcribed of the DNA templates. This part involves:(1) assembly of chromatin using the recombinant ACF-based system described and recombinant histones expressed and (2) incubation with nuclear extract and nucleoside triphosphates to allow preinitiation complex assembly and function (transcription).