Project description:Transcription of the >200 rRNA genes (rDNA) by RNA Polymerase I (RPI) determines as much as 35% of total nuclear RNA synthesis and is a major determinant of cell growth implicated in a range of hypertrophic and developmental disorders. Activation of the rDNA involves the formation of an extended nucleosome free region (NFR) by the multi-HMGbox factor UBTF, which is also implicated with the RPI specific TBP-TAFI factor SL1 in preinitiation complex formation. However, neither factor alone displays significant DNA sequence binding specificity. Here we show that in cell cooperation between SL1 and the UBTF1 splice variant creates the sequence specificity required for promoter recognition. While both UBTF1 and UBTF2 splice variants bind throughout the rDNA NFR, only UBTF1 binds at the rDNA promoters. Conditional deletion of the Taf1b subunit of SL1 depleted UBTF1 from the rDNA promoters but not from elsewhere across the rDNA NFR. We show RPI promoters are particularly poor binding sites for UBTF and suggest an induced-fit model in which promoter-specific remodelling by UBTF1 creates high affinity sites for SL1 binding. A mouse model of the UBTF-E210K pediatric neurodegeneration syndrome suggests this mutation affects cooperativity of UBTF-SL1 promoter recruitment and further supports the induced-fit model.
Project description:4C-rDNA procedure was used for analysis of genomic contacts of rDNA units in hESM01 cells. The primers for 4C were selected downstream from EcoRI site at coordinate 30487 in rDNA sequence with Accession number U13369.1.
