Tremblay, M. G. | Search bioRxiv

bioRxiv2022

Tissue-Specific Control of Ribosomal RNA Synthesis Revealed by Transcription Factor Profiling

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA Polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2200 ChIP-Seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA, and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of CEBPA (C/EBP alpha), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced Pol I occupancy. Our work identifies numerous potential rRNA regulators, and provides a template for dissection of TF roles in rRNA transcription. HIGHLIGHTSO_LIMultiple cell-type-specific transcription factors (TFs) bind canonical motifs on rDNA. C_LIO_LIThe hematopoietic TF CEBPA binds to active rDNA alleles at a conserved site. C_LIO_LICEBPA promotes Polymerase I occupancy and rRNA transcription in myeloid progenitors. C_LIO_LIWe present 47S-FISH-Flow, a sensitive assay to quantify nascent rRNA. C_LI

molecular biology↗

bioRxiv2021

Bidirectional cooperation between Ubtf1 and SL1 determines RNA Polymerase I promoter recognition in cell and is negatively affected in the UBTF-E210K neuroregression syndrome.

Transcription of the [~]200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the Taf1b subunit of SL1 causes a striking depletion UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

molecular biology↗