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Control of Ribosomal RNA Transcription by Hematopoietic Transcription Factors
Vikram R Paralkar, MD1
1 University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Background: The hematopoietic tree is composed of cells of varying sizes, functions, proliferation kinetics, and protein translation rates. Each cell type has a tightly regulated number of ribosomes, fine-tuned to meet cell-type-specific needs. The biogenesis of ribosome occurs in the nucleolus, where hundreds of rDNA repeats are simultaneously transcribed by Polymerase I (Pol I) into rRNA, the most abundant RNA in the cell. rRNA transcription accounts for the bulk of all transcription, especially in leukemic blasts, which have characteristically prominent nucleoli. Though coding genes have been dissected in detail in normal and malignant hematopoiesis, little is known about how rRNA, the most abundant RNA in the cell, is regulated. There are multiple reasons for this knowledge gap, including the historical belief that ribosome biogenesis is a "housekeeping process", and the absence of bioinformatic pipelines capable of mapping high-throughput datasets to rDNA. Consequently, rRNA has fallen within a significant blind spot in transcription studies.
Aims: We aimed to identify regulators of rRNA transcription in hematopoiesis and leukemia, with the goal of gaining insight into the principles underlying cell-type-specific control of ribosome biogenesis.
Methods: We generated customized human and mouse genome assemblies optimized for rDNA mapping with the addition of an extra “chromosome R”. We used these custom genomes to map ~2200 publicly-available human and mouse ChIP-Seq datasets encompassing 250 hematopoietic transcription factors (TFs) and chromatin factors to generate a TF-rDNA Atlas. We also developed a "47S-FISH-Flow" assay to quantify nascent rRNA on a per-cell basis. In addition, we used a degron system to degrade the hematopoietic transcription factor CEBPA to assess its role in Pol I occupancy and rRNA transcription.
Results: Through our TF-rDNA Atlas, we identified multiple distinct patterns of ChIP-Seq peaks on rDNA. We observed that several TF families such as CEBP, IRF, SPI1, RUNX families show rDNA binding patterns to conserved and previously-unrecognized motif sequences. We picked CEBPA, a critical hematopoietic and leukemic TF, for further study. Our atlas showed high-confidence CEBPA binding to human and mouse rDNA at a conserved, canonical motif sequence. We used the mouse HoxA9-ER AML cell line to dissect the context-specific role of CEBPA on rDNA, and tagged CEBPA alleles with the FKBPV degron domain, allowing us to rapidly degrade endogenous CEBPA protein on addition of dTag ligand. We then used 47S-FISH-Flow to precisely quantify nascent 47S rRNA using fluorescent in-situ hybridization (FISH) probes. We observed that degradation of CEBPA led to reduction of 47S rRNA levels, reduction in nucleolar size, reduction in mature ribosome subunit abundance, accumulation of cells in G1, and reduced growth. Timecourse ChIP-Seq showed that CEBPA degradation was followed within hours by reduction in binding of Pol I and its initiation factor partner RRN3 to rDNA, without any effect on occupancy of upstream factors such as UBTF and the SL-1 complex.
Conclusion: Numerous hematopoietic and leukemic TFs bind to rDNA at conserved motif sequences, indicating that, far from being a housekeeping process, rRNA transcription is likely regulated in a combinatorial and context-specific manner. CEBPA binds rDNA at a conserved motif sequence, and is required for optimal Pol I occupancy and rRNA transcription in myeloid and leukemic cells. Our results shed light on an important and under-explored aspect of transcriptional biology: the cell-type-specific regulation of rRNA transcription in a complex organ system like the hematopoietic tree.
This research was funded by grant R35-GM13803 from the NIGMS (NIH), R01-HL155144 from the NHLBI (NIH), and an American Society of Hematology Faculty Scholar Award.