CEBPA Regulates Molecular Kinetics of RNA Polymerase I in Myeloid Progenitors
Authors: Antony Charles , George Subin S , Blum Justin , Somers Patrick , Wu-Corts Dexter , Adhikari Santosh , Pimkin Maxim , Mir Mustafa , Paralkar Vikram R 
Published: December 2024

Ribosomal RNA (rRNA) is the most abundant cellular RNA, and its transcription from 300-600 copies of rDNA repeats by RNA polymerase I (Pol I) is the most energy intensive transcriptional process in the cell. Different normal cell types within the hematopoietic tree have different rRNA transcription rates. Leukemic “blast” cells have enlarged nucleoli, indicating abundant rRNA transcription. Thus, there is rRNA transcriptional variation in normal and malignant hematopoiesis, but because ribosome biogenesis is often considered a housekeeping process, little is known about the factors controlling Pol I activity in different cell types.
We previously published the first ever atlas of hematopoietic transcription factor (TF) binding to rDNA, and identified conserved binding of key hematopoietic TF families including CEBP, RUNX, and IRF to rDNA (Antony et al, Molecular Cell 2022). We showed that degradation of the master myeloid TF CEBPA in a mouse myeloid cell line led to rapid reduction (within hours) of Pol I occupancy on rDNA, followed by reduced 47S rRNA levels, ribosome subunit abundance, and growth. Notably, the total level of Pol I in the cell was not changed, indicating a role for CEBPA in Pol I recruitment and loading on rDNA repeats. This would be a novel role for CEBPA, which has historically been extensively studied for its role in Pol II regulation.
To dissect how CEBPA regulates Pol I molecular activity in myeloid progenitors, we utilized a live cell single molecule imaging approach. We fused HaloTag to POLR1A (core catalytic subunit of Pol I) in the mouse ER-HoxA9 myeloid cell line to permit labeling in live cells using cell-permeable fluorescent dye. Though there are tens of thousands of Pol I molecules in a cell, we used very low ligand concentration (5 nM) and sparse photoactivation to allow us to image individual molecules. Imaging at 10 ms/frame speed allowed us to capture Halo-Pol I molecules displaying a range of kinetic behaviors (from free to immobile). Analysis of thousands of compiled tracks showed that Pol I exists in two populations within the nucleus: (i) Slow-moving (rDNA-loaded), and (ii) Fast-moving (not loaded on rDNA). We observed that most (80%) of Pol I tracks reside in 2 to 3 hubs within the nucleus, likely representing nucleoli. Pol molecules residing within these hubs showed a very low diffusion coefficient of ~0.011 um^2/s, in contrast to molecules outside the hub, which had a high diffusion coefficient of 0.1509 um^2/s. We estimate that ~70% of Pol I molecules reside in nucleoli at any given time, likely loaded on rDNA and engaged in rRNA transcription.
To assess the effect of CEBPA loss on Pol I molecular behavior, we used a degron approach with the FKBP degron integrated into the endogenous Cebpa locus. Addition of dTagV-1 led to full degradation of CEBPA protein within 30-45 minutes, allowing us to assay immediate effects of its absence. We observed that CEBPA degradation led to a rapid shift in the balance of Pol I molecular populations, with a reduction in the fraction of “slow-moving” population noted at 2 hrs (and likely earlier) after dTagV-1 addition. This indicates reduced Pol I loading onto rDNA, consistent with our published findings of reduced Pol I ChIP-Seq signal.
To further dissect effects of CEBPA degradation on the duration of time spent by CEBPA on rDNA, we performed imaging at 500 ms/frame with low laser power to minimize photobleaching. This allowed us to record long videos (minutes) of individual molecules, and determine the duration of time spent by molecules in the rDNA-loaded (“slow-moving”) state. We observed that CEBPA degradation caused a progressive increase (starting within 2 hrs of dTagV-1 addition) in the duration of slow-moving tracks, indicating that CEBPA loss leads individual Pol I molecules that are loaded on rDNA to spend longer periods of time in the loaded state. This points to either Pol I pausing at rDNA promoters or reduced Pol I transcription speed along the rDNA gene. We will uncouple these two possibilities through future studies.
Collectively, our single molecule tracking studies reveal that the master myeloid TF CEBPA regulates the loading rate and pausing/speed of RNA Polymerase I. Our work indicates that one of the critical roles of CEBPA in myeloid progenitors is to boost Pol I activity, promoting rRNA transcription and ribosome production to support their rapid proliferation.