# Investigation into the function of RNA polymerase II promoter proximal pausing during terminal erythroid maturation

> **NIH NIH R01** · UNIVERSITY OF ROCHESTER · 2022 · $338,800

## Abstract

Terminal erythroid maturation involves rapid changes in gene expression in the context of a nucleus that
is progressively condensing in preparation for enucleation. Each stage of erythroid maturation is associated with
a distinct gene expression profile, however the distribution of epigenetic marks associated with both active
promoters and repressed heterochromatin is relatively static between successive stages of erythroid maturation.
In contrast, our preliminary data demonstrates that histone marks associated with active transcriptional
elongation, such as H3K36me3, change dramatically during terminal maturation, suggesting erythroblasts
preferentially regulate transcription at the level of elongation. RNA Polymerase II pausing (Pol II pausing) is a
highly regulated mechanism of transcriptional regulation whereby transcription is initiated, but “pauses” 30-60bp
downstream of the transcription start site. Pausing is a critical checkpoint in gene expression, as pol II cannot
transition into active elongation without being phosphorylated by pTEFb. pTEFb can associate with tissue
specific transcription factors, including GATA1, to facilitate pol II pause release at specific loci, or it can be
sequestered by Hexim1 in the 7sk small nuclear ribonucleoprotein (snRNP) complex rendering it in active and
unable to facilitate Pol II release. Both our preliminary data and the published literature suggest that Pol II pausing
is a critical regulator of erythroid maturation, however the mechanisms by which Pol II pausing is regulated in
maturing erythroblasts are poorly understood. Supporting a central role for Pol II pausing in maturing
erythroblasts, mass spectrometry demonstrates that terminal erythroid maturation is associated with a decrease
in the abundance of multiple histone marks associated with active transcriptional elongation, coupled with
changes in marks suggestive of increased Pol II pausing, without an associated increase in heterochromatin.
ChIP-seq studies confirm that the decrease in abundance of H3K36me3 is correlated with loss of H3K36me3
enrichment at >1600 loci. In addition, Hexim1, a central driver of pol II pausing, is highly expressed in erythroid
cells compared to other cell types and its expression is maintained at both the RNA and protein level throughout
terminal erythroid maturation. In contrast, the expression of pTEFb declines, as does the level of elongation
competent Pol II. Lastly, induction of hexim1 promotes terminal erythroid maturation, and specifically impacts
the expression of genes that lose enrichment for H3K36me3 during maturation. Together our preliminary data
support our central hypothesis that a shift in Pol II pause dynamics that increasingly favors the “paused”
state is a critical regulator of terminal erythroid maturation. In aim1, we will delineate the dynamics of Pol II
pausing in maturating erythroblasts and we will determine the consequences of altering Pol II pausing dynamics
on multiple facets of termin...

## Key facts

- **NIH application ID:** 10375479
- **Project number:** 5R01DK124777-03
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** LAURIE A. STEINER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $338,800
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10375479

## Citation

> US National Institutes of Health, RePORTER application 10375479, Investigation into the function of RNA polymerase II promoter proximal pausing during terminal erythroid maturation (5R01DK124777-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10375479. Licensed CC0.

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