Erythropoietin Regulates Transcription and YY1 Dynamics in a Pre-established Chromatin Architecture

Abstract

The three-dimensional architecture of the genome plays an essential role in establishing and maintaining cell identity. However, the magnitude and temporal kinetics of changes in chromatin structure that arise during cell differentiation remain poorly understood. Here, we leverage a murine model of erythropoiesis to study the relationship between chromatin conformation, the epigenome, and transcription in erythroid cells. We discover that acute transcriptional responses induced by erythropoietin (EPO), the hormone necessary for erythroid differentiation, occur within an invariant chromatin topology. Within this pre-established landscape, Yin Yang 1 (YY1) occupancy dynamically redistributes to sites in proximity of EPO-regulated genes. Using HiChIP, we identify chromatin contacts mediated by H3K27ac and YY1 that are enriched for enhancer-promoter interactions of EPO-responsive genes. Taken together, these data are consistent with an emerging model that rapid, signal-dependent transcription occurs in the context of a pre-established chromatin architecture.

Keywords: Developmental Biology; Genomics; Molecular Biology.

Graphical abstract

Figure 1

The FVA Murine System Faithfully Recapitulates Erythroid Differentiation during Erythropoiesis (A) The workflow for generating and isolating highly purified EPO-responsive ProEBs from a mouse injected with the Friend virus that induces anemia (FVA). (B) Microscopy images highlighting morphological changes of ProEBs isolated using the FVA system during differentiation. (C) Heatmap of RNA-seq gene expression through erythroid differentiation.

Figure 2

EPO Stimulation Results in Acute Transcriptional Changes in Proerythroblasts (A) Scatterplot comparing Pol II RPKM before and after 1 h EPO stimulation. Pearson's correlation value R = 0.94. (B) Volcano plot showing significant (p value < 0.05) differential occupancy of increased (red) and decreased (blue) Pol II after 1-h EPO stimulation. (C–E) (C) Metagene plot comparing the position of Pol II peaks relative to transcription start site (TSS) (paired Wilcoxon ranked-sign test, p = 4.882 × 10⁻¹¹). Genome browser view of ChIP-exo signal for Pol II at the up-regulated Cish locus (D) and down-regulated Jund locus (E).

Figure 3

EPO Dynamically Regulates YY1 Occupancy Genome-wide (A) Heatmap of CTCF peaks pre- and post-EPO stimulation, ranked by 1 h CTCF max peak. (C) Heatmap of YY1 and H3K27ac peaks pre- and post-EPO stimulation, ranked by 1 h YY1 max peak. (B and D) Composite plots below each heatmap quantifying the normalized tag density. (E and F) Representative genome browser view of CTCF, YY1, and H3K27ac occupancy in response to EPO stimulation, highlighted in light gray bars and red dashed box.

Figure 4

EPO Regulates Transcription in a Pre-established Chromatin Conformation (A) TF-binding motifs overrepresented in HiChIP loop anchors. (B) A schematic of chromatin features. (C) Proportion of HiChIP interactions with UCSC-annotated TSS within anchor regions compared with random sequences in mm10 genome (gray bars) (∗p < 0.0001). The hypergeometric test was applied to compare HiChIP anchors found in annotated TSSs to expected ratios. The chi-squared test was applied to compare TSS occupancy between H3K27ac and YY1 anchors, as well as comparing HiChIP anchors to randomly generated sequences in the mouse genome. (D) Proportion of HiChIP interactions with promoters of EPO-responsive genes within H3K27ac HiChIP anchor regions. (E) Representative genome browser view of overlap described in (D) with anchor regions highlighted in orange boxes. (F) Proportion of HiChIP interactions with differential H3K27ac or YY1 ChIP-exo peaks within H3K27ac HiChIP anchor regions. Dark green bars represent differential peaks, and light green bars represent invariant peaks (∗p < 0.0001). The chi-squared test was applied to compare YY1 differential and invariant peaks in H3K27ac anchors. (G) Representative genome browser view of overlap described in (F) with anchor regions highlighted in orange boxes. (H) Proportion of HiChIP interactions with differential YY1 peaks at promoters of EPO-responsive genes within H3K27ac HiChIP anchor regions. (I) Representative genome browser view of overlap described in (H) with anchor regions highlighted in orange boxes.