606 - Transcription Drives H1 Linker Histone Depletion in the Promoters of Murine Erythroid Cells.

Publication Number: 4593.606

Michael A. Willcockson, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Josephine Thrasher, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Julia Kesack, Children's Hospital of Philadephia, Philadelphia, PA, United States; Cheryl A. Keller, The Pennsylvania State University, University Park, PA, United States; Belinda Giardine, Pennsylvania State University., Huntingdon, PA, United States; Ross C. Hardison, Pennsylvania State University, University Park, PA, United States; Gerd A. Blobel, Childrens Hospital of Philadelphia, Philadephia, PA, United States

Background: H1 linker histones are the most abundant chromatin-binding proteins in eukaryotic cells. H1 stoichiometry controls chromatin compaction and deposition of epigenetic marks including H3K36me2 (active chromatin) and H3K27me3 (repressed chromatin), forming an H1-H3K36me2-H3K27me3 axis. Despite this central role, the molecular mechanism controlling H1 stoichiometry remains unclear. Understanding H1 stoichiometry is critical because mutations in H1 cause a pediatric overgrowth syndrome (Rahman syndrome), as do mutations in NSD1 affecting H3K36me2 (Sotos syndrome) and PRC2 affecting H3K27me3 (PRC2-related overgrowth), both directly regulated by H1 stoichiometry.

Objective: To determine the molecular mechanisms that control H1 stoichiometry across the genome.

Design/Methods: We engineered G1e-ER4, a well-characterized erythroid cell line, to express only the three most widely expressed H1 subtypes (H1c, H1d, and H1e), each separately tagged with a high-affinity FKBP-2×HA tag. To examine the relationship between H1 and transcription, we compared occupancy of each H1 subtype with 1) Pol II ChIP-seq (measuring polymerase binding), 2) TT-seq (measuring nascent RNA production), and 3) the TT-seq/Pol II ratio (measuring RNA production per polymerase, a surrogate for elongation efficiency). To determine if transcription is necessary for H1 depletion, we compared H1 binding between interphase and mitotic cells (where Pol II is evicted from chromatin) and inhibited Pol II with triptolide, a small molecule that blocks transcription initiation.

Results: Occupancy of each H1 subtype (ChIP-seq) is strongly inversely correlated with RNA Pol II occupancy (ChIP-seq), RNA transcription (TT-seq) and elongation efficiency (TT-seq/ChIP-seq) at promoters genome-wide (Figure 1). Comparison of H1 occupancy between interphase and prometaphase showed global loss of H1 depletion at enhancers and promoters in prometaphase when Pol II binding is lost (Figure 2). Direct inhibition of transcription with triptolide also led to global loss of H1 depletion at promoters (Figure 3).

Conclusion(s): These findings indicate that H1 occupancy is inversely associated with Pol II occupancy and that transcription is necessary for H1 depletion within promoters. This work establishes a transcription-dependent mechanism controlling H1 stoichiometry which has been shown to directly control the H1-H3K36me2-H3K27me3 axis. This study provides the necessary foundation for understanding how disruption of this axis at different entry points (H1, NSD1, PRC2) leads to overlapping overgrowth syndromes and for strategies to pharmacologically rebalance this axis.

H1 occupancy inversely correlates with transcriptional activity and Pol II occupancy within promoters and gene bodies genome-wide
Using FKBP-2×HA H1c, H1d and H1e reporter lines (Fig. 1), we mapped H1 occupancy by ChIP-seq (IP/Input; N=2 for each H1 subtype). (A) Genes were sorted by TT-seq signal (nascent RNA output) and divided into 25 equal-sized groups from lowest to highest; each point is one group, and the y-value is the median H1 signal for that group within promoters (top) or gene bodies (bottom). Within this panel, H1 was rescaled so the lowest-TT-seq group (inactive) = 1.0. (B) Genes were sorted by Pol II ChIP-seq signal (polymerase occupancy) and divided into 25 groups; H1 values were rescaled so the lowest-Pol-II group = 1.0. (C) Genes were sorted by TT-seq/Pol II (an approximation of per-polymerase productivity) and divided into 25 groups; H1 values were rescaled so the lowest-productivity group = 1.0. Across all panels, greater transcriptional activity or productivity corresponds to lower H1, yielding strong inverse trends for H1c, H1d, and H1e (colors per legend). TT-seq and Pol II ChIP-seq: GEO GSE264584 (Aboreden et al., Mol Cell, 2025).


H1 depletion is reduced during prometaphase in promoters and enhancers.
Endogenous FKBP-2×HA H1c, H1d and H1e reporter lines were profiled by ChIP-seq (IP/Input) in interphase and prometaphase G1E-ER4 cells (N=2 for each H1 subtype within each phase of the cell cycle). Prometaphase cells were enriched by nocodazole arrest and FACS using DNA content, MPM-2 (phospho-Ser/Thr-Pro) positivity, and an mCherry mitotic reporter (Zhang et al., Nature, 2019). Each panel (H1c, H1d, H1e) plots prometaphase H1 (y-axis) versus interphase H1 (x-axis) for chromatin-state bins (numbers label states); red triangles = promoters, orange triangles = enhancers, gray/black circles = other states. The dashed diagonal marks no change; the blue curve is a smoothed fit with 95% CI. Promoter and enhancer bins lie above the identity line, indicating higher H1 in prometaphase, consistent with reduced depletion of H1 at active regulatory elements during mitosis when Pol II is broadly displaced from chromatin.


Transcription is required for H1 depletion in active promoters
G1E-ER4 cells were untreated (0 h) or treated with triptolide (8 h). Promoters were ranked by baseline (0 h) promoter Pol II ChIP-seq signal and divided into 15 equal-sized n-tiles from low to high Pol II; each point is one n-tile, and the y-value is the median promoter signal for that n-tile (Pol II or H1c, as indicated), expressed as fold-change relative to the lowest-Pol-II n-tile at the same timepoint. (A) Pol II ChIP-seq: triptolide nearly abolishes promoter Pol II across all n-tiles (orange), whereas 0 h shows the expected monotonic increase with Pol II rank (green). (B) H1c ChIP-seq: triptolide raises promoter H1, with the largest increases in high-Pol-II n-tiles, indicating that transcription is required for promoter H1 depletion.