Epigenetic Remodeling Increases Plasticity of Bovine Fibroblasts Without Improving Embryo and Stem Cell Development
- 1Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada
Abstract
Epigenetic memory, reinforced by redundant heterochromatic networks, is a major barrier to somatic cell reprogramming. In cattle, single-agent chromatin-modifying treatments show inconsistent efficacy, likely due to compensatory crosstalk among repressive marks. Here, we evaluated a multi-target small-molecule cocktail, GUSTC, combining GSK343 (EZH2 inhibitor), UNC0638 (G9a/GLP inhibitor), Sodium Butyrate (HDAC inhibitor), Trichostatin A (HDAC inhibitor), and Chaetocin (SUV39H1 inhibitor) to simultaneously target facultative and constitutive heterochromatin in bovine fibroblasts. At optimized, non-cytotoxic concentrations, GUSTC treatment reduced H3K9me2, H3K9me3, H3K27me3, and HP1-alpha levels while increasing H3K9 acetylation by immunofluorescence. Using GUSTC-treated fibroblasts as donor cells for somatic cell nuclear transfer did not significantly change cleavage (83.6% vs. 92.2%; p = 0.15), blastocyst rate (28.4% vs. 39.0%; p = 0.20), bovine embryonic stem cell line derivation efficiency (26.3% vs. 33.3% of seeded blastocysts; p = 0.57), or the core pluripotency marker profile (OCT4, NANOG, SOX2) of the resulting lines. In a separate, serum-free, pluripotency-permissive culture system, GUSTC-treated cells adopted a colony-like morphology and showed increased POU5F1 and NANOG expression, along with increased DNMT1, EHMT2, HDAC1, HDAC2, and SUV39H1 transcripts. GUSTC thus reduces repressive heterochromatin marks in donor fibroblasts and is associated with a potentially more reprogramming-permissive state, without detectable improvement in preimplantation developmental competence. It provides a starting point for chemical reprogramming strategies in cattle.
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BibTeX
@misc{ambrizi2026epigenetic,
title = {{Epigenetic Remodeling Increases Plasticity of Bovine Fibroblasts Without Improving Embryo and Stem Cell Development}},
author = {D. R. Ambrizi and D. Moreno and J. R. Sangalli and R. P. Nociti and J. B. Cruz and L. V. F. Pessoa and D. Kendall and F. F. Bressan and M. R. Chiaratti and L. C. Smith and P. J. Ross and F. V. Meirelles and R. V. Sampaio},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.28.762563},
url = {https://www.biorxiv.org/content/10.64898/2026.10.28.762563v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Epigenetic Remodeling Increases Plasticity of Bovine Fibroblasts Without Improving Embryo and Stem Cell Development
AU - D. R. Ambrizi
AU - D. Moreno
AU - J. R. Sangalli
AU - R. P. Nociti
AU - J. B. Cruz
AU - L. V. F. Pessoa
AU - D. Kendall
AU - F. F. Bressan
AU - M. R. Chiaratti
AU - L. C. Smith
AU - P. J. Ross
AU - F. V. Meirelles
AU - R. V. Sampaio
PY - 2026
DA - 2026/10/28
PB - bioRxiv
DO - 10.64898/2026.10.28.762563
UR - https://www.biorxiv.org/content/10.64898/2026.10.28.762563v1
AB - Epigenetic memory, reinforced by redundant heterochromatic networks, is a major barrier to somatic cell reprogramming. In cattle, single-agent chromatin-modifying treatments show inconsistent efficacy, likely due to compensatory crosstalk among repressive marks. Here, we evaluated a multi-target small-molecule cocktail, GUSTC, combining GSK343 (EZH2 inhibitor), UNC0638 (G9a/GLP inhibitor), Sodium Butyrate (HDAC inhibitor), Trichostatin A (HDAC inhibitor), and Chaetocin (SUV39H1 inhibitor) to simultaneously target facultative and constitutive heterochromatin in bovine fibroblasts. At optimized, non-cytotoxic concentrations, GUSTC treatment reduced H3K9me2, H3K9me3, H3K27me3, and HP1-alpha levels while increasing H3K9 acetylation by immunofluorescence. Using GUSTC-treated fibroblasts as donor cells for somatic cell nuclear transfer did not significantly change cleavage (83.6% vs. 92.2%; p = 0.15), blastocyst rate (28.4% vs. 39.0%; p = 0.20), bovine embryonic stem cell line derivation efficiency (26.3% vs. 33.3% of seeded blastocysts; p = 0.57), or the core pluripotency marker profile (OCT4, NANOG, SOX2) of the resulting lines. In a separate, serum-free, pluripotency-permissive culture system, GUSTC-treated cells adopted a colony-like morphology and showed increased POU5F1 and NANOG expression, along with increased DNMT1, EHMT2, HDAC1, HDAC2, and SUV39H1 transcripts. GUSTC thus reduces repressive heterochromatin marks in donor fibroblasts and is associated with a potentially more reprogramming-permissive state, without detectable improvement in preimplantation developmental competence. It provides a starting point for chemical reprogramming strategies in cattle.
ER -