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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

W18 - Epigenetic Regulation of Nuclear Mechanics: Modulate DNA Methylation, Heterochromatin, and Mechanotransduction in Mouse Embryonic Stem Cells

Friday, October 10, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Kajol Harsh, PhD

Graduate Research Assistant
Texas A&M
Houston, Texas, United States

Primary Investigator(s)

Yun Huang

Professor, Associate Director
Texas A&M University
HOUSTON, Texas, United States

Co-Author(s)

Tingting Hong

Assistant Research Scientist
Texas A&M University, Texas, United States

Last Author(s)

Yubin Zhou

Professor
Texas A&M University
Houton, Texas, United States

Introduction: : The complex regulatory crosstalk between epigenetic modifications and chromatin architecture plays a pivotal role in transcriptional control and genome stability in pluripotent systems. In mouse embryonic stem cells (mESCs), perturbations in DNA methylation and chromatin compaction are closely associated with impaired differentiation and altered mechanical properties. This study investigates how disruptions in DNA methylation and demethylation pathways affect heterochromatin organization, specifically histone H3 lysine 9 trimethylation (H3K9me3), and their downstream impact on nuclear stiffness and mechanotransduction. Using genetically engineered mESC lines deficient in key DNA methylation regulators, we demonstrate that loss of methylation leads to distinct alterations in nuclear stiffness, potentially mediated by differential expression of mechanosensitive genes and components of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. Ultra-resolution microscopy reveals structural changes in nuclear envelope integrity and chromatin compaction, further supporting the mechanical consequences of epigenetic disruption. Lamin knockout models underscore the role of the nuclear lamina in maintaining nuclear architecture and stiffness. Through microfluidic based microphysiological systems such as confined migration assays and embryoid body formation embedded in extracellular matrices (ECMs) of varying stiffness, we show that epigenetic state modulates stem cell responses to mechanical cues, influencing lineage commitment and morphogenesis. Our findings propose a mechanistic model in which DNA methylation status governs heterochromatin integrity and nuclear mechanics via modulation of the LINC complex and ECM interactions. This integrative framework bridges epigenetics, nuclear architecture, and mechanobiology, offering new insights into stem cell engineering and the design of biomimetic environments for regenerative applications.

Materials and

Methods: : Microfluidic devices were fabricated using 2-step photolithography and soft lithography. SU-8 layers (7 μm and 250 μm) were patterned on silicon wafers, developed, and silanized. PDMS replicas were cast, cured, and bonded to fluorodishes via oxygen plasma. Polyacrylamide hydrogels of varying stiffness were synthesized and functionalized with fibronectin. mESCs were cultured on feeder layers in LIF-supplemented media. For migration assays, fibronectin-coated devices were seeded with cells and exposed to serum gradients. Time-lapse and confocal imaging tracked migration over 12–48 hours, and cell passage through constrictions was quantified.
Immunofluorescence: Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in 2% BSA, and stained with anti-Lamin-A and anti-SUN2 antibodies overnight. Secondary antibodies (Alexa Fluor 488/564), phalloidin (Alexa Fluor 647), and DAPI were used for visualization. AFM: mESCs were seeded on fibronectin-coated dishes. Live-cell stiffness and topography were measured using spherical and conical probes. RT-qPCR: cDNA was synthesized and amplified using SYBR Green with a 3-step cycling protocol. Western blot: Proteins were extracted with RIPA buffer, separated via SDS-PAGE, transferred, and probed with antibodies. CMS-IP-seq: 5hmC-enriched DNA was immunoprecipitated and sequenced using Illumina.

Results, Conclusions, and Discussions::

Results: Genetically engineered mESC lines deficient in DNA methylation regulators exhibited significant alterations in nuclear mechanics and chromatin organization. Loss of DNA methylation led to reduced H3K9me3 levels and disrupted heterochromatin compaction. These changes correlated with decreased nuclear stiffness, particularly in Tet knockout (TetKO) cells, which also showed reduced Lamin A/C expression. In contrast, Dnmt triple knockout (Dnmt TKO) cells retained higher nuclear stiffness and Lamin levels. Confined migration assays revealed that TetKO cells migrated more slowly than Dnmt TKO cells. Embryoid body formation in ECMs of varying stiffness further demonstrated that epigenetic state modulates mechanosensitive lineage commitment.

Conclusion: Our findings establish a mechanistic link between DNA methylation status, heterochromatin integrity, and nuclear mechanics in mESCs. Loss of methylation disrupts chromatin compaction and nuclear envelope structure, leading to altered mechanical properties and impaired cellular responses to physical cues. Lamin A/C and the LINC complex emerge as key mediators of this epigenetic-mechanical interface.

Discussions: This study highlights the critical role of epigenetic regulation in maintaining nuclear architecture and mechanotransduction in pluripotent stem cells. The contrasting phenotypes of TetKO and Dnmt TKO cells underscore the nuanced effects of methylation versus demethylation pathways on nuclear stiffness and migration. Reduced Lamin levels in TetKO cells likely contribute to their softer nuclei and impaired migration, while Dnmt TKO cells maintain structural integrity and responsiveness to ECM cues. These insights advance our understanding of how chromatin state influences stem cell behavior and provide a framework for engineering biomimetic environments that guide stem cell fate through mechanical and epigenetic cues.

Acknowledgements and/or References (Optional): : Manuscript under consideration/review
1.Confinement on a Chip platform (published): Brendan McKee, TingChing Wang, Kajol Harsh et al ,Excess surface area of the nuclear lamina enables unhindered cell migration through constrictions. Sci. Adv.11,eads6573(2025).DOI:10.1126/sciadv.ads6573
2. Hong, T., Li, J., Guo, L. et al. TET2 modulates spatial relocalization of heterochromatin in aged hematopoietic stem and progenitor cells. Nat Aging 3, 1387–1400 (2023). https://doi.org/10.1038/s43587-023-00505-y