DNA replication is of paramount importance in cell proliferation, the process by which cells grow and divide to produce new cells. During this critical phase of the cell cycle, a cell duplicates its DNA, ensuring that each daughter cell receives a complete set of genetic information. Beyond mere duplication of genetic material, DNA replication also involves the crucial task of recovering the epigenome. The epigenome plays a fundamental role in regulating gene expression without changing the underlying DNA sequence. Epigenetic modifications need to be precisely and efficiently duplicated and transferred onto newly synthesized DNA post-replication, allowing the preservation of cellular identity and function across generations. It is within this context that cell fate transitions may occur. As the cell duplicates its genome and epigenome, modifications can be introduced or removed, and chromatin architecture can change, leading to alteration in gene expression that could potentially trigger a cell to leave its original state and transition into a different cell type. Therefore, DNA replication represents a critical window for the study of cell fate decisions in human embryonic stem cells and offers unique insights into the dynamic nature of the epigenome and its influence on cellular identity and development.Due to the underexplored nature of this phase of the cell cycle, we lack the technologies to be able to reliably assay changes during and post-DNA replication. In this work, technologies were developed to provide a means to this end. First, Repli-ATAC-seq was developed to understand how the chromatin landscape is reestablished in post-replication time by assaying chromatin accessibility. This assay shed light on nucleosome dynamics after replication, bringing to light the potential timing of accessibility changes that may be reflected in biological processes such as DNA mismatch repair, transcription factor accessibility binding dynamics, and an association between increased accessibility and high gene expression. Furthermore, an S-phase fractioned single cell 10X Multiome ATAC and Gene Expression assay was produced to understand if and how chromatin architecture and gene expression are related during S-phase, and whether or not there are clues to explore cell state identities throughout S-phase. Remarkably, a neuroectodermal contributor DACH1 revealed itself as a contributor to a potentially primed cell identity in S-phase. This work establishes techniques that could be used to identify cell cycle dynamics of chromatin accessibility and associated biological changes.

Cells are known to sense and respond to their mechanical microenvironment in profound ways. Various evidence has implicated the adhesome, a body of adhesion associated proteins, in several cell fate decisions, including differentiation and proliferation. Furthermore, recent work has demonstrated that substrate topography can modulate the epigenetic state of fibroblasts, facilitate cell reprogramming, and temper inflammatory activation. However, the effectors responsible for such phenomena remain poorly understood. Here we explore the effect of biomaterial design, adhesion, and intracellular forces of the epigenetic and transcriptional regulation of cell state during macrophage activation and somatic cell reprogramming.

Regarding macrophage activation, we relied entirely on murine bone marrow-derived macrophages (BMDMs). To study the effect of adhesion on BMDM behavior, we utilized microcontact printing to produce fibronectin patterned substrates ontop of which we seeded BMDMs. Next, we induced macrophage activation using lipopolysaccharide (LPS) and interferon-γ (INFγ) and found patterned substrates reduce inflammatory gene expression and histone-3 acetylation (H3Ac) while also increasing cellular elongation. We incorporated these results into a bioinformatic reanalysis of transcriptional and epigenetic data derived from a study of H3Ac protein binding during macrophage polarization. This analysis allowed us to identify epigenetic mechanisms linking the adhesome and inflammatory gene expression.

In our investigation of somatic cell reprogramming, we found extracellular matrix binding (ECM) and mechanosensitive ion channel activation reduce reprogramming efficiency. In addition to these results, we discovered that 104 adhesome genes are dynamically regulated during the reprogramming process. Subsequently, we performed an shRNA knockdown of each of these genes and found over 90% of our knockdowns increased the number of TRA-1-60 positive pluripotent colonies. Our knockdown of SHROOM3, a gene associated with apical cellular constriction, increased reprogramming efficiency by 27-fold. Further investigation into SHROOM3 identified a mechano-sensitive critical state transition, which may be necessary for successful reprogramming.

These observations establish adhesome gene expression and mechanical signaling as influential regulators of cell fate during macrophage activation and reprogramming. Moreover, our findings may guide future attempts to regulate cell state and fate using biophysical stimuli. They may also help shed light on cell behavior in various disease states involving cancer and inflammation.

Due to the mechanosensitive nature of stem cells, intense research has also focused on the role of the cell microenvironment in directing cell fate decisions and stemness acquisition. It has been demonstrated that modulation of biophysical cues such as lateral confinement or substrate stiffness can enhance induced pluripotent stem cell (iPSC) and cancer stem cell (CSC) generation. While biophysical studies commonly involve modulating cell-cell and/or cell-ECM adhesions, a systematic study on the effect of perturbing cell adhesion during reprogramming has not been performed. Therefore, we propose to study the effect of a collection of cell-cell and cell-ECM associated genes, known as the Adhesome, on stemness acquisition of reprogramming cells, in the context of iPSC and CSC reprogramming.

Here, we report on a custom RNAi screen targeting 103 adhesome genes which demonstrated that adhesome expression is largely refractory to somatic reprogramming and revealed two novel top hits not previously associated with stemness acquisition: SHROOM3 and CSRP1. scRNA-seq was performed on reprogramming fibroblasts and A549 cells with SHROOM3 and CSRP1 knockdowns, respectively. Adhesome expression knockdown in both reprogramming models decreased PCP and EMT signatures, increased stemness acquisition, and, as revealed by CellChat inferred communication patterns, utilized similar cell-environment signaling during stemness acquisition. Furthermore, trajectory inference of the adhesome gene knockdown in both reprogramming models revealed novel pathways to stem-like states and cell fate transitions. Together, our findings support the adhesome as a novel target during somatic and oncogenic dedifferentiation for the development of more effective stem cell therapies and cancer treatments.

DNA replication plays an important part in allowing cells to proliferate and develop into complex tissues. The advent of multicellular organisms, however, has been theorized to be intertwined with the tradeoff of aging and disease. These events are highly associated with drastic changes in gene expression across a cell population, often regulated by the epigenome. The set of heritable modifications that make up the epigenetic landscape are known to be altered by cell fate, aging, and disease. However, the dynamic processes by which the changes in the epigenome, and subsequently transcriptome, lead to these modified cell states are not clearly understood. In this dissertation, we demonstrate that DNA replication leads to a transient window of epigenetic entropy, providing the first evidence of a molecular link between cell fate, aging, and disease. In order to elucidate this link, we made use of replication-associated bisulfite sequencing (Repli-BS) and replication-associated assay for transposase-accessible chromatin sequencing (Repli-ATAC) datasets in human embryonic stem cells (hESCs). Our results suggest that the temporality of this window for both the chromatin architecture and DNA methylation differs across the genome. Specifically, we identified that the regions with the most prolonged window of epigenetic entropy are located at regulatory features, associate with expression variability, and are susceptible to age- and disease-related epigenetic drift. Additionally, this dissertation explores the impact of individual LMNA mutations on the epigenome that lead to unique disease outcomes of dilated cardiomyopathy (DCM) and brachydactyly using patient-derived fibroblasts and induced pluripotent stem cells (iPSCs). Analyses combining multiple epigenetic features and transcriptomic data suggest that differentially methylated regions (DMRs) are associated with the misregulation of regulatory elements, and that, in combination with chromatin remodeling, could lead to gene dysregulation ending in DCM. Ultimately, our results provide evidence that somatic and reprogrammed patient cells could serve as models to understand the mechanism behind which disease-related regulatory abnormalities lead to laminopathies like DCM and brachydactyly.

Macrophages are innate immune cells that respond to pathogens in our body with inflammatory activation that brings about their destruction and removal. Additionally, macrophages can also play critical roles in tissue healing that follows inflammation. While many studies describe the effects of biochemical cues on macrophage inflammatory activation, the effects of biomechanical cues have not been well explained mechanistically. This dissertation aims to describe epigenetic mechanisms that empower macrophage mechanomodulation to guide inflammatory activation. Chapter 1 describes the mechanomodulatory effects of biomaterials used as negative pressure wound therapy wound dressings. We find that wound dressings that are non-adhesive suppress macrophage inflammatory activation. Chapter 2 describes the effects of biophysical cues that enable macrophage elongation. Using microcontact printing to facilitate macrophage elongation, we observe inflammatory downregulation, and concomitant reduction in histone 3 acetylation. Gene correlation analysis of high throughput gene expression data suggested the influence of the adhesome protein Src in mediating the cell elongation induced suppression of histone acetylation. This observation was verified by Src genetic knockdown and pharmacological inhibition, which both resulted in reduced histone 3 acetylation. In addition, Src activation phosphorylates and suppresses PKCδ, in turn a suppressor of the prominent histone acetyltransferase p300. Probing the mechanism further, we find that Src activation that happens in macrophages exposed to lipopolysaccharide (LPS)/interferon gamma (IFNγ), is curtailed in micropatterned macrophages, leading to inflammatory downregulation through deficient p300 HAT activity and subsequent histone 3 hypoacetylation. Chapter 3 describes the inflammatory effects of the mechanosensitive transcriptional coactivators Yes associated protein (YAP), and Transcriptional Coactivator With PDZ-Binding Motif (TAZ/WWTR1) that is caused by their interplay with the histone acetylation reader BRD4. We find all three proteins colocalize in the same immunocomplexes, and the shRNA-mediated knockdown of these three genes results in a significantly overlapping subset of differentially expressed genes. Upregulation of YAP/TAZ activity by overexpressing their respective constitutively active forms (YAP-5SA and TAZ-S89A) results in increased inflammation that can be offset by addition of BRD4 inhibitors (BETi). We also find that constitutive activity of YAP/TAZ results in increased oxLDL uptake, an event that is closely linked to atherogenesis. oxLDL uptake in macrophages was offset by either YAP, TAZ or BRD4 knockdown using shRNA, or using BETi treatment. Finally, we find that BETi drug treatment is atheroprotective in AAV9-D377Y-mPCSK9 mice that were fed with high fat diet (HFD) and functions by suppressing YAP/TAZ targets like CD36 and OLR1, both of which are oxLDL uptake receptors in macrophages.

Thousands of patients require esophageal replacement annually. While immune activation is critical for wound-healing processes and tissue regeneration, chronic non-resolving inflammation can cause fibrosis, tissue damage, and disease. Given recent evidence that immune regulation becomes dysfunctional with aging, there is a vital need to better understand the host immune responses involved in tissue regeneration and inflammation. Previous studies identified signaling pathways involved in tissue regeneration with acellular graft implants such as the phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and hepatocyte growth factor receptor (c-MET) signaling pathways. However, whether and how epigenetic mechanisms influence tissue regeneration has not been studied. A better understanding of the epigenetic mechanisms involved in regeneration and how they are connected to disease can help identify novel therapeutic targets and improve our ability to determine when these biological systems have gone awry.In chapter 2, we discuss a potential novel mechanism for how the loss of DNA methylation during aging could contribute to immune dysfunction through a novel analysis of previously published epigenetic data and a review of the literature. In chapter 3, we characterized DNA methylation dynamics during tissue regeneration and present novel findings that suggest epigenetic mechanisms influence key signaling pathways involved in growth, inhibition of apoptosis, and immune activation in regenerating esophageal epithelial tissues. Our results showed that hypomethylation of CpGs occurs within the promoters of genes associated with immune activation, while hypermethylation of CpGs occurs in the promoters of genes involved in PI3K-Akt activation. Taken together, the findings in chapter 2 and chapter 3 provide insights into how epigenetics changes are important regulators of immune responses and may influence immune dysfunction during aging when cells exhibit a notable loss in regenerative potential. The results of this work can help identify novel opportunities to improve the efficacy of biomedical implants by manipulating epigenetic mechanisms involved in immune activation to promote wound healing and tissue regeneration. Finally, in chapter 4, we discuss findings from a collaborative project that showed the gram-negative bacteria, Pseudomonas aeruginosa, responds to the presence of stimulated neutrophils by activating the fro system, which regulates the expression of antioxidative genes to mitigate the bactericidal effects of HOCl. Our results suggest that fro activation could function as a bacterial defense mechanism against a neutrophil attack mechanism which could promote colonization in human tissues.

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