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Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.

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Yes / The maintenance of a proper nuclear architecture and 3D organization of the genes, enhancer elements and transcription machinery plays an essential role in tissue development and regeneration. Here we show that in the developing skin, epidermal progenitor cells of mice lacking p63 transcription factor display alterations in the nuclear shape accompanied by marked decrease in expression of several nuclear envelop-associated components (Lamin B1, Lamin A/C, SUN1, Nesprin-3, Plectin) compared to controls. Furthermore, ChIP-qPCR assay showed enrichment of p63 on Sun1, Syne3 and Plec promoters, suggesting them as p63 targets. Alterations in the nuclei shape and expression of nuclear envelope-associated proteins were accompanied by altered distribution patterns of the repressive histone marks H3K27me3, H3K9me3 and heterochromatin protein 1- alpha in p63-null keratinocytes. These changes were also accompanied by downregulation of the transcriptional activity and relocation of the keratinocyte-specific gene loci away from the sites of active transcription towards the heterochromatin-enriched repressive nuclear compartments in p63-null cells. These data demonstrate functional links between the nuclear envelope organization, chromatin architecture and gene expression in keratinocytes and suggest nuclear envelope-associated genes as important targets mediating p63-regulated gene expression programme in the epidermis.