The brain is one of the more complexly patterned organs of the body where specific regions of the brain is designated for various functions such as memory, speech, cognitive learning and muscle movement. Each discrete region of the brain has different proportion of neural cell types contributing to structural and functional differences. I am very fascinated in understanding how gene regulatory networks modulate the development of specific regions of the brain in neurogenesis. I am using a combination of advance gene editing technology, CRISPR and high throughput chromatin interaction approaches to unravel the genetic regulatory machinery that upholds the early neural lineage commitment of embryonic stem cells. I am also investigating the impact of the genetic variations in noncoding region that can lead to neurological disorders. For this study, I am generating CRISPR mediated enhancer deletions in human induced pluripotent stem cells and propagating them to cerebral organoids to understand the functional and structural changes in the brain organoids.
There is substantial interest in the evolutionary forces that shaped developmental gene regulation in humans, rodents, and other mammals. Early embryos are patterned and specialized into different cell types by inductive signaling molecules and transcription factors. How these signaling pathways are integrated at transcriptional enhancers to specify embryonic cell fates in mammalian development is not well understood. We posit that in many cases, evolutionary divergence in gene expression patterns and embryonic cell fates are caused by alterations in enhancer sequences. We integrate functional genomics, computational data mining with mouse and human embryonic stem cell (ESC) models of early embryonic tissues to determine species-specific patterns of enhancer activity. Our studies will identify cases of enhancer sequence evolution that lead to regulatory innovations in developmental gene expression programs.
Transcription factors are key elements in lineage commitment and tissue specification during development. To better understand the transcriptional regulatory network that governs early cell fate, we have focused on the role of Myelin regulatory factor (MYRF) during embryonic development. MYRF is well known to regulate myelin genes in Oligodendrocytes which control myelination in the central nervous system. Even though myelination occurs after birth, MYRF is expressed in mouse embryonic stem cells and in various tissues including the heart, lung, liver, and intestine by mid-gestation. Using a Cre-Lox system, we have generated a mouse line that ubiquitously depletes MYRF in the progeny. Our studies have determined that gene ablation leads to embryonic death. This shows that MYRF plays a broader transcriptional role than originally expected. By applying a variety of in-vitro and in-vivo approaches, we are in the process of determining the underlying cause of embryonic death.
In my PhD at the Mitchell Lab, I have focused on using computational, experimental genomics and molecular biology approaches to better understand the mammalian (mouse and human) regulatory genome. I have used bioinformatics, computational biology, machine learning, next-generation sequencing, molecular evolution and comparative genomics to identify the genome sequence code that confers enhancer activity in embryonic stem (ES) cells. Moreover, I confirmed this novel enhancer model based on TFBS threshold, using molecular biology experiments by creating first synthetic enhancers for Embryonic Stem Cells which have enhancer activity equivalent to the natural enhancers. Using this enhancer model, I have deciphered transcription factor binding code in multiple tissues (heart, brain and liver).
Preterm labour is a global concern, lacking effective treatments due to the poorly understood molecular mechanisms underlying contractions in the uterine muscle tissue known as the myometrium. Gene regulation occurs via a complex interplay between transcriptional factors and regulatory regions that allow the uterus to transition from a quiescent to a contractile phenotype. My research project focuses on the role of transcriptional regulation in the myometrium to better understand the process of pregnancy and labour. For this, I commonly carry out experiments such as qPCR and those involving next generation sequencing techniques, namely ATAC-seq and ChIP-seq, on myometrium tissue.
SOX2 is a pluripotency-associated transcription factor that is often upregulated in multiple types of cancer, where it has been linked with increased tumourigenesis, metastasis, proliferation and poor patient outcome. My current goal is to identify (1) the epigenetic mechanisms that result in SOX2 upregulation in cancer, (2) which molecular players are involved in regulating SOX2 in cancer cells, and (3) what is the regulatory role of this transcription factor in a cancer context. To investigate these questions, I have generated CRISPR-mediated enhancer deletions in human cancer cell lines, which have shown significant loss of SOX2 expression. Through next generation sequencing, I identified SOX2 downstream targets that rely on this transcription factor to sustain transcription in cancer cells.
How uterine smooth muscle cells turn on genes required for childbirth-associated contractions is the overarching question guiding my doctoral research project. I aim to establish the ways in which these cells’ gestational age-specific epigenomic and transcriptomic profiles contribute to the myometrium tissue’s adoption of either a quiescent or a contractile state. Using in vitro cell culture models, I am currently investigating how select transcription factors work together to up-regulate the promoters of labor-driving genes.
Gene transcription is controlled in a precise spatial and temporal manner during development and this is known to be controlled by non-coding regulatory elements that can be located at kilobase to megabase distances from the genes they regulate. To properly regulate their cognate genes, enhancers need to be brought into close spatial proximity to them, however, the precise signals directing these long-range chromatin interactions are currently unknown. The goal of my project is to understand at the sequence level, what regulatory code is needed to direct these long-range interactions. To do this I will be using species sequence conservation data to direct CRISPR based chromatin modifications to alter chromatin topography.
Embryonic development relies on stem cells and their differentiation into varying cell types, this process works by turning genes on or off in a specific time and space. Since all the cells in the body have an identical genome, there are regulatory elements within the DNA controlling gene expression and therefor cell identity. These elements are termed enhancers, and my project works to further understand these regions by assessing and modifying their sequences, epigenetic landscape, and surrounding chromatin in mouse embryonic stem cells. I have been able to create synthetic regulatory sequences that were confirmed in vitro, and I am now working towards testing these within a genomic context to uncover the regulatory code that enhancers follow. Common experiments I execute in the lab include CRISPR, ChIP-qPCR, cloning, and reporter assays.
Trophoblast stem-cells are the first lineage commitment that the early embryo makes after fertilization and go on to become the placenta and extra-embryonic tissues of the fetus. Our collaborators have previously discovered a critical, maternally imprinted gene, Sfmbt2, which has seen to be required for the normal development of fetus by allowing the trophoblast stem-cells to divide indefinitely. My project aims to identify what cis-regulatory elements regulate Sfmbt2 and whether they are also responsible for its parent-of-origin expression. To understand its regulation, I employ CRISPR-Cas9 mediated deletions, gene expression using qPCR, Site-Directed Mutagenesis, enhancer reporter assays, and in vivo studies using genome edited mice.
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Past Lab Members
Julie Chih-yu Chen