Project1: Mechanisms of Zygotic Genome Activation - A major Nuclear Reprogramming Event
How does the naive genome in the early embryo progressively undergo a series of modifications to control gene expression in “time and space” such that proper cellular differentiation programs are correctly implemented? We investigate the mechanisms of the earliest genome activation to understand how the control of cell fates is transferred from maternal genes to zygotic genes during this period, and when transcription factors specify cell fates and establish epigenetic marks on the embryonic genome. We investigate how the chromatin dynamics are influenced by the coordinated action of maternal and zygotic transcription factors.
During embryonic endoderm formation, Foxh1 transcription factor selectively binds the genome, premarking developmental genes for activation beginning with the onset of zygotic gene activation. A cohort of maternal transcription factors interacts with Foxh1 around the CRMs harboring OVF (Otx/Tbox/Fox) triple binding motifs, prior to the deposition of enhancer marks. The OVF motifs cluster around endoderm specific genes, forming super enhancers, which is the docking sites for other zygotically active mesendodermal transcription factors. Our data support the view that the coordinated binding of maternal transcription factors drives early lineage specification to select CRM clusters frequently associated with super-enhancers to initiate specific developmental gene regulatory programs.
Paraiso, K,, I L Blitz, Coley M, Cheung, Sudoh N., Taira, M., and Cho K.W.Y. (2019).Endodermal maternal transcription factors establish super enhancers during zygotic genome activation. Cell Reports 27:2962-2977. PMID: 31167141
Paraiso, K,, Blitz, IL., Zhou, J., and Cho K.W.Y. (2019). Morpholinos do not elicit an innate immune response during early Xenopus embryogenesis. Developmental Cell 49:643-650. PMID: 31112700
Charney, R.M., Forouzmand, E., Cho, J.S., Cheung, J., Paraiso, K.D., Yasuoka, Y., Takahashi, S., Taira, M., Blitz, I.L., Xie, X. and. Cho K.W.Y. (2017). Foxh1 occupies cis-regulatory modules prior to dynamic transcription factor interactions controlling the mesendoderm gene program. Developmental Cell 40:1-13. PMID:28325473.
Owens, N.D.L. 1, Blitz, I.L.1, Lane, M.A., Overton, J.D., Gilchrist, M.J#, Cho, K.W.Y#.,Khokha, M.K.#(2016). Embryogenesis kinetics measured by high-resolution absolute quantitation of transcripts. 1: co-first authros, contributed equally. # co-senior authors. Cell Reports 14, 632-647.PMID: 26774488
Yasuoka, Y., Suzuki, Y., Takahashi, S., Sudou, N., Haramoto, Y., Cho, KW., Asashima, M., Sugano, S., and Taira, M. (2014). Otx2 and TLE/Groucho occupancy marks tissue-specific cis-regulatory modules for head specification. Nature Communications, 9:4322.
Project 2: Transcriptomics and Epigenomics Controlling Endoderm Development: An Integrative Whole-organism Single-cell Analysis
Gene regulatory networks consist of interactions between transcription factors and enhancers, and are responsible for the control of gene expression throughout the genome, in every biological process. We view that understanding the logic and function of GRNs is fundamental to biology, and have two major projects in this area. First, we perform and collect hundreds of bulk RNA-seq and ChIP-seq, ATAC-seq and DNase-seq datasets, and apply the state of the art Self-Organizing Map analysis to create interactome graph linking transcription factor inputs and target gene expression outputs, generating thousands of genomic links to understand the network structures that control endoderm development. Second, due to recent technological advances to analyze the complexities of biological systems at the single-cell level, we examine how different cell states arise to create various endodermal organ buds during embryonic endoderm development by applying single cell RNA-seq and ATAC-seq in both Xenopus and ES cells.
Charney RM, Paraiso KD, Blitz IL, Cho KWY. (2017). A gene regulatory program controlling early Xenopus mesendoderm formation: Network conservation and motifs. Seminars in Cell Dev Biol.66:12-24. PMID: 28341363
Project 3: Genome Editing by CRISPR/Cas9 in Whole Animal
The CRISPR/Cas9 system relies upon the formation of double-stranded hybrids between synthetic guide RNA and genomic DNA and confers mutations in the genome. With Xenopus, the ability to microinject hundreds of synchronous, in vitro-fertilized embryos represents a significant advantage over other systems for examining mutations in the F0 generation. We use Xenopus tropicalis and develop methods to eliminate a large genomic region and induce a homologous recombination at a desired locus within the genome.
Project 4: Mammalian Preimplantation Development: Diagnosis and Predication
The standard non-invasive method to assess embryo quality and viability relies on the visual inspection of embryo morphology according to predefined criteria. Development of more quantitative and objective means for assessing embryo quality that are simpler, safer, and faster could provide significant advantages in assisted reproduction. We apply the non-invasive and label-free phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) approach to preimplantation mouse embryos. FLIM produces an image based on the differences in the exponential decay rate of the fluorescence from a sample. We identify endogenous fluorescent biomarkers (metabolic signatures) of preimplantation embryos for embryo quality assessment. Our goals are to determine the best parameters in phasor-FLIM for predicting preimplantation embryo quality.
We also examine the role of TGF-b signaling during preimplantation development using single cell genomic approaches and single cell resolution imaging approaches. We map and quantitate the dynamic activities of TGF-b signaling activities in preimplantation mouse embryos and uncover the biological function. We also combine modeling and experimentation to develop a stochastic modeling platform on both mouse and human development to predict the outcome of physical and chemical manipulations of the mammalian embryos.
Ning Ma, Nabora Reyes de Mochel, Paula Pham Tae Yoo, Ken Cho#, Michelle Digman#.(2019). Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach. (# shared senior authors). Scientific Reports, In press
Holmes,W.R., Mochel, S., Wang, Q., Du, H., Cinquin, O., Cho, K.W*., Nie, Q*. *contributed equally, co-senior authors. (2017). Intracellular noise aids construction of early embryonic structures..PLoS Comput Biol. 13(1):e1005320.PMID: 28114387
Reyes de Mochel, NS., Luong, M., Chiang, M., Javier, A.L., Luu, E., Cinquin, O., and Cho, KWY. (2015). BMP signaling regulates both cell proliferation and ICM lineage commitment in preimplantation-stage mouse embryos. Dev Biol. 397: 45-55.