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Toshihiro Shioda, MD, PhDAssociate Professor of MedicineHarvard Medical School
DirectorMolecular Profiling Laboratory
The Shioda laboratory is interested in how exposure of pregnant women to various types of stresses such as drugs, toxic chemicals, or malnutrition affects health of their children throughout their lifespan and even beyond generations. There is mounting evidence that exposure of fetuses in the womb to stresses may cause long-lasting damages to the mechanisms regulating gene expression in human and animal cells, resulting in increased risk of adult-onset diseases including cancer. If such damages are introduced into cells generating gametes (sperms and eggs), heritable disorders might emerge without involving DNA mutations. To detect and prevent such disease-causing, heritable damages in gene expression regulation, our laboratory has been developing human and mouse cell culture models of the gametes-generating stem cells. Our recently published study has established that our mouse gametes-generating stem cell culture model faithfully reflects gene-regulating molecular events occurring in the natural stem cells in mouse embryos, opening the door to examine effects of stresses on gene regulation, efficiently and in-depth without exposing pregnant animals or human subjects.
Toshihiro Shioda, MD, PhDPrincipal Investigator Group Members
In vitro production of primordial germ cells from human and mouse pluripotent stem cells
Evidence is accumulating that in utero exposure of mammalian fetuses to various types of environmental stresses such as drugs, toxic industrial chemicals, or nutritional problems may cause adult-onset disorders that are heritable beyond multiple generations without involving nucleotide base mutations.
Epidemiological studies on human subjects and laboratory animal experiments support the existence of transgenerational epigenetic inheritance of acquired traits in mammals, which is a phenomenon firmly established to occur in C. elegans and also likely the case in Drosophila. However, the germline genome of mammals is known to be subjected to genome-wide erasure of normal or abnormal epigenetic marks followed by re-establishment of correct, sex-specific epigenomes. This process, Germline Epigenetic Reprogramming (GER), occurs in the Primordial Germ Cells (PGCs), which are the precursors of all germline cells. Whereas most protein-coding genes lose their epigenetic marks such as DNA methylation in PGCs during GER, recent studies suggest that certain types of repetitive sequences associated with heterochromatin formation may escape GER. Apparently, GER is a major hurdle for epimutations acquired in one generation to be transmitted to the subsequent generations, and the exceptional genomic elements that escape the epigenetic erasure during GER are of special interest as possible vehicles of transgenerational epigenetic inheritance in mammals.
One of the most challenging aspects of studying mechanisms of mammalian GER is that mammalian PGCs are rare and hard to access because they emerge only temporarily in the body of mammalian embryos/fetuses during the early stages of in utero development. It is impossible to expose pregnant women or their fetuses to potentially disease-causing stresses for research purposes, and animal experiments using rodent models often results in total lack of PGCs or embryonic lethality after exposure to stresses or genetic manipulations. In an effort to establish credible and effective in vitro surrogate models of PGCs for mechanistic studies on GER, we are developing methods for efficient production of human and mouse PGC-Like Cells (PGC-LCs), which are cell culture models of PGCs differentiated from the pluripotent embryonic stem cells or iPS cells. Deep sequencing analyses of mouse PGC-LCs and gonadal natural PGCs for mRNA expression, DNA methylation, DNA hydroxymethylation, and histone modifications have demonstrated significant epigenomic and transcriptomal similarities between our mouse PGC-LCs and natural embryonic mouse PGCs.
Our mouse PGC-LCs effectively recapitulate GER, erasing most of the epigenetic marks at protein-coding genes but preserving DNA hypermethylation at known GER-escaping repeat sequences such as the IAP class endogenous retroviruses. Mouse PGC-LCs were also able to erase aberrant DNA hypermethylation at the Dlk1-Dio3 imprinting control region specifically introduced in the precursor iPS cells generated under a vitamin C-deficient cell culture condition. This is the first experimental demonstration that a specific epimutation is erased during GER. Furthermore, using mouse PGC-LCs, we have identified novel GER-resistant sequences, including the γ-satellite heterochromatin repeats. These observations support the usefulness of mouse PGC-LCs as a model for epigenomic research on germline cells. We have been producing PGC-LCs from human iPS cells as well. Our transcriptomal analyses suggest that human PGC-LCs appear to reflect an early-stage PGCs before initiation of GER. Comprehensive characterization of human PGC-LCs is presently underway.
To examine whether transgenerational epigenetic inheritance involve monoallelic gene expression, we have generated mouse iPS cells whose paternal and maternal chromosomes are derived from Mus spretus and Mus musculus, respectively, by interspecific in vitro fertilization. Taking advantage of the rich (~ 1%) SNPs evenly distributed throughout the genomes of these two distant species of Mus, we are developing a cell culture model and a computational data analysis pipeline for sensitive and quantitative determination of monoallelic gene expression in these iPS cells and their PGC-LC products.
Erasure of aberrant DNA hypermethylation at the imprinting control regions IG-DMR and Gtl2-DMR in the pluripotency-compromised, Gtl2-negative mouse iPS cells during differentiation to primordial germ cell-like cells (PGCLCs) via epiblast-like cells (EpiLCs). Deep sequencing traces for DNA methylation determined using the Methyl- CpG Binding Domain protein-enriched genome sequencing (MBD-seq) technique are shown. Red trace, Gtl2-negative iPS cells and their derivatives; blue trace, Gtl2-positive iPS cells and their derivatives; green trace, natural primordial germ cells isolated from E12.5 mouse embryos.
View a list of publications by researchers at the Shioda Laboratory
Miyoshi N, Stel JM, Shioda K, Qu N, Odajima J, Mitsunaga S, Zhang X, Nagano M, Hochedlinger K, Isselbacher KJ, and Shioda T. Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells. Proc Natl Acad Sci USA. 113(34):9545-50 (2016).
Miyoshi N, Wittner BS, Shioda K, Hirota T, Ito T, Ramaswamy S., Isselbacher KJ, Sgroi DC, and Shioda T. Nodes-and- connections RNAi knockdown screening: Identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis. Oncogenesis 4:e172 (2015).
Tajima K, Yae T, Javaid S, Tam O, Comaills V, Morris R, Wittner BS, Liu M, Engstrom A, Takahashi F, Black JC, Ramaswamy S, Shioda T, Hammell M, Haber DA, Whetstine JR, and Maheswaran S. SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes. Nature Communications 6:8257 (2015).
Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu H, Broderick KT, Desai R, Fox DB, Brannigan BW, Trautwein J, Arora KS, Desai N, Dahl DM, Sequist LV, Smith MR, Kapur R, Wu CL, Shioda T, Ramaswamy S, Ting DT, Toner M, Maheswaran S, and Haber DA. RNA-seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science 349:1351-6 (2015).
National Research Council Committee (Berg AO, Bailor III JC, Gandolfi AJ, Kriebel D, Morris JB, Pinkerton KE, Rusyn I, Shioda T, Smith TJ, Wetzler M, ZeiseL, and Zweidler-McKay P). Review of the Formaldehyde Assessment in the National Toxicology Program 12th Report on Carcinogens. The National Academies. The National Academies Press (2014).
Janesick AS, Shioda T, Blumberg B. Transgenerational inheritance of prenatal obesogen exposure. Molecular and Cellular Endocrinology 398:31-35 (2014).
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