Project description:We develop a theoretical-computational framework for inferring cell state transition dynamics, and apply it to mouse embryonic stem cells states defined by expression levels of Esrrb, Tbx3, and Zscan4. RNA-seq was performed to characterize the larger transcriptional differences between states expressing combinations of these three specific genes, and proceed to explore their dynamic interconversion. Overall design: A double knock-in reporter for Esrrb and Tbx3 with distinct fluorescent proteins was constructed to enable purification of substates defined by their relative expression levels (Esrrb-/Tbx3-; Esrrb+/Tbx3-; Esrrb+/Tbx3+). A second line was constructed using a promoter-fragment reporter to isolate Zscan4+ from Zscan4- cells. Following FACS isolation, the subpopulations were sequenced on an Illumina HiSeq2500. Biological replicates were collected on different days.
Project description:Mouse embryonic stem (ES) cell cultures exhibit heterogeneity and recently are discovered to sporadically enter the 2-cell (2C)-embryo state, critical for ES potency. Zscan4 could mark the sporadic 2C-state of ES cells. However, factors that regulate the Zscan4(+)/2C state remain to be elucidated. We show that Tbx3 plays a novel role in regulation of Zscan4(+)/2C state. Tbx3 activates 2-cell genes including Zscan4 and Tcstv1/3, but not vise versa. Ectopic expression of Tbx3 results in telomere elongation, consistent with a role for Zscan4 in telomere lengthening. Mechanistically, Tbx3 decreases Dnmt3b and increases Tet2 protein levels, and reduces binding of Dnmt3b to subtelomeres, resulting in reduced DNA methylation and derepression of genes at subtelomeres, e.g. Zscan4. These data suggest that Tbx3 can activate Zscan4(+)/2C state by negative regulation of DNA methylation at repeated sequences, linking to telomere maintenance and self-renewal of ES cells.
Project description:We previously reported that Zscan4 showed heterogeneous expression patterns in mouse embryonic stem (ES) cells. To identify genes that show similar expression patterns, we carried out high-throughput in situ hybridization assays on ES cell cultures for 244 genes. Most of the genes are involved in transcriptional regulation, and were selected using microarray-based comparisons of gene expression profiles in ES and embryonal carcinoma (EC) cells versus differentiated cell types. Pou5f1 (Oct4, Oct3/4) and Krt8 (EndoA) were used as controls. Hybridization signals were detected on ES cell colonies for 147 genes (60%). The majority (136 genes) of them showed relatively homogeneous expression in ES cell colonies. However, we found that two genes unequivocally showed Zscan4-like spotted expression pattern (spot-in-colony pattern; Whsc2 and Rhox9). We also found that nine genes showed relatively heterogeneous expression pattern (mosaic-in-colony pattern: Zfp42/Rex1, Rest, Atf4, Pa2g4, E2f2, Nanog, Dppa3/Pgc7/Stella, Esrrb, and Fscn1). Among these genes, Zfp42/Rex1 showed unequivocally heterogeneous expression in individual ES cells prepared by the CytoSpin. These results show the presence of different types or states of cells within ES cell cultures otherwise thought to be undifferentiated and homogeneous, suggesting a previously unappreciated complexity in ES cell cultures.
Project description:Zinc finger and SCAN domain containing 4 (Zscan4) is a gene that is specifically expressed during zygotic genome activation (ZGA) in mouse preimplantation embryos, and a reduction of Zscan4 transcripts leads to developmental failure. In mouse embryonic stem cells (ESCs), Zscan4 is expressed transiently in as little as 1-5% of the cell population. Zscan4 has also been shown to enhance the efficiency of mouse induced pluripotent stem cells (iPSCs) generation and their quality. Although ZSCAN4 plays important roles in murine embryos and stem cells, its expression and role in bovine embryos is unknown. This study examines ZSCAN4 transcripts in bovine embryos at various developmental stages and attempts to elucidate the functions of ZSCAN4 during bovine preimplantation development. ZSCAN4 transcripts were found to be upregulated at the 8- and 16-cell stages. We next attempted ZSCAN4 downregulation in bovine early embryos by RNA interference and evaluated developmental competency and transcripts levels of genes involved in ZGA and iPSCs generation. Although the bovine embryos injected with ZSCAN4-siRNA could develop to the 8-cell stage, very few were developing beyond the 16-cell stage. PIWIL2 expression was reduced in ZSCAN4 downregulated embryos. It is possible that ZSCAN4 downregulated embryos fail to regulate gene expression during ZGA. Our results indicate that ZSCAN4 is an important factor for the preimplantation development of bovine embryos.
Project description:The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo, is known to deteriorate during long-term cell culture. Previously, we have shown that ES cells oscillate between Zscan4(-) and Zscan4(+) states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency.
Project description:Mouse zinc finger and SCAN domain containing 4 (Zscan4) proteins, which are encoded by multiple copies of Zscan4 genes, are expressed specifically in preimplantation embryos in vivo and embryonic stem (ES) cells in vitro. However, the expression patterns of mouse Zscan4 in vivo have been largely elusive. Here, we show that Zscan4 proteins are expressed in adult ovaries and testes. In ovaries, Zscan4 proteins were detected in germinal vesicle (GV) stage oocytes in antral follicles, indicating that Zscan4 genes are activated during the diplotene/dictyate stage in meiotic prophase I. Remarkably, Zscan4 showed different spatial localization patterns between two distinct GV oocytes, which can be distinguished by global chromatin organization-surrounded nucleolus (SN) and non-surrounded nucleolus (NSN). These spatiotemporal differences in Zscan4 localizations correlated with the transition of RNA polymerase II-mediated transcriptional status during GV oocyte maturation. In testes, Zscan4 proteins were detected in spermatocytes at late pachytene/diplotene stages and in Sertoli cells. These results suggest that Zscan4 may play critical roles during late meiotic prophase in both males and females.
Project description:The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110? catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2.
Project description:T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self-renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both NF?BIB and p14(ARF) , known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, and RAX). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both hESCs and mouse ESCs.
Project description:Orphan nuclear receptor estrogen related receptor ? (Esrrb or ERR?) is well known in stem cells and early embryonic development. However, little is known about its function in cancer.We investigated the mRNA profile alterations induced by Esrrb expression and its synthetic ligand DY131 in human prostate cancer DU145 cells via RNA-Seq analysis.We distinguished 67 mRNAs differentially expressed by Esrrb alone. Although DY131 alone did not change any gene, treatment of DY131 in the presence of Esrrb altered 1161 mRNAs. These observations indicated Esrrb had both ligand-independent and ligand-dependent activity. When Esrrb was expressed, DY131 treatment further regulated 15 Esrrb-altered mRNAs. DY131 acted as an antagonist for 11 of 15 mRNAs (wdr52, f13a1, pxdn, spns2, loc100506599, tagln, loc441454, tkel1, sema3f, zcwpw2, sdc2) and as an agonist for 4 of the 15 mRNAs (rarres3, oasl, padi2, ddx60). Gene ontology analyses showed altered genes are related to transcription and translation regulation, cell proliferation and apoptosis regulation, and cellular metabolism.Our results characterized mRNA profiles in DU145 prostate cancer cells driven by Esrrb expression and Esrrb ligand DY131, and provided multiple markers to characterize Esrrb's function in Esrrb research.
Project description:Uncontrolled self-renewal plays a direct function in the progression of different types of carcinomas. The same molecular pathway that manages self-renewal in normal stem cells also seems to manage cancer stem cells. Here, we examine the expressions of self-renewal regulatory factors Oct4, Nanog, Sox2, nucleostemin, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 in tissue samples of colon, prostate, and bladder carcinomas as well as cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2. We used reverse transcriptase polymerase chain reaction to examine expressions of the above mentioned regulatory factors in cancer cell lines HT-29, Caco-2, HT-1376, LNCaP, and HepG2 and in 20 tumor tissue samples. Total RNA was isolated by the ISOGEN method. RNA integrity was checked by agarose gel electrophoresis and spectrophotometry. Expressions of Oct4 and nucleostemin at the protein level were determined by immunocytochemistry. A significant relationship was found between tumor grade and self-renewal gene expression. Expressions of stem cell specific marker genes were detected in all examined cancer cell lines, in 40% to 100% of bladder cancer samples, and in 60% to 100% of colon and prostate cancer samples. Oct4 expressed in 100% of tumor tissue samples. Our data show that stem cell markers Oct4, Nanog, Sox2, nucleostemin, Bmi, Zfx, Esrrb, Tcl1, Tbx3, and Dppa4 significantly express in cancer cell lines and cancer tissues. Hence, these markers might be useful as potential tumor markers in the diagnosis and/or prognosis of tumors.