Kotaro J. Kaneko

Regulation of gene expression at the beginning of mammalian development and the TEAD family of transcription factors

In mouse development, transcription is first detected in late 1-cell embryos, but translation of newly synthesized transcripts does not begin until the 2-cell stage. Thus, the onset of zygotic gene expression (ZGE) is regulated at the level of both transcription and translation. Chromatin-mediated repression is established after formation of a 2-cell embryo, concurrent with the developmental acquisition of enhancer function. The most effective enhancer in cleavage stage mouse embryos depends on DNA binding sites for TEF-1, the prototype for a family of transcription factors that share the same TEA DNA binding domain. Mice contain at least four, and perhaps five, genes with the same TEA DNA binding domain (mTEAD genes). Since mTEAD-2 is the only one expressed during the first 7 days of mouse development, it is most likely responsible for the TEAD transcription factor activity that first appears at the beginning of ZGE. All four mTEAD genes are expressed at later embryonic stages and in adult tissues; virtually every tissue expresses at least one family member, consistent with a critical role for TEAD proteins in either cell proliferation or differentiation. The 72-amino acid TEA DNA binding domains in mTEAD-2, 3, and 4 are approximately 99% homologous to the same domain in mTEAD-1, and all four proteins bind specifically to the same DNA sequences in vitro with a Kd value of 16-38 nM DNA. Since TEAD proteins appear to be involved in both activation and repression of different genes and do not appear to be functionally redundant, differential activity of TEAD proteins must result either from association with other proteins or from differential sensitivity to chromatin-packaged DNA binding sites.

Developmental acquisition of enhancer function requires a unique coactivator activity

Enhancers are believed to stimulate promoters by relieving chromatin‐mediated repression. However, injection of plasmid‐encoded genes into mouse oocytes and embryos revealed that enhancers failed to stimulate promoters prior to formation of a two‐cell embryo, even though the promoter was repressed in the maternal nucleus of both oocytes and one‐cell embryos. The absence of enhancer function was not due to the absence of a required sequence‐specific enhancer activation protein, because enhancer function was not elicited even when these proteins either were provided by an expression vector (GAL4:VP16) or were present as an endogenous transcription factor (TEF‐1) and shown to be active in stimulating promoters. Instead, enhancer function in vivo required a unique coactivator activity in addition to enhancer‐specific DNA binding proteins and promoter repression. This coactivator activity first appeared during mouse development in two‐ to four‐cell embryos, concurrent with the major onset of zygotic gene expression. Competition between various enhancers was observed in these embryos, but not competition between enhancers and promoters, and competition between enhancers was absent in one‐cell embryos. Moreover, enhancer function in oocytes could be partially restored by pre‐injecting mRNA from cells in which enhancers were active, the same mRNA did not affect enhancer function in two‐ to four‐cell embryos.

TEAD4 establishes the energy homeostasis essential for blastocoel formation

It has been suggested that during mouse preimplantation development, the zygotically expressed transcription factor TEAD4 is essential for specification of the trophectoderm lineage required for producing a blastocyst. Here we show that blastocysts can form without TEAD4 but that TEAD4 is required to prevent oxidative stress when blastocoel formation is accompanied by increased oxidative phosphorylation that leads to the production of reactive oxygen species (ROS). Both two-cell and eight-cell Tead4-/- embryos developed into blastocysts when cultured under conditions that alleviate oxidative stress, and Tead4-/- blastocysts that formed under these conditions expressed trophectoderm-associated genes. Therefore, TEAD4 is not required for specification of the trophectoderm lineage. Once the trophectoderm was specified, Tead4 was not essential for either proliferation or differentiation of trophoblast cells in culture. However, ablation of Tead4 in trophoblast cells resulted in reduced mitochondrial membrane potential. Moreover, Tead4 suppressed ROS in embryos and embryonic fibroblasts. Finally, ectopically expressed TEAD4 protein could localize to the mitochondria as well as to the nucleus, a property not shared by other members of the TEAD family. These results reveal that TEAD4 plays a crucial role in maintaining energy homeostasis during preimplantation development.

Activation of zygotic gene expression in mammals

Abstract Preimplantation development in the mouse involves expression of about 11,000 genes, only a few hundred of which appear during the transition from maternal to zygotic gene expression. Transcription begins in most, if not all, mammals during the late 1-cell stage (phase I), but expression of most zygotic genes is delayed until the 2-cell to the 16-cell stages, depending on the mammal. In mice, a small group of genes are expressed immediately after the first mitosis, while a larger group of genes are expressed during the subsequent G2 phase. Zygotic gene activation (ZGA) is delayed by a time dependent mechanism (“zygotic clock”) that regulates both transcription and translation. It appears to involve post-translational modification of RNA polymerases, translational control of maternal gene expression, developmental acquisition of chromatin mediated repression as well as the ability to alleviate this repression with sequence-specific enhancers, and changes in DNA methylation. This delay allows remodeling of chromatin into a form that globally represses gene activity so that selected genes can then be activated in a temporally and spatially specific program. Some transcription factors such as Sp1 and TBP function from oocyte to embryo, while others are selectively expressed during ZGA. OCT-4(OCT-3) regulates the production and subsequent differentiation of embryonic stem cells, a prerequisite for embryo development. TEAD-2(TEF-4), whose activity is regulated by the transcriptional coactivator YAP65, appears to regulate contact inhibition during cell proliferation, a prerequisite for tissue formation. Thus, identification and characterization of genes whose expression is activated by fertilization opens the door to understanding how mammalian development begins.

The dual roles of geminin during trophoblast proliferation and differentiation.

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

The acrosomal protein Dickkopf-like 1 (DKKL1) facilitates sperm penetration of the zona pellucida.

OBJECTIVE To determine the role of Dkkl1 in mouse development, viability, and fertility. DESIGN Prospective experimental study. SETTING Government research institution. ANIMAL(S) Mice of C57BL/6, B6D2F1/J, and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S) Expression of the Dkkl1 gene was characterized during early mouse development, and the effects of Dkkl1 ablation on reproduction and fertility were characterized in vitro and in vivo. MAIN OUTCOME MEASURE(S) Dkkl1 RNA expression was determined by Northern blotting hybridization as well as quantitative reverse transcriptase-polymerase chain reaction assays. In vitro fertilization assays were used to assess fertility of sperm from male mice lacking functional Dkkl1. RESULT(S) Dkkl1 is a gene unique to mammals that is expressed primarily in developing spermatocytes and its product localized in the acrosome of mature sperm. Here we show that Dkkl1 also is expressed in the trophectoderm/placental lineage. Surprisingly, embryos lacking DKKL1 protein developed into viable, fertile adults. Nevertheless, the ability of sperm that lacked DKKL1 protein to fertilize wild-type eggs was severely compromised in vitro. Because this defect could be overcome either by removal of the zona pellucida or by the presence of wild-type sperm, Dkkl1, either directly or indirectly, facilitates the ability of sperm to penetrate the zona pellucida. Penetration of the zona pellucida by Dkkl1(-) sperm was delayed in vivo as well as in vitro, but the delay in vivo was compensated by other factors during preimplantation development. Accordingly, Dkkl1-/- males offer an in vitro fertilization model for identifying factors that may contribute to infertility. CONCLUSION(S) DKKL1 is a mammalian-specific, acrosomal protein that strongly affects in vitro fertilization, although the effect is attenuated in vivo.

Geminin is Essential to Prevent DNA Re‐Replication‐Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells

Geminin is a dual‐function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re‐replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re‐replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self‐renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self‐renewal or differentiation rapidly induced DNA re‐replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re‐replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re‐replication‐dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability. Stem Cells 2015;33:3239–3253

The acrosomal protein Dickkopf-like 1 (DKKL1) is not essential for fertility

OBJECTIVE To determine the role of Dkkl1 on mouse development, viability, and fertility. DESIGN Prospective experimental study. SETTING Government research institution. ANIMAL(S) Mice of C57BL/6 and 129X1/SvJ strains, as well as transgenic mice of mixed C57BL/6 and 129X1/SvJ strains were used for the studies. INTERVENTION(S) Mice were constructed that lacked a functional Dkkl1 gene. MAIN OUTCOME MEASURE(S) Deletion of the gene was confirmed by DNA, RNA, and protein analyses; in vivo fertility was examined by continuous mating scheme. RESULT(S) Previous studies have shown that Dkkl1, a gene unique to mammals, is expressed predominantly, if not exclusively, in developing spermatocytes, and the DKKL1 protein accumulates in the acrosome of mature sperm. Subsequent studies (reported in the accompanying article) demonstrate that Dkkl1 also is expressed in the trophectoderm/placental lineage. Taken together, these results strongly suggested that DKKL1 protein is required for terminal differentiation either of trophoblast giant cells or of sperm, both of which are directly involved in fertility. To challenge this hypothesis, conditional targeted mutagenesis was used to ablate the Dkkl1 gene in mice. Surprisingly, Dkkl1 nullizygous embryos developed into viable, fertile adults, despite the fact that they failed to produce any portion of the DKKL1 protein. CONCLUSION(S) DKKL1 is a mammalian-specific acrosomal protein that is not essential either for development or fertility.