The regulation of the expression and activation of the essential ATF1 transcription factor in the mouse preimplantation embryo

The co-expression of the CREB and ATF1 transcription factors is required for the development of preimplantation embryos. Embryotropin-mediated, calcium/calmodulin-dependent signalling activates CREB-induced transcription in the two-cell embryo, but the regulation of ATF1 in the embryo is not known. This study demonstrates that ATF1 begins to accumulate within both pronuclei of the mouse zygote by 20 h post-human chorionic gonadotrophin. This did not require new transcription (not blocked by α-amanitin), but was dependent upon protein synthesis (blocked by puromycin) and the activity of P38 MAP kinase. ATF1 becomes an active transcription factor upon being phosphorylated. A marked accumulation of phosphorylated ATF1 was evident in two-cell embryos and this persisted in subsequent stages of development. This phosphorylation was enhanced by the actions of autocrine embryotropic mediators (including Paf) and required the mutual actions of P38 MAP kinase and calmodulin-dependent pathways for maximum levels of phosphorylation. The combined inhibition of these two pathways blocked embryonic genome activation (EGA) and caused embryos to enter a developmental block at the two-cell stage. The members of the CREB family of transcription factors can generate one of the most diverse transcriptomes of any transcription factor. The demonstration of the presence of activated CREB and ATF1 within the embryonic nucleus at the time of EGA places these transcription factors as priority targets as key regulators of EGA.

Download Full-text

cAMP-responsive element-binding protein expression and regulation in the mouse preimplantation embryo

Reproduction ◽

10.1530/rep-07-0249 ◽

2007 ◽

Vol 134 (5) ◽

pp. 667-675 ◽

Cited By ~ 10

Author(s):

X L Jin ◽

C O’Neill

Keyword(s):

Transcription Factors ◽

Preimplantation Embryo ◽

Binding Protein ◽

Dependent Manner ◽

Cell Stage ◽

Embryonic Genome ◽

Element Binding Protein ◽

Cell Embryo ◽

Responsive Element ◽

Camp Responsive Element Binding

Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.

Download Full-text

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

International Journal of Molecular Sciences ◽

10.3390/ijms22010460 ◽

2021 ◽

Vol 22 (1) ◽

pp. 460

Author(s):

Huan Ou-Yang ◽

Shinn-Chih Wu ◽

Li-Ying Sung ◽

Shiao-Hsuan Yang ◽

Shang-Hsun Yang ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Fluorescent Protein ◽

Mouse Embryos ◽

Cell Nuclei ◽

Cell Stage ◽

Promoter Assay ◽

Maternal To Zygotic Transition ◽

Mouse Zygotes ◽

Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Download Full-text

Transcript profiling of bovine embryos implicates specific transcription factors in the maternal-to-embryo transition

Biology of Reproduction ◽

10.1093/biolre/ioz209 ◽

2019 ◽

Vol 102 (3) ◽

pp. 671-679 ◽

Cited By ~ 3

Author(s):

Yanina S Bogliotti ◽

Nhi Chung ◽

Erika E Paulson ◽

James Chitwood ◽

Michelle Halstead ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Gene Activation ◽

Preimplantation Embryos ◽

Maternal Mrna ◽

Embryonic Genome ◽

Maternal Mrnas ◽

Low Levels ◽

Genome Activation ◽

Further Development

Abstract Full-grown oocytes are transcriptionally quiescent. Following maturation and fertilization, the early stages of embryonic development occur in the absence (or low levels) of transcription that results in a period of development relying on maternally derived products (e.g., mRNAs and proteins). Two critical steps occur during the transition from maternal to embryo control of development: maternal mRNA clearance and embryonic genome activation with an associated dramatic reprogramming of gene expression required for further development. By combining an RNA polymerase II inhibitor with RNA sequencing, we were able not only to distinguish maternally derived from embryonic transcripts in bovine preimplantation embryos but also to establish that embryonic gene activation is required for clearance of maternal mRNAs as well as to identify putative transcription factors that are likely critical for early bovine development.

Download Full-text

Transcription of paternal Y-linked genes in the human zygote as early as the pronucleate stage

Zygote ◽

10.1017/s0967199400002100 ◽

1994 ◽

Vol 2 (4) ◽

pp. 281-287 ◽

Cited By ~ 76

Author(s):

Asangla Ao ◽

Robert P. Erickson ◽

Robert M.L. Winston ◽

Alan H Handysude

Keyword(s):

Mammalian Species ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

Human Embryos ◽

Cell Stage ◽

Gonad Differentiation ◽

Embryonic Genome ◽

Human Preimplantation Embryos

SummaryGlobal activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

Download Full-text

Parallel Profiling of Active Transcription Factors Using an Oligonucleotide Array-Based Transcription Factor Assay (OATFA)

Journal of Proteome Research ◽

10.1021/pr050053l ◽

2005 ◽

Vol 4 (4) ◽

pp. 1451-1456 ◽

Cited By ~ 13

Author(s):

Wei Shao ◽

Hua-Jiang Wei ◽

Ji-Ying Qiao ◽

Yong-Chao Zhao ◽

Yi-Min Sun ◽

...

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Oligonucleotide Array ◽

Active Transcription ◽

Factor Assay

Download Full-text

rDNA nascent transcripts promote a unique spatial organization during mouse early development

10.1101/2021.04.20.440564 ◽

2021 ◽

Author(s):

Martine Chebrout ◽

Maimouna Coura Kone ◽

Habib U. Jan ◽

Marie Cournut ◽

Martine Letheule ◽

...

Keyword(s):

Early Development ◽

Spatial Organization ◽

Ribosomal Genes ◽

Cell Stage ◽

Major Satellite ◽

Cell Cycles ◽

Embryonic Genome ◽

Active Transcription ◽

Polymerase I ◽

Nascent Transcripts

AbstractDuring the first cell cycles of the early development, the chromatin of the embryo is highly reprogrammed alongside that embryonic genome starts its own transcription. The spatial organization of the genome is a major process that contributes to regulating gene transcription in time and space, however, it is poorly studied in the context of early embryos. To study the cause and effect link between transcription and spatial organization in embryos, we focused on the ribosomal genes, that are first silent and begin to transcribe during the 2-cell stage in mouse. We demonstrated that ribosomal sequences are spatially organized in a very peculiar manner from the 2-cell to the 16-cell stage with transcription and processing of ribosomal RNAs excluding mutually. Using drugs inhibiting the RNA polymerase I, we show that this organization, totally different from somatic cells, depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors major satellite sequences transcription after the 4-cell stage.

Download Full-text

Uridine and guanosine incorporation by the mouse one-cell embryo

Development ◽

10.1242/dev.44.1.133 ◽

1978 ◽

Vol 44 (1) ◽

pp. 133-148

Author(s):

R. J. Young ◽

K. Sweeney ◽

J. M. Bedford

Keyword(s):

Cell Stage ◽

Trichloracetic Acid ◽

Insoluble Material ◽

Embryonic Genome ◽

Internal Position ◽

Cell Embryo ◽

The One ◽

Sugar Derivatives ◽

Hydrolysis Of ◽

Derivatives Of

The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1–9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1–3 h than during the period 4–9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H] uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H] guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1–3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H] uridine 1–3 h post fertilization. Very little of the [3H] uridine taken up by the embryos is present as [3H] UTP, or [3H] CTP; most is found as [3H] UMP or [3H] UDP or as the sugar derivatives. Alkaline or ribonuclease (A, T1 and T2) hydrolysis of the 3H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H] uridine 1–3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demonstrates that [3H] uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1–3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H] uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.

Download Full-text

Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo

Circulation Research ◽

10.1161/res.113.suppl_1.a267 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Joachim Altschmied ◽

Nicole Büchner ◽

Sascha Jakob ◽

Sabrina Farrokh ◽

Christine Goy ◽

...

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Transcription Factors ◽

Target Genes ◽

Splice Variants ◽

Luciferase Reporter ◽

Dependent Manner ◽

Active Transcription ◽

Human Transcription Factor

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis. Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns. With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes. In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect. In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

Download Full-text

Embryonic gene activation in in vitro produced embryos of the domestic cat (Felis catus)

Reproduction ◽

10.1530/rep-10-0298 ◽

2010 ◽

Vol 140 (4) ◽

pp. 531-540 ◽

Cited By ~ 34

Author(s):

R Waurich ◽

J Ringleb ◽

B C Braun ◽

K Jewgenow

Keyword(s):

Mrna Expression ◽

Gene Activation ◽

Transcript Abundance ◽

Dna Methyltransferases ◽

Domestic Cat ◽

Preimplantation Embryos ◽

Embryonic Genome ◽

Junction Protein ◽

Cell Embryo

Accurate embryonic gene activation (EGA) is essential for the embryo's developmental potency and reflects the quality ofin vitroproduced embryos. To describe the dynamic and temporal patterns of EGA in the cat, the mRNA expression of developmentally important genes (DNA methyltransferases 1 and 3A,DNMT1andDNMT3A; gap junction protein α 1,GJA1; transcription factor octamer 4,POU5F1(OCT4); insulin-like growth factor (IGF) 1 and 2 receptors,IGF1RandIGF2R) was examined by RT-PCR techniques in preimplantation embryos obtained afterin vitromaturation and IVF. Furthermore, influences of ICSI and sperm cryopreservation on the relative mRNA abundance in 4–5-days-old morulae were analyzed. Total RNA was obtained from immature and matured oocytes, 2-cell embryos, 4-cell embryos, and 8–16-cell embryos, morulae, and blastocysts. RNA was transcribed into single-stranded cDNA by reverse transcriptase. After amplification, a nonfelid standard RNA was used for semiquantitative analysis. Our results showed an increase in transcript abundance from the matured oocyte to the 2-cell embryo for all examined genes except forIGF2R, indicating that,in vitro, the embryonic genome is activated shortly after fertilization. However, the activation pattern varied markedly between the different genes. We also found different patterns of mRNA expression for the examined genes in morulae produced either by IVF or ICSI, and using fresh or cryopreserved sperm. Owing to high variations within the single groups of compared morulae, we were able to observe only a tendency toward higher relative mRNA expression in embryos derived by IVF with fresh sperm in comparison to all other groups.

Download Full-text

Preovulatory oocyte aging in mice affects fertilization rate and embryonic genome activation

10.1101/209437 ◽

2017 ◽

Author(s):

Hannah Demond ◽

Debora Dankert ◽

Ruth Grümmer ◽

Bernhard Horsthemke

Keyword(s):

Fold Increase ◽

Fertilization Rate ◽

Developmental Competence ◽

Preimplantation Embryos ◽

Cell Stage ◽

Embryonic Genome ◽

Oocyte Aging ◽

And Control ◽

Genome Activation ◽

Zygotic Genome

AbstractDelayed ovulation, or preovulatory aging, can seriously compromise the developmental competence of oocytes. In the present study, we have investigated the effect of preovulatory aging on preimplantation embryos. Delaying ovulation with the gonadotropin releasing hormone (GnRH) antagonist Cetrorelix led to a decline in 2-cell rate from 76 to 46%. From control mice, an average of 17 embryos per mouse was retrieved. This number decreased to a mean of 5 embryos per mouse after preovulatory aging, suggesting that fertilization is impaired by aging. For analysis of zygotic genome activation, 2-cell embryos were incubated with BrUTP, which was incorporated into nascent RNA and detected by immunohistochemistry. A 2.85-fold increase in fluorescence intensity was detected after aging, pointing to a precocious activation of the genome. A possible effect of preovulatory aging on genomic imprint maintenance was investigated at the 8-cell stage. Deep amplicon bisulfite sequencing of Igf2r, Snrpn, H19 and Pou5f1 showed no significant changes between embryos derived from preovulatory-aged oocytes and control embryos, indicating stable imprint maintenance throughout epigenetic reprogramming. We conclude that preovulatory aging of the oocyte affects fertilization and embryonic genomic activation.

Download Full-text