The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing
2015; Springer Nature; Volume: 34; Issue: 11 Linguagem: Inglês
10.15252/embj.201490648
ISSN1460-2075
AutoresKen‐ichiro Abe, Ryoma Yamamoto, Vedran Franke, Minjun Cao, Yutaka Suzuki, Masataka G. Suzuki, Kristian Vlahoviček, Petr Svoboda, Richard M. Schultz, Fugaku Aoki,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle20 April 2015free access Source Data The first murine zygotic transcription is promiscuous and uncoupled from splicing and 3′ processing Ken-ichiro Abe Ken-ichiro Abe Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Ryoma Yamamoto Ryoma Yamamoto Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Vedran Franke Vedran Franke Bioinformatics Group, Division of Biology, Faculty of Science, Zagreb University, Zagreb, Croatia Search for more papers by this author Minjun Cao Minjun Cao Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Yutaka Suzuki Yutaka Suzuki Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan The University of Tokyo, Tokyo, Japan Search for more papers by this author Masataka G Suzuki Masataka G Suzuki Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Kristian Vlahovicek Kristian Vlahovicek Bioinformatics Group, Division of Biology, Faculty of Science, Zagreb University, Zagreb, Croatia Department of Informatics, University of Oslo, Oslo, Norway Search for more papers by this author Petr Svoboda Corresponding Author Petr Svoboda Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Search for more papers by this author Richard M Schultz Corresponding Author Richard M Schultz Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Fugaku Aoki Corresponding Author Fugaku Aoki Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Ken-ichiro Abe Ken-ichiro Abe Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Ryoma Yamamoto Ryoma Yamamoto Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Vedran Franke Vedran Franke Bioinformatics Group, Division of Biology, Faculty of Science, Zagreb University, Zagreb, Croatia Search for more papers by this author Minjun Cao Minjun Cao Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Yutaka Suzuki Yutaka Suzuki Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan The University of Tokyo, Tokyo, Japan Search for more papers by this author Masataka G Suzuki Masataka G Suzuki Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Kristian Vlahovicek Kristian Vlahovicek Bioinformatics Group, Division of Biology, Faculty of Science, Zagreb University, Zagreb, Croatia Department of Informatics, University of Oslo, Oslo, Norway Search for more papers by this author Petr Svoboda Corresponding Author Petr Svoboda Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Search for more papers by this author Richard M Schultz Corresponding Author Richard M Schultz Department of Biology, University of Pennsylvania, Philadelphia, PA, USA Search for more papers by this author Fugaku Aoki Corresponding Author Fugaku Aoki Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan Search for more papers by this author Author Information Ken-ichiro Abe1,‡, Ryoma Yamamoto1,‡, Vedran Franke2,‡, Minjun Cao1, Yutaka Suzuki3,4, Masataka G Suzuki1, Kristian Vlahovicek2,5,‡, Petr Svoboda 6, Richard M Schultz 7 and Fugaku Aoki 1 1Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan 2Bioinformatics Group, Division of Biology, Faculty of Science, Zagreb University, Zagreb, Croatia 3Department of Medical Genome Science, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan 4The University of Tokyo, Tokyo, Japan 5Department of Informatics, University of Oslo, Oslo, Norway 6Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic 7Department of Biology, University of Pennsylvania, Philadelphia, PA, USA ‡These authors contributed equally to this work ‡Corresponding author for computational biology *Corresponding author. Tel: +420 241063147; E-mail: [email protected] *Corresponding author. Tel: +1 215 898 7869; E-mail: [email protected] *Corresponding author. Tel: +81 471 36 3695; Fax: +81 471 36 3698; E-mail: [email protected] The EMBO Journal (2015)34:1523-1537https://doi.org/10.15252/embj.201490648 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Initiation of zygotic transcription in mammals is poorly understood. In mice, zygotic transcription is first detected shortly after pronucleus formation in 1-cell embryos, but the identity of the transcribed loci and mechanisms regulating their expression are not known. Using total RNA-Seq, we have found that transcription in 1-cell embryos is highly promiscuous, such that intergenic regions are extensively expressed and thousands of genes are transcribed at comparably low levels. Striking is that transcription can occur in the absence of defined core-promoter elements. Furthermore, accumulation of translatable zygotic mRNAs is minimal in 1-cell embryos because of inefficient splicing and 3′ processing of nascent transcripts. These findings provide novel insights into regulation of gene expression in 1-cell mouse embryos that may confer a protective mechanism against precocious gene expression that is the product of a relaxed chromatin structure present in 1-cell embryos. The results also suggest that the first zygotic transcription itself is an active component of chromatin remodeling in 1-cell embryos. Synopsis Transcriptome analysis in mouse 1-cell embryos reveals widespread transcription of intergenic regions devoid of core-promoter elements. The resulting RNAs, possibly involved in chromatin remodeling, are poorly processed to prevent aberrant expression. The first round of zygotic transcription occurs at low levels and is genome-wide. Zygotic transcription is opportunistic and can occur without defined core-promoter elements. Gene transcription in the zygote is uncoupled from splicing and 3′ processing, leaving most transcripts nonfunctional. Genes transcribed in zygotes often yield functional mRNAs in 2-cell embryos. Introduction The oocyte-to-embryo transition (OET) entails a dramatic reprogramming of gene expression and conversion of a differentiated transcriptionally quiescent oocyte to totipotent blastomeres (De La Fuente & Eppig, 2001; Abe et al, 2010). The timing of zygotic genome activation (ZGA) is species dependent. Genome activation in mice is the earliest for mammals studied to date; the first wave of transcription (also referred to as the minor ZGA wave) starts at the mid-1-cell stage shortly after pronucleus formation, as evidenced by BrdU incorporation (Bouniol et al, 1995; Aoki et al, 1997) and expression of sperm-borne transgenes (Matsumoto et al, 1994). Although repetitive elements, for example, B2-containing sequences (Vasseur et al, 1985) and MuERV-L (Kigami et al, 2003), are expressed in 1-cell mouse embryos, transcription of single-copy genes is poorly understood, as is their function, which is not necessary for cleavage to the 2-cell stage (Warner & Versteegh, 1974). Studies using plasmid-borne reporter genes have provided insights regarding mechanisms that govern transcription in 1-cell embryos. These studies demonstrated that reporter gene expression in 1-cell embryos does not require an enhancer for efficient expression, whereas an enhancer is required for efficient expression in 2-cell embryos (Wiekowski et al, 1991; Majumder et al, 1993). Thus, 1-cell embryos are transcriptionally permissive, but development to the 2-cell stage is accompanied by formation of a transcriptionally repressive state (DePamphilis, 1993) and genome-wide accumulation of repressive histone modification marks (Santos et al, 2005). This developmental change in transcriptional regulation involves DNA replication at the 2-cell stage because an enhancer is not required for efficient transcription when 2-cell embryos were treated with aphidicolin, which inhibits DNA replication (Wiekowski et al, 1991; Majumder et al, 1993; Henery et al, 1995; Forlani et al, 1998). The transcriptionally repressive state likely stems from chromatin structure because increasing histone acetylation in 2-cell embryos by treating the embryos with butyrate, an inhibitor of histone deacetylase, relieves the requirement for an enhancer for efficient expression of the reporter gene (Majumder et al, 1993; Wiekowski et al, 1993). Similar conclusions were reached using expression of the 2-cell transiently expressed endogenous Eif1a gene (Davis et al, 1996). Microarray profiling identified zygotic mRNA expression at the 2-cell (major ZGA wave) but not the 1-cell stage (Hamatani et al, 2004; Wang et al, 2004; Zeng & Schultz, 2005). This finding suggests that mRNAs are either not expressed or microarray profiling is not sensitive enough to detect mRNAs produced during minor ZGA. More recent high-throughput sequencing (HTS) experiments identified hundreds of different mRNAs with increased abundance in 1-cell embryos (Park et al, 2013; Xue et al, 2013; Deng et al, 2014). However, due to increased RNA adenylation in 1-cell embryos (Piko & Clegg, 1982), poly(A) RNA sequencing results are difficult to interpret as they might reflect polyadenylation changes and not 1-cell transcription per se (Xue et al, 2013; Deng et al, 2014). The most comprehensive study regarding transcription in 1-cell embryos to date used SOLiD sequencing after ribosomal RNA depletion and identified ~600 genes upregulated > 1.5-fold between the oocyte and 1-cell embryo (Park et al, 2013). In this study, we employed total RNA sequencing to identify sequences transcribed in 1-cell embryos and to gain understanding about mechanisms that govern their expression. We show that pervasive transcription occurs in intergenic regions including many transposons whose transcription continues far into their genomic flanks; transcription can occur independently of defined core-promoter elements; over four thousand genes are transcribed in 1-cell embryos with ~5% being transcribed transiently; the majority of genes transcribed in 1-cell embryos are also transcribed in 2-cell embryos when the major wave of genome activation occurs; and mRNAs transcribed at the 1-cell stage are mostly nonfunctional because their 3′ end processing and splicing are highly inefficient. Results Global analysis of the first wave of transcription To explore the first wave of zygotic transcription, we characterized total RNA in metaphase II-arrested eggs (MII eggs) and embryos by HTS. We sequenced total RNA rather than selecting poly(A)-containing RNA (poly(A) RNA) because extensive RNA polyadenylation that occurs post-fertilization would be difficult to distinguish from bona fide zygotic mRNA synthesis (Piko & Clegg, 1982; Oh et al, 2000; Meijer et al, 2007). In addition, the RNA was not amplified to further minimize any deviation from the initial distribution of mRNAs. We isolated RNA from two sets of MII eggs and 1-cell embryos and one set of 2-cell and 4-cell embryos, morulae, and blastocysts. We also prepared RNA from 1-cell embryos in which transcription was inhibited by 5,6-dichlorobenzimidazole riboside (DRB), an inhibitor of RNA polymerase II activity (Sehgal et al, 1976), and 2-cell embryos treated with the DNA replication inhibitor aphidicolin (Bucknall et al, 1973) (Supplementary Table S1). Libraries from two sets of MII eggs and 1-cell embryos were subjected to 35-nt single-end sequencing (35SE); one set from each stage was subjected to 76-nt paired-end (76PE) sequencing with depths of 33–58 × 106 (Supplementary Table S1). Analysis of the sequencing results showed a high degree of reproducibility among the duplicate sets as well as for biological replicates (Supplementary Fig S1A). Comparison of our 76PE Illumina data and 50SE SOLiD data obtained by sequencing of total RNA depleted of ribosomal RNA from identical stages (Park et al, 2013) showed a high concordance of the signal distribution along chromosomes (r2 = ~0.8, Supplementary Fig S1B) and a good similarity of transcriptome changes between different stages (r2 = ~0.6, Supplementary Fig S1C), confirming the overall reproducibility of our data. The relative abundance of rRNA, repetitive sequences, annotated mRNAs, and unique sequences in the individual libraries were consistent with previous measurements (Fig 1A and Supplementary Fig S1D) (Piko & Clegg, 1982). For example, the fraction of mRNA in MII eggs and early cleavage-stage embryos is greater than that in somatic cells—for example, mRNA makes 2.5% of total RNA mass in HeLa cells (Jackson et al, 2000)—and the decline in the relative abundance of mRNA between the MII egg and 2-cell embryo correlates with the known degradation of maternal mRNA during this developmental period (Bachvarova & De Leon, 1980; Piko & Clegg, 1982). The fraction of mRNA was somewhat higher than previously reported (Piko & Clegg, 1982), which likely reflects that total RNA for HTS was size-selected for RNAs > 200 nt, causing a higher incidence of mRNAs in sequenced libraries. The small increase in the representation of rRNA and repeat-derived sequences at the expense of mRNA-derived reads in 1-cell embryos (Fig 1A) could be a consequence of ongoing maternal mRNA degradation in 1-cell embryos and/or new transcription of rRNA and retrotransposons. The frequency of 5′ external transcribed spacer (ETS)-derived reads from the 45S rRNA precursor, an indirect proxy of rRNA transcription, did not suggest robust rRNA transcription in 1-cell embryos because the amount of 5′ ETS-derived reads in MII eggs was actually greater than that in 1-cell embryos (Supplementary Fig S2A). However, in agreement with rDNA transcription initiating during the 2-cell stage (Zatsepina et al, 2003), we observed a ~fivefold increase in the number of 5′ ETS-derived reads between the 1-cell and 2-cell stages and a further more dramatic increase between 2-cell and 4-cell stages (Supplementary Fig S2B). Figure 1. General features of sequenced total RNA during ZGA A. Composition of selected libraries produced from total RNA. Shown is the proportion of reads matching rRNA-derived transcripts (rRNA), transcripts produced from repeats identified by RepeatMasker (repeat), annotated mRNAs (mRNA), and other sequences (other). B. Hierarchical clustering of 76-nt paired-end datasets from different stages of preimplantation development performed on Spearman correlations between log2 of RPKM values for transcripts. C, D. Comparison of MII and 1-cell transcriptomes (C) and 1-cell and 1-cell + DRB transcriptomes (D). The graph was made by plotting expression values for mRNAs (log2[RPKM + 1]) from 76PE sequencing data. Each point represents one transcript whose position indicates its abundance value in indicated stages. RPKM values were increased by adding 1 in order to obtain log2 values above zero. Download figure Download PowerPoint Unsupervised clustering of the samples based on reads mapping to exon sequences showed that the 1-cell embryo mRNA transcriptome was very similar to that of MII eggs and that DRB treatment had little apparent effect on the transcriptome (Fig 1B). These results are consistent with previous transcriptome studies (Hamatani et al, 2004; Wang et al, 2004; Zeng & Schultz, 2005) and are further supported by quantification of reads mapping to exons in MII eggs, 1-cell, and 1-cell + DRB samples (Fig 1C and 1D) lending confidence that new insights obtained from our HTS approach are warranted and not an experimental artifact. The most notable change in the 1-cell RNA population relative to that of MII eggs was a widespread occurrence of individual, rarely overlapping, DRB-sensitive reads whose density rarely exceeded a few reads per locus (~0.1 counts per million (CPM), hence referred to as low CPM reads hereafter). In a display of a larger genomic region, low CPM reads appeared as an unevenly distributed ‘grass in a forest’ (Fig 2 and Supplementary Fig S3). Low CPM reads appeared more frequently within gene-rich regions but were readily found in intergenic regions (Fig 2A and Supplementary Fig S3A). Although low CPM reads were also observed at later embryonic stages, their appearance was not as uniform and striking as in 1-cell embryos. Figure 2. Global view of the minor wave of ZGA Transcription at the 1-cell stage manifests as increased number of low CPM reads scattered across the genome. Shown are HTS data [a customized screenshot from the UCSC Genome browser (Karolchik et al, 2012)] from different stages mapped into a 5-Mb region of the genome with a variable gene density. The vertical scale was trimmed at 0.5 CPM; trimming is indicated by horizontal dashed lines. Note the appearance of low-density reads in 1-cell embryos (indicated by the black arrow), which are not observed in unfertilized oocytes and 1-cell embryos treated with DRB. Low CPM reads also appear in intergenic regions. Masking maternally transcribed regions reveals chromosome-wide presence of low CPM reads in 1-cell embryos. A UCSC browser screenshot of the chromosome 16 shows 76PE raw data mapped to the genome upon masking reads derived from oocytes. The vertical scale was trimmed at 20 reads; trimming is indicated by horizontal dashed lines. A small number of reads detected in MII eggs comes from repetitive sequences, which otherwise do not yield signal because of multi-mapping restrictions. Zygotic transcription from the Y chromosome initiates at the 1-cell stage. Shown are HTS data from different stages mapped into the ˜3-Mb annotated region of the chromosome Y. The vertical scale was trimmed at 0.5 CPM; trimming is indicated by horizontal dashed lines. The residual signal on the chromosome Y in MII eggs and 1-cell embryos treated with DRB is an artifact caused by common retrotransposon-derived sequences (mainly MT-derived). Download figure Download PowerPoint Because the presence of low CPM reads became readily apparent when sequences present in MII eggs were masked (Fig 2B), we examined the annotated proximal end of the Y chromosome (~3 Mb) (Fig 2C). Transcripts detected from this region must be of zygotic origin, and thus, low CPM reads would not be obscured by maternal mRNAs. Indeed, Y-chromosome-derived, DRB-sensitive low CPM reads were readily observed in the 76PE dataset (Fig 2C), and low CPM reads were also found in 50SE data (Park et al, 2013) (Supplementary Fig S3B). Given that the low CPM reads originated from genes as well as intergenic regions, we explored these two transcript categories separately. Widespread intergenic transcription in 1-cell embryos The occurrence of low CPM reads in intergenic regions was first confirmed by quantitative analysis. We divided intergenic regions across the entire genome into 1-kb segments and determined the number of segments to which at least a single read was uniquely mapped. The number of 1-kb loci harboring uniquely mapped reads was threefold higher in 1-cell embryos than in MII eggs (Fig 3A). This number decreased gradually with development and reached levels similar to those of MII eggs at the morula stage (Fig 3A), while the number of loci harboring uniquely mapped reads in DRB-treated 1-cell embryos remained similar to that in MII eggs. Furthermore, aphidicolin treatment of 2-cell embryos to inhibit the DNA replication restored the number of loci to the level observed in 1-cell embryos. These results suggest the presence of a transcriptionally permissive state in 1-cell embryos might be governed by the same mechanisms that regulate expression of plasmid-borne reporter genes during this period of development and the 2-cell stage (Wiekowski et al, 1991; Majumder et al, 1993). Figure 3. Analysis of genome-wide intergenic transcription in 1-cell embryos A. Quantitative analysis of transcription from intergenic regions in oocytes and preimplantation embryos. Intergenic regions were divided into 1-kb segments across the whole genome, and the number of segments to which at least a single read was uniquely mapped was determined. Results suggest that treatment of 1-cell embryos with DRB reduced the number of segments yielding intergenic transcripts. In contrast, treatment of 2-cell embryos with aphidicolin (Aph.) prevented reduction of the number of segments yielding intergenic transcripts. Bars representing samples treated with DRB and aphidicolin are gray to distinguish results from treated embryos and from the normal course of preimplantation development. An asterisk indicates a significant difference by chi-square test (P < 0.05). B. Frequency of reads derived from mobile elements. Association of reads with a particular class of mobile DNA in each sample was determined by RepeatMasker and is displayed as a percentage of those reads in each library. C. Relative changes of expression of four specific retrotransposons. Relative expression for each retrotransposon was calculated from RPKMs where expression in 1-cell embryos was set to one. D. MuERV-L retrotransposons asymmetrically neighbor higher frequency of low CPM reads in large intergenic regions. Shown is an example of MuERV-L located in an intergenic region, which is not maternally expressed and becomes highly transcribed during zygotic genome activation at the 2-cell stage. A part of its transcription apparently invades almost 150 kb of its genomic flank, whereas the same region downstream of the MuERV-L yields higher frequency of low CPM reads in 1-cell stage in DRB-dependent manner. This pattern is most apparent for MuERV (see also Supplementary Fig S4B), but MT2 and ORR1AO insertions can also produce a similar asymmetric pattern (data not shown). The vertical scale was trimmed at 0.5 CPM; trimming is indicated by horizontal dashed lines. E, F. Validation of intergenic transcription at the 1-cell stage. Two intergenic regions harboring low CPM reads on chromosome 2 in 1-cell embryos were analyzed by RT–PCR. Both loci are annotated in Supplementary Fig S4C. Shown in (E) is RT–PCR analysis of intergenic transcription of two loci (shown in detail in Supplementary Fig S4C) in MII eggs and 1-cell embryos treated with and without DRB. ctrl = RT–PCR of a spiked α-rabbit globin mRNA demonstrating consistent RT–PCR efficiency across samples. Shown in (F) is the effect of inhibiting the second round of DNA replication in intergenic transcription at the 2-cell stage as examined by RT–PCR. Two-cell embryos were treated with aphidicolin (Aph.) 15 h after insemination to inhibit the second round of DNA replication. ctrl = RT–PCR of a spiked rabbit α-globin mRNA demonstrating consistent RT–PCR efficiency across samples. Experiments were performed three times with reproducible results; a representative result is shown (E, F). Source data are available online for this figure. Source Data for Figure 3 [embj201490648-sup-0006-SDataFig3e-f.pdf] Download figure Download PowerPoint Intergenic transcripts yielding low CPM reads could be either short (i.e., one read or pair of reads would represent one short transcription unit) or they could be fragments of rare long transcripts (i.e., one transcript would yield multiple sequenced fragments, most of which would be far from the actual transcription start site). Two observations suggest some intergenic low CPM reads were derived from fragmented long transcripts. First, when the sequenced libraries were combined to achieve greater depth, low CPM reads representing discrete short transcriptional units did not overlap but rather were more densely populated over a larger area (Supplementary Fig S3A). Second, numerous transcribed intergenic regions at the 1-cell stage correlated with downstream regions of several different retrotransposons whose transcription at the 2-cell stage apparently invaded their neighborhood. Repetitive sequences represent a source of potential promoters for intergenic transcription. Accordingly, we analyzed repetitive element-derived reads and found that the 1-cell stage contained the highest frequency of retrotransposon-derived reads among all the samples (Fig 3B). The increase in retrotransposon-derived read abundance between MII and 1-cell stage was DRB-sensitive and involved all major classes of retrotransposons (LINE, LTR, and SINE). Consistent with the development of a DNA replication-dependent transcriptionally repressive environment in 2-cell embryos, aphidicolin treatment resulted in higher frequency of retrotransposon-derived reads (Fig 3B). When examined individually, different retrotransposons showed diverse patterns of expression and various levels of transcription at the 1-cell stage (Fig 3C and Supplementary Fig S4A). The best example of a retrotransposon-supplied promoter producing long intergenic transcripts was the type L mouse endogenous retrovirus (MuERV-L). Transcription of MuERV-L is detected in 1-cell embryos and is very high in 2-cell embryos (Kigami et al, 2003; Svoboda et al, 2004), and was confirmed by our HTS data (Supplementary Fig S4B). Strikingly, a genomic flank on one side of MuERV-L appeared transcribed up to 200 kb downstream of the element (Fig 3D and Supplementary Fig S4B). In 1-cell embryos, we did not observe high read density over the retrotransposon but saw low CPM reads within the same area as in 2-cell embryos (Fig 3D, Supplementary Fig S4B and C). This feature of MuERV-L elements seemed a general feature that yielded a prominent pattern that became apparent when sequencing data from larger genomic regions were displayed (Supplementary Fig S4B). To confirm intergenic transcription in 1-cell embryos, we selected two intergenic regions for which reads were uniquely mapped in 1-cell embryos but not MII eggs and examined their expression by RT–PCR. One of these intergenic regions was not MuERV-associated (locus #1), whereas the other was downstream of an MuERV-L element (locus #2) (Supplementary Fig S4C). We first confirmed that transcription of these intergenic regions occurred only in 1-cell embryos but not in MII eggs or 1-cell embryos treated with DRB (Fig 3E). We also observed that their expression decreased by the 2-cell stage but remained high when DNA replication was inhibited (Fig 3F). Taken together, these results suggest that a transcriptionally permissive state fosters promiscuous expression from intergenic regions in 1-cell embryos and includes retrotransposon transcription. Subsequently, promiscuous expression at the 2-cell stage is inhibited in DNA replication-dependent manner. Core-promoter element-independent transcription in 1-cell embryos To identify sequence features controlling 1-cell transcription, we constructed pGL3 luciferase vectors containing the promoter region of the Zp3 or Tktl1 gene (pGL3-Zp3 or pGL3-Tktl1, respectively), which are expressed in growing oocytes or embryos at the 1- and 2-cell stages, respectively (Hamamoto et al, 2014). These constructs, in principle, allow to test whether transcription in 1-cell embryos can initiate at ‘maternal’ (active only in the oocyte) and/or at ‘zygotic’ (active in preimplantation embryos) promoters. As a negative control, we used the original pGL3 luciferase vector lacking the gene promoter (pGL3-Basic). Growing oocytes microinjected with pGL3-Zp3 showed a significant level of luciferase activity as expected, whereas 1-cell embryos did not (Fig 4A). Surprisingly, the pGL3-Basic vector supported luciferase activity in 1-cell embryos but not in oocytes at a level comparable to that of pGL3-Zp3 in oocytes (Fig 4A). A corresponding observation was made when the transcriptional activity of pGL3-Tktl1 and pGL3-Basic was examined in 1- and 2-cell embryos (Fig 4B). Figure 4. Transcription from a microinjected plasmid lacking an annotated promoter Transcriptional activity in pGL3-Basic vector lacking a promoter in oocytes and 1-cell embryos. pGL3 vector with the Zp3 or no promoter sequence (pGL3-Zp3 or pGL3-Basic vector, respectively) was injected into the nuclei of growing oocytes or the male pronucleus of 1-cell embryos. After 8 h, luciferase activity was measured. Data are expressed as mean ± SEM (n = 3) and were analyzed using Student's t-test. Asterisks indicate significant differences (P < 0.05). Transcriptional activity in pGL3-Basic vector lacking a promoter in 1-cell and 2-cell embryos. The analysis was performed similarly as in (A), except a pGL3 vector with the Tktl1 promoter (pGL3-Tktl1) replaced pGL3-Zp3 and the experiment was performed in 1-cell and 2-cell embryos. Data are expressed as mean ± SEM (n = 3) and were analyzed using Student's t-test. Asterisks indicate significant differences (P < 0.05). Determination of
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