Information

Activation Of Embryonic Genome


Embryonic gene activation is a process by which the embryo begins to transcribe its newly formed genome.As the embryonic gene activation occurs during early stages the paternal genome may not have any influence at that stage.

Now, My question is why does only the maternal genome play a role in embryonic gene activation? Does paternal genome have a role in prior/post embryonic genome activation?


The pronuclei fuse and decondensation happens and now the cell is called a zygote. There is a stage called the midblastula transition (MBT), until which only the maternal products (cytoplasmic factors such as RNAs, proteins and ribosomes) are used. After MBT the zygote starts expressing its own genes from both the paternal and maternal alleles. However, certain genomic regions are imprinted which means that one of the alleles is epigenetically silenced. It can be either paternal or maternal.


Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells

H3K27ac is well recognized as a marker for active enhancers and a great indicator of enhancer activity. However, its functional impact on transcription has not been characterized. By substituting lysine 27 in histone variant H3.3 with arginine in mouse embryonic stem cells, we diminish the vast majority of H3K27ac at enhancers. However, the transcriptome is largely undisturbed in these mutant cells, likely because the other enhancer features remain largely unchanged, including chromatin accessibility, H3K4me1, and histone acetylation at other lysine residues. Our results clearly reveal that H3K27ac alone is not capable of functionally determining enhancer activity.


The Maternal-to-Zygotic Transition

Célia Baroux , Ueli Grossniklaus , in Current Topics in Developmental Biology , 2015

Abstract

The maternal-to-zygotic transition (MZT) defines a developmental phase during which the embryo progressively emancipates itself from a developmental control relying largely on maternal information. The MZT is a functional readout of two processes: the clearance of maternally derived information and the de novo expression of the inherited, parental alleles enabled by zygotic genome activation (ZGA). In plants, for many years the debate about whether the MZT exists at all focused on the ZGA alone. However, several recent studies provide evidence for a progressive alleviation of the maternal control over embryogenesis that is correlated with a gradual ZGA, a process that is itself maternally controlled. Yet, several examples of zygotic genes that are expressed and/or functionally required early in embryogenesis demonstrate a certain flexibility in the dynamics and kinetics of the MZT among plant species and also intraspecific hybrids.


Histone modifications are the influencers of zygotic genome awakening

Fab-based imaging to monitor the dynamics of active transcription and histone modifications during ZGA in living zebrafish embryos revealed spatiotemporal coordination within single nuclei, where H3K27 acetylation was concentrated at distinct foci on the miR-430 gene cluster prior to its transcription. These observations, together with inhibitor assays, suggest that H3K27ac precedes active transcription during ZGA. Credit: Tokyo Tech

The zebrafish is an important model organism in biology. Humans and zebrafish share 70 percent of their genes, and more than 80 percent of human genes associated with disease are known to have a zebrafish counterpart. Additionally, their entire genome sequence has been identified, and it is more efficient to grow fish than mice.

Their development, however, is a little different from that of mammals. Although fish reproduce sexually, like all vertebrates, fertilization of the egg is external, as both the egg and sperm are released into the water. Despite this important difference, the majority of sequences involved in protein synthesis associated with embryonic development and regulation is conserved across species.

This means that some of the basic building blocks that give cells the instructions of how to build a living organism are conserved across species. Instead of getting rid of these sequences, evolution regulates their expression through modifications of genomic DNA and histone proteins, around which DNA is bound. These "epigenetic factors," as scientists call them, determine whether genes become functional or not because they alter the chemical and/or structural composition of DNA. This regulation of what gets to become what is particularly important when an organism develops.

A group of scientists from Tokyo Tech, led by Professor Hiroshi Kimura, set out to study epigenetic changes during zebrafish development. Professor Kimura says, "The structural conservation of histones and crucial enzymes for transcription (i.e., RNA polymerases II), as well as the fact that zebrafish embryos are transparent, makes them a perfect model to study the role of these modifications in the development of living organisms."

(A) Scheme of experiments. Fluorescently labeled Fabs prepared from modification-specific antibodies are injected into 1-cell-stage zebrafish embryos, which are then mounted for a lightsheet (SiMView) or a confocal (FV1000) microscope. (B) Representative images taken with a SiMView microscope. Fabs specific to active, phosphorylated RNA polymerase II (RNAP2 Ser2ph) and histone H3 acetylated at the 9th lysine (H3K9ac) were simultaneously injected and imaged. RNAP2 Ser2ph Fabs were clearly concentrated in nuclei around the 1k-cell stage, whereas H3K9ac Fabs were enriched in nuclei from the 8-cell stage. Scale bar: 100 μm. Credit: Tokyo Tech, Development

Using fab-based live endogenous modification labeling, or FabLEM, a technique to visualize the dynamics of post-translational modifications using specific antibodies in live cells (Figure 2), the team observed active RNA polymerase II during zygotic genome activation (ZGA), or the process during which the development of the embryo comes under control of the zygote or the developing embryo itself instead of the maternally deposited material. Although the timing of this transition is species-specific, it also occurs in mammals.

At the 512-cell stage embryos, H3K27 acetylation is accumulated (H3K27ac arrow in magenta) before the onset of transcription as indicated by active phosphorylated RNA polymerase II (RNAP2 Ser2ph arrow in green). Magnified images of miR-430 locus are also shown. Scale bar: 10 μm. Credit: Tokyo Tech, Development

Then the team compared the changes in histone modification with the timing of genome activation. One of the main findings of their studies was that acetylation that occurs on the 27th lysine in histone H3, or H3K27, becomes concentrated prior to active transcription (Figure 3). Visualizing transcripts of the earliest activated gene miR-430 together with H3K27 acetylation, confirmed the findings, and transcription inhibitor did not prevent the accumulation of H3K27 acetylation. What the results suggest is that H3K27 acetylation is an epigenetic factor that awakes the genome for transcription.

Because RNA polymerase II is present in all species, the method can also be applied to study the development in other living organisms. "This opens the possibility of further research on the link between transcription regulation and nuclear organization," remarks Professor Kimura.


Regulation of zygotic gene activation in the preimplantation mouse embryo: global activation and repression of gene expression

Superimposed on the activation of the embryonic genome in the preimplantation mouse embryo is the formation of a transcriptionally repressive state during the two-cell stage. This repression appears mediated at the level of chromatin structure, because it is reversed by inducing histone hyperacetylation or inhibiting the second round of DNA replication. We report that of more than 200 amplicons analyzed by mRNA differential display, about 45% of them are repressed between the two-cell and four-cell stages. This repression is scored as either a decrease in amplicon expression that occurs between the two-cell and four-cell stages or on the ability of either trichostatin A (an inhibitor of histone deacetylases) or aphidicolin (an inhibitor of replicative DNA polymerases) to increase the level of amplicon expression. Results of this study also indicate that about 16% of the amplicons analyzed likely are novel genes whose sequence doesn't correspond to sequences in the current databases, whereas about 20% of the sequences expressed during this transition likely are repetitive sequences. Lastly, inducing histone hyperacetylation in the two-cell embryos inhibits cleavage to the four-cell stage. These results suggest that genome activation is global and relatively promiscuous and that a function of the transcriptionally repressive state is to dictate the appropriate profile of gene expression that is compatible with further development.


Materials and methods

Mouse models, human CRC patients and tumor collection

Mouse tumors

All tumors were isolated as spontaneously occurring lesions in Apc Min/+ [57], Smad3 -/- [58], and Tgfb1 -/- Rag2 -/- , collected at three-to-nine months of age depending on the model (for a review, see [6]). The only exceptions were two Apc Min/+ tumors, UW_3_2778 and UW_6_2748, that were 13 and 14 months and the three Tgfb1 -/- Rag2 -/- tumors, all five of which had histological features of locally invasive carcinoma [7]. Three- to four-month old mice from various AXB recombinant inbred lines were treated with AOM doses chosen for enhancement of inter-strain differences in susceptibility [11]. Mice were given four weekly i.p. injections of 10 mg AOM per kg body weight, and tumors were collected six months after the first injection. Animals were euthanized with CO2, colons removed, flushed with 1× phosphate-buffered saline (PBS), and laid out on Whatman 3 MM paper. A summary of the mouse strains, mutant alleles and source laboratories is presented in Table 5. All tumors were obtained from the colon only, the particular segment of which is indicated in the Gene Expression Omnibus (GEO) database [59] reposited sample information (GSE5261). The majority of Tgfb1 -/- Rag2 -/- and Smad3 -/- tumors occur in the cecum and proximal colon and all samples isolated for characterization were obtained from there. In contrast, tumors isolated from Apc Min/+ and AOM mice occurred predominantly in the mid- and distal colon. A small portion of the tumor was placed in formalin for histology, with the remainder finely dissected into RNAlater (Ambion Inc., Austin, TX, USA) and stored at -20°C. Normal adult colon RNA for reference was obtained from whole colon samples harvested from ten eight-week-old C57BL/6 male mice. The tissue was lysed in Trizol Reagent (Invitrogen Systems Inc., Carlsbad, CA, USA) and homogenized. Total RNA was purified using a Qiagen kit (USA-Qiagen Inc., Valencia, CA, USA).

Human samples: collection/biopsies, regulatory aspects, compliance and informed consents

Sample collection protocol and analyses at the H Lee Moffitt Cancer Center and Research Institute have been described previously [37]. Information collected with the samples for this study includes solid tumor staging criteria for tumor, nodes, and metastases (TNM), Dukes staging/presentation criteria, pathological diagnosis, and differentiation criteria.

RNA isolation

All RNA samples were purified using Trizol Reagent from finely dissected tumors and were subjected to quality control screening using the Agilent BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

Microarray procedures and data analysis

Mouse cDNA arrays

Mouse tumors were analyzed on Vanderbilt University Microarray Core (VUMC)-printed 20 K mouse cDNA arrays, composed principally of PCR products derived from three sources: the 15 K National Institute of Aging mouse cDNA library the Research Genetics mouse 5 K set and an additional set of cDNAs mapped to RefSeq transcripts. Labeling, hybridization, scanning, and quantitative evaluation of these two-color channel arrays were performed according to VUMC protocols [60] using a whole mouse Universal Reference standard (E17.5 whole fetal mouse RNA). Arrays were analyzed by GenePix version 3.0 (MDS Inc., Sunnyvale, CA, USA), flagged and filtered for unreliable measurements, with dye channel ratios corrected using Lowess and dye-specific correction normalization as previously described [15].

Human Affymetrix oligonucleotide arrays

Human RNA samples were labeled for hybridization to Affymetrix HG-U133plus2 microarrays using the Affymetrix-recommended standard labeling protocol (Small-scale labeling protocol version 2.0 with 0.5 μg of total RNA Affymetrix Technical Bulletin). Microarrays were scanned with MicroarraySuite version 5.0 to generate 'CEL' files that were processed using the RMA algorithm as implemented by Bioconductor [15].

Analysis strategy

The four different mouse models of CRC were compared for model-specific differences, then compared to mouse colon development stages, and then to human CRC samples (Figure 1). The mouse tumor sample array data are composed of Lowess-normalized Cy3:Cy5 labeling ratios of each individual tumor sample versus a universal E17.5 whole fetal mouse reference RNA (described using MIAME guidelines in the NCBI GEO database under series accession number GSE5261). The first approach to referencing was to compare normalized ratios across the tumor series. To do this, for each gene, the Lowess-corrected ratio for each probe element (sample versus E17.5 whole fetal mouse reference) was divided by the median ratio for that probe across the entire tumor sample series. This is termed the median-per-tumor expression ratio and was useful for identifying, clustering and visualizing differences that occur between the different tumor samples. Since we previously collected mouse expression data for normal E13.5-E18.5 colon samples from inbred C57BL/6J and outbred CD-1 mice [15] using the identical E17.5 whole fetal mouse reference, this allowed us to combine the data directly. Differential expression profiles in the tumors were combined with relative developmental gene expression levels by direct comparisons of ratios determined within each experimental series. Initial comparisons were made between median normalized tumor data to gene expression levels observed in the E13.5-E18.5 and adult (eight week post-natal) colon samples, which were referenced to either E13.5 samples or to the adult colon. The latter approach subsequently allowed for the broadest comparison of mouse and human data using gene ortholog mapping. Correlated phenomena could be observed from any of the different referencing strategies.

Inter-organism gene ortholog and inter-platform comparison strategy

Pairs of human and mouse ortholog genes (12,693) were curated using the Mouse Genome Informatics (MGI The Jackson Laboratory) [61] and National Center for Biotechnology Information (NCBI) Homologene [62] databases. Individual microarray elements or features were mapped to these. The concatenated human and mouse RefSeq IDs was used as the composite ID for the orthologous gene pair in the ortholog genome definition. NIA/Research Genetics mouse cDNAs were mapped to human orthologs using a variety of resources, usually via the Stanford Online Universal Reference resource [63]. Gene transcript assignments were made unique by choosing the longest corresponding transcript. To map the Affymetrix human and mouse array data into the ortholog genome, we used a sequence matching approach. First, we obtained human and mouse transcript sequences from RefSeq [64] and probe sequences from the manufacturer's website [65]. Next, we computed all perfect probe-transcript pairs. We excluded probes that matched multiple gene symbols but accepted probes that matched multiple transcripts. Probe sets were assigned to represent a given transcript if at least 50% of the perfect match probes of the probe set matched to that transcript. The newly assigned transcript identifiers were then used to map probe sets to ortholog genes. Since some transcripts have multiple probe-set representations on both the Affymetrix and cDNA microarrays to one ortholog identifier, we employed an ad hoc strategy to use the average of those probe sets or cDNAs that exhibited consistent regulation across a sample series. In such cases, the signals of the regulated probe sets that were interpreted as being in agreement were averaged and assigned to the corresponding ortholog. We excluded probe sets or cDNAs that we were aware corresponded to non-transcript genomic sequence as tested using BLAT at the UCSC Goldenpath website [66].

Mouse-human RefSeq gene ortholog assignments can be found at GenomeTrafac [67, 68]. All ortholog assignments and cross-species mapping annotations were incorporated into annotations associated with the Affymetrix HG-U133 plus2.0 genome. Gene expression ratios obtained for the mouse samples were then represented as expression values within the human platform for all of the probe sets that mapped to the corresponding mouse gene ortholog. Data for the primary human sample series, as well as the combined mouse-human data sets, are available in the Cincinnati Children's Hospital Medical Center microarray data server [69] in the HG-U133 genome under the KaiserEtAl_2006 folders ('guest' login all cross-platform ortholog gene identifiers are contained as annotation fields within the HG-U133 genome table).

Statistical and data visualization approaches

Most normalization, expression-level referencing, statistical comparisons, and data visualization were performed using GeneSpring v7.0 (Silicon Genetics-Agilent (part of Agilent Technologies). Fisher's exact test was performed online at the MATFORSK Fisher's Exact Test server [70]. To identify differentially expressed features between two or more classes, we applied GeneSpring's Wilcoxon-Mann-Whitney or the Kruskal-Wallis test, respectively. For three or more classes, the initial non-parametric test was followed by the Student-Newman-Keuls post-hoc test. Results from the primary analyses were corrected for multiple testing effects by applying Benjamini and Hochberg false discovery rate (FDR) correction [71]. In general, due to the referencing strategies, good platform technical performances, and moderately low within-group biological variation of gene expression, stringent cutoffs could be used, that is, the FDR level of significance was set between FDR < 5.10 -5 and FDR < 5.10 -4 . K-means clustering was performed using the GeneSpring K-means tool and the Pearson correlation similarity measure.

Ontology-based analysis of gene cluster-associated functional correlates

Gene expression clusters were analyzed for the occurrence of multiple genes involved in related gene function categories by comparing each list of coordinately regulated clustered genes to categories within Gene Ontology, pathways, or literature-based gene associations using GATACA [72], Ontoexpress [73], and Ingenuity Pathway Analysis, version 3 (IPA, Ingenuity Systems, Redwood City, CA, USA) [74]. To do this, each cluster indicated in Figures 2, 4, 5, 6 and 8 was converted to a list of gene identifiers, uploaded to the application, and examined for over-representation of multiple genes from one or more molecular networks, or functional or disease associations as developed from literature mining. Networks of these focus genes were algorithmically generated based on the relationships of individual genes as derived from literature review and used to identify the biological functions and/or associated pathological processes most significant for each gene cluster. Fisher's exact test was used to calculate a p value estimating the probability that a particular functional classification or category of genes is associated with a particular pattern or cluster of gene expression more than would be expected by chance. For each cluster, only the top significant functional classes and canonical pathways are shown. Figure 7a shows a diagram of the canonical WNT signaling pathway and an associated-gene network that was a top-ranked association of the clusters that exhibited significant over-expression in AOM and Apc Min/+ versus Smad3 -/- and Tgfb1 -/- Rag2 -/- mouse models. Genes or gene products are represented as nodes, and biological relationships between nodes are represented as edges (lines). All edges are supported by at least one literature reference from a manuscript, or from canonical information stored in the Ingenuity Pathways Knowledge Base.

QRT-PCR

To confirm the validity of data normalization and referencing procedures as well as the cDNA gene assignments of the printed arrays used in the microarray analyses, we used qRT-PCR to measure relative levels of nine genes found by microarray data analysis to be differentially expressed (FDR < 5.10 -5 ) in tumors from Apc Min/+ and Smad3 -/- mice. Total RNAs from C57BL6 Apc Min/+ and 129 Smad3 -/- tumor samples (20 μg) were reverse-transcribed to cDNA using the High Capacity cDNA Archive Kit (oligo-dT primed Applied Biosystems, Foster City, CA, USA). qRT-PCR reactions (20 μl) were set up in 96-well MicroAmp Reaction Plates (Applied Biosystems) using 10 ng of cDNA template in Taqman Universal PCR Master Mix and 6-FAM-labeled Assays-on-Demand primer-probe sets (Applied Biosystems). Reactions were run on an MX3000P (Stratagene, a division of Agilent Technologies) with integrated analysis software. Threshold cycle numbers (Ct) were determined for each target gene using an algorithm that assigns a fluorescence baseline based on measurements prior to exponential amplification. Relative gene expression levels were calculated using the ΔΔCt method [75], with the Gusb gene as a control. Fold-change was determined relative to expression in normal adult colon from two C57BL/6J mice.

Immunohistochemistry

Immunohistochemical procedures were performed as described [15]. Apc Min/+ and Smad3 -/- colon tumors were rapidly dissected, fixed in 4% paraformaldehyde, and embedded in paraffin before cutting 10 μm thick sections. Antigen retrieval was performed by boiling for 20 minutes in citrate buffer, pH 6.0. Sections were treated with 0.3% hydrogen peroxide in PBS for 30 minutes, washed in PBS, blocked in PBS plus 3% goat serum and 0.1% Triton X-100, and then incubated with primary antibodies and HRP-conjugated goat anti-rabbit secondary antibody (Sigma, St Louis, MO, USA). Antigen-antibody complexes were detected with a DAB peroxidase substrate kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's protocol.


Acknowledgements

The authors thank members of the Harrison laboratory and the reviewers for helpful feedback on the manuscript. K.N.S. was supported in part by the National Institutes of Health (NIH) National Research Service award T32 GM007215. M.M.H. was supported by grant R01GM11694 from the National Institute of General Medical Sciences and a Vallee Scholar Award.

Reviewer information

Nature Reviews Genetics thanks B. Cairns, K. Kuznetsova, N. Vastenhouw and the other anonymous reviewer(s) for their contribution to the peer review of this work.


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Activation of human embryonic gene expression in cytoplasmic hybrid embryos constructed between bovine oocytes and human fibroblasts

Cross-species somatic all number transfer (SCNT) provides a potential solution to overcome the problem of oocyte shortage for therapeutic cloning. To further characterize the system, we constructed cytoplasm hybrid embryos between bovine oocytes and human fibroblasts and examined dynamics of human gene activation during preimplantation stages. Data from this study showed that human embryonic genes, OCT4, SOX2, NANOG, E-CADHERIN, as well as beta-ACTIN, were activated by enucleated bovine oocytes. Activation of human genes was correlated with developmental potential of the embryos. The extent of human gene activation varied drastically and was incomplete in a large proportion of the embryos. Activation of human genes in the human-bovine cytoplasm hybrid embryos occurs in a temporal pattern resembling that of the bovine species. Results from this study suggest that human gene products are required for hybrid embryos to develop to later preimplantation stages. Facilitating human genome activation may improve successful rates in cross-species SCNT.


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