Chromatin profiling and state predictions reveal insights into epigenetic regulation during early porcine development.

BACKGROUND: Given their physiological similarities to humans, pigs are increasingly used as model organisms in human-oriented biomedical studies. Additionally, their value to animal agriculture across the globe has led to the development of numerous studies to investigate how to improve livestock welfare and production efficiency. As such, pigs are uniquely poised as compelling models that can yield findings with potential implications in both human and animal contexts. Despite this, many gaps remain in our knowledge about the foundational mechanisms that govern gene expression in swine across different developmental stages, particularly in early development. To address some of these gaps, we profiled the histone marks H3K4me3, H3K27ac, and H3K27me3 and the SWI/SNF central ATPase BRG1 in two porcine cell lines representing discrete early developmental time points and used the resulting information to construct predicted chromatin state maps for these cells. We combined this approach with analysis of publicly available RNA-seq data to examine the relationship between epigenetic status and gene expression in these cell types. RESULTS: In porcine fetal fibroblast (PFF) and trophectoderm cells (PTr2), we saw expected patterns of enrichment for each of the profiled epigenetic features relative to specific genomic regions. H3K4me3 was primarily enriched at and around global gene promoters, H3K27ac was enriched in promoter and intergenic regions, H3K27me3 had broad stretches of enrichment across the genome and narrower enrichment patterns in and around the promoter regions of some genes, and BRG1 primarily had detectable enrichment at and around promoter regions and in intergenic stretches, with many instances of H3K27ac co-enrichment. We used this information to perform genome-wide chromatin state predictions for 10 different states using ChromHMM. Using the predicted chromatin state maps, we identified a subset of genomic regions marked by broad H3K4me3 enrichment, and annotation of these regions revealed that they were highly associated with essential developmental processes and consisted largely of expressed genes. We then compared the identities of the genes marked by these regions to genes identified as cell-type-specific using transcriptome data and saw that a subset of broad H3K4me3-marked genes was also specifically expressed in either PFF or PTr2 cells. CONCLUSIONS: These findings enhance our understanding of the epigenetic landscape present in early swine development and provide insight into how variabilities in chromatin state are linked to cell identity. Furthermore, this data captures foundational epigenetic details in two valuable porcine cell lines and contributes to the growing body of knowledge surrounding the epigenetic landscape in this species.

Chromatin remodeling in mammalian embryos.

The mammalian embryo undergoes a dramatic amount of epigenetic remodeling during the first week of development. In this review, we discuss several epigenetic changes that happen over the course of cleavage development, focusing on covalent marks (e.g., histone methylation and acetylation) and non-covalent remodeling (chromatin remodeling via remodeling complexes; e.g., SWI/SNF-mediated chromatin remodeling). Comparisons are also drawn between remodeling events that occur in embryos from a variety of mammalian species.

ARID1A, a component of SWI/SNF chromatin remodeling complexes, is required for porcine embryo development.

Mammalian embryos undergo dramatic epigenetic remodeling that can have a profound impact on both gene transcription and overall embryo developmental competence. Members of the SWI/SNF (Switch/Sucrose non-fermentable) family of chromatin-remodeling complexes reposition nucleosomes and alter transcription factor accessibility. These large, multi-protein complexes possess an SNF2-type ATPase (either SMARCA4 or SMARCA2) as their core catalytic subunit, and are directed to specific loci by associated subunits. Little is known about the identity of specific SWI/SNF complexes that serve regulatory roles during cleavage development. ARID1A, one of the SWI/SNF complex subunits, can affect histone methylation in somatic cells; here, we determined the developmental requirements of ARID1A in porcine oocytes and embryos. We found ARID1A transcript levels were significantly reduced in 4-cell porcine embryos as compared to germinal vesicle-stage oocytes, suggesting that ARID1A would be required for porcine cleavage-stage development. Indeed, injecting in vitro-matured and fertilized porcine oocytes with double-stranded interfering RNAs that target ARID1A, and evaluating their phenotype after seven days, revealed that the depletion of ARID1A results in significantly fewer cells than their respective control groups (p < 0.001).

Dynamic changes in nuclear import of a nuclear localisation signal-bearing substrate in 8-cell stage porcine embryos.

Coordinated intracellular trafficking is critically important for proper timing of major cellular events during embryogenesis. Nuclear import mediated by the karyopherin α/ß (importin α/ß) heterodimer is perhaps the best characterised nuclear trafficking system in eukaryotic cells. Seven karyopherin α subtypes have been identified in the domestic pig, and although each karyopherin α subtype transports proteins bearing classical nuclear localisation signals (NLSs), individual karyopherin α subtypes have been shown to preferentially transport specific cargoes. The aim of the present study was to determine the mechanism by which BRN2, a transcription factor previously reported to be transported by the karyopherin α/ß heterodimer, gains access to the nucleus in porcine oocytes and embryos. Using a combination of in vivo and in vitro assays, we tested the hypothesis that discrete karyopherin α subtypes transport BRN2 into the nuclei of porcine oocytes and cleavage stage embryos. Our results show that ectopically expressed BRN2 adopts a nuclear localisation in all nuclei through the 4-cell stage of development, whereas only a subset of blastomeres in 8-cell stage embryos possess nuclear BRN2. This pattern is unique to BRN2 because another ectopically expressed NLS-containing protein is able to adopt a nuclear localisation in all blastomeres of 8-cell stage embryos.

Mother's exercise during pregnancy programmes vasomotor function in adult offspring.

The intrauterine environment is influenced by maternal behaviour and programmes atherosclerotic disease susceptibility in offspring. The aim of this investigation was to test the hypothesis that mothers' exercise during pregnancy improves endothelial function in 3-, 5- and 9-month-old porcine offspring. The pregnant sows in the exercise group ran for an average of 39.35 ± 0.75 min at 4.81 ± 0.35 km h(-1) each day for 5 days per week for all but the last week of gestation. This induced a significant reduction in resting heart rate (exercised group, 89.3 ± 3.5 beats min(-1); sedentary group, 102.1 ± 3.1 beats min(-1); P < 0.05) but no significant differences in gestational weight gain (65.8 ± 2.1 versus 63.3 ± 1.9%). No significant effect on bradykinin-induced vasorelaxation with and without l-NAME was observed. A significant main effect was identified on sodium nitroprusside-induced vasorelaxation (P = 0.01), manifested by a reduced response in femoral arteries of all age groups from exercised-trained swine. Nitric oxide signalling was not affected by maternal exercise. Protein expression of MYPT1 was reduced in femoral arteries from 3-month-old offspring of exercised animals. A significant interaction was observed for PPP1R14A (P < 0.05) transcript abundance and its protein product CPI-17. In conclusion, pregnant swine are able to complete an exercise-training protocol that matches the current recommendations for pregnant women. Gestational exercise is a potent stimulus for programming vascular smooth muscle relaxation in adult offspring. Specifically, exercise training for the finite duration of pregnancy decreases vascular smooth muscle responsiveness in adult offspring to an exogenous nitric oxide donor.

The polycomb group protein EED varies in its ability to access the nucleus in porcine oocytes and cleavage stage embryos.

Chromatin-modifying complexes serve essential functions during mammalian embryonic development. Polycomb group proteins EED, SUZ12, and EZH2 have been shown to mediate methylation of the lysine 27 residue of histone protein H3 (H3K27), an epigenetic mark that is linked with transcriptional repression. H3K27 trimethylation has been shown to be present on chromatin in mature porcine oocytes, pronuclear and 2-cell stage embryos, with H3K27 trimethylation decreasing at the 4-cell stage and not detectable in blastocyst stage embryos. The goals of this study were to determine the intracellular localization of the polycomb group protein EED in porcine oocytes and cleavage stage porcine embryos produced by in vitro fertilization and to determine the binding abilities of karyopherin α subtypes toward EED. Our results revealed that EED had a strong nuclear localization in 4-cell and blastocyst stage embryos and a strong perinuclear staining in GV-stage oocytes; EED was not detectable in the nuclei of pronuclear or 2-cell stage embryos. An in vitro binding assay was performed to assess the ability of EED to interact with a series of karyopherin α subtypes; results from this experiment revealed that EED can interact with several karyopherin α subtypes, but with varying degrees of affinity. Together these data indicate that EED displays a dynamic change in intracellular localization in progression from immature oocyte to cleavage stage embryo and that EED possess differing in vitro binding affinities toward individual karyopherin α subtypes, which may in part regulate the nuclear access of EED during this window of development.

IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage porcine embryo.

Global patterns of histone methylation are remodelled during cleavage development. Of the five histone methyltransferases known to mediate methylation of the lysine 9 residue of histone H3 (H3K9), euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) has been shown to be a primary mediator of H3K9 dimethylation; BIX-01294 has been shown to be a specific inhibitor of EHMT2. The objective of the present study was to determine the effect of BIX-01294 treatment on global H3K9 dimethylation in porcine embryos. We hypothesised that inhibition of EHMT2 by BIX-01294 would result in reduced levels of H3K9 dimethylation and compromised embryo development. Our results showed that incubation in 5µM BIX-01294 markedly reduced global levels of H3K9 dimethylation at the pronuclear, 2-cell and 4-cell stages of development and resulted in developmental arrest before blastocyst formation. Although transient exposure of embryos to BIX-01294 did not alter in vitro development, embryos transiently exposed to BIX-01294 did not establish pregnancy. These data demonstrate that BIX-01294 is a potent inhibitor of H3K9 dimethylation and that transient alterations in global histone modifications can have profound effects on embryo developmental potential.

Identification of karyopherin α1 and α7 interacting proteins in porcine tissue.

Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors) must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway. To this end, we performed a GST pull-down assay using porcine orthologs of karyopherin α1 (KPNA1) and karyopherin α7 (KPNA7) and prey protein derived from porcine fibroblast cells and used a liquid chromatography and tandem mass spectrometry (LC-MS/MS) approach to determine the identity of KPNA1 and KPNA7 interacting proteins. Our screen revealed that the proteins that interact with KPNA1 and KPNA7 are generally nuclear proteins that possess nuclear localization signals. We further validated two candidate proteins from this screen and showed that they are able to be imported into the nucleus in vivo and also interact with members of the karyopherin α family of proteins in vitro. Our results also reveal the utility of using a GST pull-down approach coupled with LC-MS/MS to screen for protein interaction partners in a non-traditional model system.

KPNA7, an oocyte- and embryo-specific karyopherin α subtype, is required for porcine embryo development.

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/ß heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherinαsubtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherinα7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherinαsubtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherinαsubtypes and that these karyopherinαsubtypes differentially transport intracellular cargo during cleavage development.

Differential developmental requirements for individual histone H3K9 methyltransferases in cleavage-stage porcine embryos.

Dimethylated H3K9 is a heritable epigenetic mark that is closely linked with transcriptional silencing and known to undergo global remodelling during cleavage development. Five mammalian histone methyltransferases (HMTases), namely Suv39H1, Suv39H2, SetDB1, EHMT1 and EHMT2, have been shown to mediate the methylation of H3K9. The aim of the present study was to determine the developmental requirements of these HMTases during cleavage development in porcine embryos. We hypothesised that knockdown of the abovementioned HMTases would differentially affect porcine cleavage development. To test this hypothesis, IVM and IVF porcine oocytes were divided into one of three treatment groups, including non-injected controls, oocytes injected with a double-stranded interfering RNA molecule specific for one of the HMTases and oocytes injected with a corresponding mutated (control) double-stranded RNA molecule. Nuclei were counted in all embryos 6 days after fertilisation. Although no significant difference in total cell number was detected in embryos injected with EHMT1 and EHMT2 interfering RNAs (compared with their respective control groups), embryos injected with interfering RNAs that targeted Suv39H1, Suv39H2 and SetDB1had significantly lower cell numbers than their respective control groups (P<0.05). This suggests that individual HMTases differentially affect in vitro developmental potential.

Global H3K9 dimethylation status is not affected by transcription, translation, or DNA replication in porcine zygotes.

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked to transcriptional repression. The observed structure of chromatin in porcine and murine embryos is different with regard to H3K9 dimethylation status, leading to our hypothesis that the intracellular mechanisms responsible for H3K9 methylation would also differ between these two species. The objectives of this study were: (1) to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes, (2) determine the extent to which the intracellular localization of individual pronuclei correlated with H3K9 dimethylation status, and (3) to determine the abundance of transcripts encoding the histone methyltransferases, with H3K9 methylation activity, in porcine oocytes and embryos. Our findings are that (1) H3K9 dimethylation status is not affected by DNA replication, transcription, or protein synthesis, (2) the location of a pronucleus does not significantly affect the H3K9 dimethylation status of the chromatin within that pronucleus, and (3) the histone methyltransferases with activity for H3K9 differ in transcript abundance in porcine oocytes and cleavage stage embyros. These results support our hypothesis that there is a difference in intracellular mechanisms affecting dimethylation status of H3K9 between porcine and murine embryos.

Differential remodeling of mono- and trimethylated H3K27 during porcine embryo development.

Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage porcine embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2-cell, 4-cell, 8-cell, and blastocyst stage embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage embryos produced by fertilization; the signals were faint or absent in nuclei of two-cell through blastocyst stage embryos. In addition, EED transcripts were increased from the four-cell stage (P < 0.05) in embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV-stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four-cell stage (P < 0.05) in parthenogenetic and in vivo derived embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during porcine embryogenesis.

STIM1 regulates store-operated Ca2+ entry in oocytes.

The single transmembrane-spanning Ca(2+)-binding protein, STIM1, has been proposed to function as a Ca(2+) sensor that links the endoplasmic reticulum to the activation of store-operated Ca(2+) channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca(2+) entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca(2+) stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca(2+) stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca(2+) channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca(2+) levels in the store-depleted oocytes after Ca(2+) add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca(2+) store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca(2+) entry.

Gene expression and development of early pig embryos produced by serial nuclear transfer.

During nuclear transfer, reprogramming makes the donor nucleus capable of directing development of the reconstructed embryo. In most cases reprogramming is incomplete, which leads to abnormal expression of early embryonic genes and subsequently, to reduced developmental potential. In the present study, we monitored the expression of Oct4, Nanog, and Sox2 in cloned porcine embryos and evaluated whether serial nuclear transfer, the transfer of nuclei of cloned embryos into enucleated oocytes, has the potential to provide a more complete reprogramming of the donor genome. The data suggested that Nanog and Sox2 expression is properly reactivated after nuclear transfer, but the relative abundance of Oct4 transcripts is abnormally low in cloned porcine blastocysts compared to control embryos produced by in vitro fertilization. When the nuclei of 8- to 16-cell stage cloned embryos were introduced into enucleated oocytes to expose the chromosomes repeatedly to the ooplasmic factors, the resulting embryos showed poor developmental potential: a significantly lower percentage of embryos developed to the 4-cell (12.0% vs. 31.8%), 8-cell (3.1% vs. 15.0%) and blastocyst (0% vs. 8.7%) stages compared to those produced following a single round of nuclear transfer (P < 0.05). The additional time for reprogramming also did not improve gene expression. By the late 4-cell stage, Oct4 and Sox2 expression levels were low in serial nuclear transfer embryos compared to those in embryos generated by in vitro fertilization or nuclear transfer. Overall, both developmental and gene expression data indicated that reprogramming of the donor nucleus could not be improved by serial nuclear transfer in the pig.

Manipulation of SMARCA2 and SMARCA4 transcript levels in porcine embryos differentially alters development and expression of SMARCA1, SOX2, NANOG, and EIF1.

Epigenetic reprogramming plays a pivotal role during embryogenesis, including both covalent and non-covalent modifications to chromatin. In this study, we investigated the role of SNF2 chromatin remodeling ATPases (SMARCA2 (previously known as BRAHMA), SMARCA4 (previously known as BRG1), SMARCA5 (previously known as SNF2H), SMARCA1 (previously known as SNF2L), CHD3, and CHD5) during porcine preimplantation embryonic development. Transcript levels for these ATPases change dynamically throughout development. We also investigated the effect of altering transcript levels of SMARCA2 and SMARCA4 via mRNA injection. Overexpression of SMARCA2 and SMARCA4 severely impaired embryo development. Results from these experiments show that embryos injected with SMARCA2 mRNA arrest between the four-cell and blastocyst stages. However, embryos injected with either wild-type SMARCA4 or a dominant negative variant or SMARCA4 arrest before zygotic genome activation. No differences in transcript abundance of SOX2, POU5F1, NANOG, and EIF1 (previously known as eIF1A) were detected after injection with SMARCA2 or its dominant negative variant at 48 h post-injection. Conversely, embryos injected with wild-type SMARCA4 and its dominant negative variant possessed altered expression of these genes. Examination of SNF2-type ATPase transcript abundance across all treatment groups revealed that only SMARCA1 was altered following injection with wild-type SMARCA2 and wild-type and dominant negative SMARCA4. We conclude that the arrest in porcine embryo development observed after injection is specific to the ATPase injected. Our data strongly support the hypothesis that SMARCA2 and SMARCA4 play different but fundamental roles controlling gene expression during early mammalian embryogenesis.

Expression of eukaryotic elongation initiation factor 1A differentially marks zygotic genome activation in biparental and parthenogenetic porcine embryos and correlates with in vitro developmental potential.

Zygotic genome activation (ZGA) is a major event during cleavage development. In vitro manipulation of mammalian embryos (including embryo culture) can result in developmental arrest around the time of ZGA. Eukaryotic elongation initiation factor 1A (eIF1A) has been used as a marker for ZGA in some mammalian species. We hypothesised expression of eIF1A can be used to assess ZGA in the pig; we also hypothesised that the expression profile of eIF1A can be used to assess developmental potential in vitro. The aims of the present study were to determine the expression pattern of eIF1A during porcine cleavage development and to assess its expression levels in embryos of different quality. We used a real-time reverse transcription-polymerase chain reaction assay to quantify eIF1A transcripts at different time points during cleavage development in porcine embryos produced by parthenogenetic activation (PA) and in vitro fertilisation (IVF). We found that eIF1A is activated at the two-cell stage in IVF embryos and at the four-cell stage in PA embryos. We showed that the increase in transcript levels observed in parthenogenetic embryos is dependent on de novo transcription. We found altered levels of eIF1A transcripts in parthenogenetic embryos that presented as either two- or eight-cell embryos 48 h after activation compared with four-cell embryos at the same time point. Our work supports the hypothesis that eIF1A is a marker of porcine ZGA and its expression profile can be used to assess embryo quality.

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development.

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2. The objective of this study was to determine the expression pattern of Oct4, Nanog, and Sox2 in cleavage stage porcine embryos obtained in vivo or by in vitro fertilization and parthenogenetic activation. We used quantitative, real time PCR to assess the relative amount of each transcript in cleavage stage embryos. We found that Oct4 was transiently activated at the 2-cell stage (P-value <0.05) while Nanog and Sox2 were activated at the 4-cell stage (P-value <0.05) in in vitro embryos. Embryos derived in vivo showed a similar but not identical pattern of expression of Nanog mRNA been in highest abundance both at the 4 cell and the blastocyst stage. The activation observed at the 4-cell stage for Nanog and Sox2 was shown to be RNA polymerase II dependent (P-value <0.05). This study showed that Oct4, Nanog, and Sox2 possess similar, but not identical, patterns of expression between in vitro and in vivo derived porcine embryos. The difference between the amount of transcripts may reflect the reduced developmental potential observed in in vitro cultured embryos.

Developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin-remodeling transcripts in donor cells.

Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT-derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2-type ATP-dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2-type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB-NT embryos and PSOS-NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast-NT embryos (23.53%, 16.98%, and 11.63%, respectively; P < 0.05). Fibroblast cells contained a significantly higher amount of SNF2L and CHD3 transcripts while Brg1 and SNF2H were the most expressed transcripts in all the cell lines analyzed. Metaphase II oocyte expression profile appeared to be unique compared to the cell lines analyzed. This work supports our hypothesis that an array of chromatin-remodeling proteins on donor cells may influence the chromatin structure, effect epigenetic reprogramming, and developmental potential.

Developmental arrest induced in cleavage stage porcine embryos following microinjection of mRNA encoding Brahma (Smarca 2), a chromatin remodeling protein.

Smarca 2 (Brahma) and Smarca 4 (Brahma related gene 1, BRG1) alternatively occupy the catalytic site of SWI/SNF chromatin remodeling complexes. Mammalian embryos undergo a dramatic amount of epigenetic remodeling during cleavage development, which plays key roles in regulating both gene transcription and the developmental potential of the embryo. In order to understand how the epigenetic state of cleavage stage embryos is regulated, it is important to identify the factors that mediate epigenetic changes during cleavage development. In this study we hypothesized that altered expression of Smarca 2 would have profound effects on embryo development. The objectives of this study were to determine the expression pattern of Smarca 2 and determine the effects of Smarca 2 overexpression in cleavage stage parthenogenetic porcine embryos. Smarca 2 transcripts are most abundant in germinal vesicle (GV) stage oocytes and decline progressively during cleavage development. At the blastocyst stage, Smarca 2 transcripts are reduced by 18-fold (GV stage oocyte vs. blastocyst stage embryo, P < 0.05). Parthenogenetic porcine embryos injected with mRNA encoding wild type human Smarca 2 exhibited a dramatic developmental arrest as compared to noninjected embryos, embryos injected with GFP mRNA, or mRNA encoding a dominant negative version of human Smarca 2 (P < 0.01). This work demonstrates the importance of Smarca 2 containing SWI/SNF chromatin remodeling complexes in preimplantation porcine embryos and how perturbing the amount of Smarca 2 in porcine embryos disrupts cleavage development.

The SNF2 domain protein family in higher vertebrates displays dynamic expression patterns in Xenopus laevis embryos.

All eukaryotes share a common nuclear infrastructure, in which DNA is packaged into nucleosomal chromatin. Its functional states, in particular the accessibility of the chromatin fiber to trans-acting factors, are determined by two classes of evolutionarily conserved enzymes, i.e. histone modifying enzymes and ATP-driven nucleosome remodeling machines. Browsing the annotated human genome database, we establish here a family of SNF2-like nuclear ATPases, which are the core enzymatic subunits of chromatin remodeling protein complexes. Homologues of those human genes are also to a large extent found in the Xenopus laevis genome, indicating a high degree of sequence conservation of this family among vertebrates. Expression analyses of the ATPase family of proteins reveal stage- and tissue-specific domains of peak RNA expression during early frog embryogenesis. These dynamic expression profiles suggest specific functional requirements for individual members of this family throughout early stages of vertebrate development.