Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos

During differentiation, somatic nuclei acquire highly specialized DNA and chromatin modifications, which are thought to result in cellular memory of the differentiated state. Upon somatic nuclear transfer into oocytes, the donor nucleus may have to undergo reprogramming of these epigenetic marks in order to achieve totipotency. This may involve changes in epigenetic features similar to those that occur in normal embryos during early development. However, there is accumulating evidence that epigenetic reprogramming is severely deficient in cloned embryos. Several reports reveal inefficient demethylation and inappropriate reestablishment of DNA methylation in quantitative and qualitative patterns on somatic nuclear transfer. Here we examine histone H3 lysine 9 (H3-K9) methylation and acetylation in normal embryos and in those created by somatic nuclear transfer. We find that H3-K9 methylation is reprogrammed in parallel with DNA methylation in normal embryos. However, the majority of cloned embryos exhibit H3-K9 hypermethylation associated with DNA hypermethylation, suggesting a genome-wide failure of reprogramming. Strikingly, the precise epigenotype in cloned embryos depends on the donor cell type, and the proportion of embryos with normal epigenotypes correlates closely with the proportion developing to the blastocyst stage. These results suggest a mechanistic link between DNA and histone methylation in the mammalian embryo and reveal an association between epigenetic marks and developmental potential of cloned embryos.     

Epigenetic inheritance of cell differentiation status

Epigenetic modifications influence gene expression pattern and provide a unique signature of a cell differentiation status. Without external stimuli or signalling events, this cell identity remains stable and unlikely to change over many cell divisions. The epigenetic signature of a particular cell fate therefore needs to be replicated faithfully in daughter cells; otherwise a cell lineage cannot be maintained. However, the mechanism of transmission of cellular memory from mother to daughter cells remains unclear. It has been suggested that the inheritance of an active or silent gene state involves different kinds of epigenetic mechanisms, e.g. DNA methylation, histone modifications, replacement of histone variants, Polycomb group (PcG) and Trithorax group (TrxG) proteins. Emerging evidence supports the role of histone variant H3.3 in maintaining an active gene status and in remodelling nucleosomal composition. Here we discuss some recent findings on the propagation of epigenetic memory and propose a model for the inheritance of an active gene state through the interaction of H3.3 with other epigenetic components.     

Dramatic replacement of histone variants during genome remodeling in nuclear-transferred embryos

The genome of differentiated somatic nuclei is remodeled to a totipotent state when they are transplanted into enucleated oocytes. To clarify the mechanism of this genome remodeling, we analyzed changes in the composition of core histone variants in nuclear-transferred embryos, since recent evidence has revealed that chromatin structure can be remodeled as a result of variant histone replacement. We found that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. Accompanying this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was DNA replication-independent. These results suggest that most core histone H2A and H3 components are dynamically exchanged between donor nuclei and recipient cytoplasm, which further suggests that replacement of donor cell histones with oocyte-stored histones may play a key role in genome remodeling in nuclear-transferred embryos. In addition, the incorporation patterns of all of the histone variants in the nuclear-transferred embryos were virtually the same as in the fertilized embryos. Only the incorporation pattern of H3.1 differed; it was incorporated into the transplanted donor nuclei, but not in the pronuclei of fertilized embryos. This result suggests that the incorporation of H3.1 has a detrimental effect on the process of genome remodeling and contributes to the low success rate of somatic nuclear cloning.     

Dramatic replacement of histone variants during genome remodeling in nuclear-transferred embryos

The genome of differentiated somatic nuclei is remodeled to a totipotent state when they are transplanted into enucleated oocytes. To clarify the mechanism of this genome remodeling, we analyzed changes in the composition of core histone variants in nuclear-transferred embryos, since recent evidence has revealed that chromatin structure can be remodeled as a result of variant histone replacement. We found that the donor cell-derived histone H3 variants H3.1, H3.2, and H3.3, as well as H2A and H2A.Z, were rapidly eliminated from the chromatin of nuclei transplanted into enucleated oocytes. Accompanying this removal, oocyte-stored histone H3 variants and H2A.X were incorporated into the transplanted nuclei, while the incorporation of H2A and H2A.Z was minimal or not detected. The incorporation of these variant histones was DNA replication-independent. These results suggest that most core histone H2A and H3 components are dynamically exchanged between donor nuclei and recipient cytoplasm, which further suggests that replacement of donor cell histones with oocyte-stored histones may play a key role in genome remodeling in nuclear-transferred embryos. In addition, the incorporation patterns of all of the histone variants in the nuclear-transferred embryos were virtually the same as in the fertilized embryos. Only the incorporation pattern of H3.1 differed; it was incorporated into the transplanted donor nuclei, but not in the pronuclei of fertilized embryos. This result suggests that the incorporation of H3.1 has a detrimental effect on the process of genome remodeling and contributes to the low success rate of somatic nuclear cloning.     

Position effect variegation and epigenetic modification of a transgene in a pig model

Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.     

Epigenetic regulation of extracellular-superoxide dismutase in human monocytes

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall and plays an important role in normal redox homeostasis. We previously showed the significant reduction or induction of EC-SOD during human monocytic U937 or THP-1 cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), respectively; however, its cell-specific expression and regulation have not been fully elucidated. It has been reported that epigenetic factors, such as DNA methylation and histone modification, are involved in several kinds of gene regulation. In this study, we investigated the involvement of epigenetic factors in EC-SOD expression and determined high levels of DNA methylation within promoter and coding regions of EC-SOD in THP-1 cells compared to those in U937 cells. Moreover, treatment with a DNA methyltransferase inhibitor, 5-azacytidine, significantly induced the expression of EC-SOD in THP-1 cells, indicating the importance of DNA methylation in the suppression of EC-SOD expression; however, the DNA methylation status did not change during THP-1 cell differentiation induced by TPA. On the other hand, we detected histone H3 and H4 acetylation during differentiation. Further, pretreatment with histone acetyltransferase inhibitors, CPTH2 or garcinol, significantly suppressed the TPA-inducible EC-SOD expression. We also determined the epigenetic suppression of EC-SOD in peripheral blood mononuclear cells. Treatment with granulocyte macrophage colony-stimulating factor (GM-CSF)/granulocyte-CSF induced that expression. Overall, these findings provide novel evidence that cell-specific and TPA-inducible EC-SOD expression are regulated by DNA methylation and histone H3 and H4 acetylation in human monocytic cells.     

A cross-talk between DNA methylation and H3 lysine 9 dimethylation at the KvDMR1 region controls the induction of Cdkn1c in muscle cells

The cdk inhibitor p57^kip2, encoded by the Cdkn1c gene, plays a critical role in mammalian development and in the differentiation of several tissues. Cdkn1c protein levels are carefully regulated via imprinting and other epigenetic mechanisms affecting both the promoter and distant regulatory elements, which restrict its expression to particular developmental phases or specific cell types. Inappropriate activation of these regulatory mechanisms leads to Cdkn1c silencing, causing growth disorders and cancer. We have previously reported that, in skeletal muscle cells, induction of Cdkn1c expression requires the binding of the bHLH myogenic factor MyoD to a long-distance regulatory element within the imprinting control region KvDMR1. Interestingly, MyoD binding to KvDMR1 is prevented in myogenic cell types refractory to the induction of Cdkn1c. In the present work, we took advantage of this model system to investigate the epigenetic determinants of the differential interaction of MyoD with KvDMR1. We show that treatment with the DNA demethylating agent 5-azacytidine restores the binding of MyoD to KvDMR1 in cells unresponsive to Cdkn1c induction. This, in turn, promotes the release of a repressive chromatin loop between KvDMR1 and Cdkn1c promoter and, thus, the upregulation of the gene. Analysis of the chromatin status of Cdkn1c promoter and KvDMR1 in unresponsive compared to responsive cell types showed that their differential responsiveness to the MyoD-dependent induction of the gene does not involve just their methylation status but, rather, the differential H3 lysine 9 dimethylation at KvDMR1. Finally, we report that the same histone modification also marks the KvDMR1 region of human cancer cells in which Cdkn1c is silenced. On the basis of these results, we suggest that the epigenetic status of KvDMR1 represents a critical determinant of the cell type-restricted expression of Cdkn1c and, possibly, of its aberrant silencing in some pathological conditions.