H3S10 PHOSPHORYLATION MARKS CONSTITUTIVE HETEROCHROMATIN DURING INTERPHASE IN EARLY MOUSE EMBRYOS, UNTIL THE 4-CELL STAGE

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H3S10P in mammalian gametes

It is now readily accepted that the chromatin structure and nuclear organization are both epigenetically regulated. Different epigenetic signatures are seen in different cell types which are basically the result of specific functions (Alcobia et al., 2000). Gametes are very specialized cells which upon fertilization undergo impressive changes in their chromatin structure and nuclear content. But firstly during gametogenesis, these cells also undergo drastic rearrangements in their chromatin. For instance in the sperm, histones are replaced by protamines in the chromatin in order to highly pack the DNA (Goldberg et al., 1977; Kimmins et al., 2007), whereas the oocyte needs to acquire competence to resume meiosis in an attempt to fully develop and therefore it undergoes a massive chromatin rearrangement (Bui et al., 2004). These two impressive events have raised the interest of many scientists in understanding how spermatogenesis and oogenesis are epigenetically regulated.
From all these events, the maturation process the oocyte goes through is the one which attracts the most attention due to its importance in establishing good quality oocytes that will be capable of originating a viable embryo. Remarkably, as the oocyte approaches prophase I (also known as GV “germinal vesicle” stage) it acquires meiotic competence. In these fully-grown oocytes the chromatin then changes from decondensed (Non-Surrounded-Nucleolus, NSN) to condensed state (Surrounded-Nucleolus, SN) (Figure 13; Hirao et al., 1995).

H3S10P in mammalian embryos

Despite all this information on these two pericentromeric heterochromatin markers, H3K9me3 and HP1•, in mammalian preimplantation embryos, nothing much is known about H3S10P. It is said that this epigenetic modification is a marker of pericentromeric heterochromatin in somatic cells and different organisms, nevertheless it is an open question as to whether this histone modification is also a marker of this type of heterochromatin in preimplantation embryos. So far only three reports have shown association of H3S10P and pericentromeric heterochromatin in preimplantation mouse embryos. They however present conflicting data.
In the work done by Wang and colleagues, they have showed for the first time that H3S10P is present during interphase of 1- and 2-cell embryos, as well as on condensed chromosomes during prophase and anaphase of the first mitosis (Wang et al., 2006). Moreover, they state that this distribution pattern for H3S10P is completely different from the one they observed in mouse oocytes. In the first oocyte meiosis, dephosphorylation of histone H3 at Ser10 is seen in the transition of anaphase to telophase, an event not observed in mouse embryos. They also suggested that H3S10P overlaps with pericentromeric heterochromatin around the NPBs in 1-cell embryos. Thereafter Huang et al. (2007), with their study on the comparison of some epigenetic modifications between in vivo and IVF embryos, found that H3S10P is detected at the pronuclear stage, with strong labelling in the perinucleolar region (around the NPBs) and diffusely detected throughout the nucleoplasm. As for the first mitosis, they observed strong H3S10P staining on the chromosomes which they say gradually decreases at anaphase. Furthermore, during the following divisions, H3S10P is obviously detected in the interphasic blastomeres from the 2-cell to morula stage, but undetectable at the blastocyst stage, except for the strong staining co-localized with the chromosomes.
As for the study carried out by Teperek-Tkacz et al. (2010), they have shown that H3S10P is detected in the condensed chromatin regions surrounding the NPBs very early during the pronuclear stage (19-21h phCG), showing a very short window of dephosphorylation of the Ser10 at the early-middle S phase (22-25h phCG) and de novo phosphorylation around 26h phCG seen only on the heterochromatin of the paternal pronucleus. H3S10P is then detected in the whole chromatin of both pronuclei at the G2 phase and all chromosomes from prophase to telophase of the first mitosis. Moreover, they say that a similar pattern of phosphorylation/dephosphorylation of H3S10 was observed in the interphase of the second cell cycle and that H3S10 remains highly phosphorylated on the chromosomes on the subsequent mitosis from prophase to telophase. These three reports have shown contradicting results for the same histone modification even though they have used the same antibody for the detection of H3S10P. In addition Wang et al. (2006) and Huang et al. (2007) seem to adopt the same approach for detection of H3S10P. Nevertheless they obtained different results. On the other hand Teperek-Tkacz and colleagues used a slightly different protocol which included pronase treatment for zona pellucid removal, fixation with a higher concentration of PFA and the use of M2 as culture medium (Teperek-Tkacz et al., 2010). These different steps could have caused some disturbance of the H3S10P distribution, leading to different conclusions. Consequently, more investigation needs to be carried out on H3S10P in preimplantation embryos in order to clarify the discrepancies found in these reports.
As it can be seen, the epigenetic regulation scenario in mammalian embryos is puzzling and the studies done so far cannot explain the biological significance and differences seen for many of these epigenetic markers. Even though it is known that there are species-specific contributions to the epigenetic regulation which could cause the differences seen for epigenetic markers in various types of mammalian embryos, it is important to further investigate to understand these divergences in an attempt to clarify even better how preimplantation embryos are epigenetically regulated.

Somatic Cell Nuclear Transfer (SCNT)

During normal development the genomes of the two very specialized cells, the sperm and the oocyte, undergo profound changes. They lose their characteristic nuclear and chromatin configuration giving way to the establishment of a zygotic nuclear and chromatin organization. It is exactly this distinctive embryonic configuration that should be achieved by the somatic nucleus after Somatic Cell Nuclear Transfer, which basically consists in injecting a somatic mature donor nucleus into an enucleated oocyte. This conversion of a more differentiated nuclear condition into an embryonic one is one of the ways of carrying out nuclear reprogramming. In this case, the mature cell is forced to setback its differentiated state to an undifferentiated immature embryonic one. It is the faithful reprogramming of the somatic genome by the complete elimination of the epigenetic memory of the donor nucleus (Hochedlinger and Jaenisch, 2006).
Nuclear reprogramming by Somatic Cell Nuclear Transfer has caught the interest of many research groups due to the opportunity of testing nuclear potency, distinguishing between genetic and epigenetic state and alteration of various donor cells, as well as the possibility of generating embryonic stem cells for the treatment of animal model disease and the prospect of generating patient-specific human ES cells (Hochedlinger and Jaenisch, 2006). Moreover, the opportunity to clone by Somatic Cell Nuclear Transfer has been achieved in a variety of mammalian species and has potential applications for human health, improvement of agriculture species, protection of exotic and endangered species and advancement of basic biological research (Murphey et al., 2008). Despite the great achievement in cloning mammals like mice, sheep, cattle and pigs using nuclear transfer, the overall efficiency of producing live cloned offspring is quite low.
It is thought that the causes for the low efficiency of this technique could be: mechanical damage of the samples during nuclear transfer, incomplete nuclear reprogramming or inappropriate in vitro culture systems (Kawasumi et al., 2007). It is known that three aspects are crucial for development: nuclear architecture, genome organization and gene expression. Therefore, if one of these factors is found irregular or faulty in embryos, the development will be compromised and this is usually true for cloned embryos.

Epigenetic reprogramming after SCNT

It is also believed that incomplete epigenetic reprogramming of the somatic nucleus, in other words, the incomplete re-establishment of the embryonic epigenetic patterns may be one of the causes of development failure of cloned animals (Mann and Bartolomei, 2002). The nuclear-transferred donor genome should be reprogrammed to activate the appropriate embryonic genes at the appropriate time, just as occurs in normal fertilized embryos (Kawasumi et al., 2007). It is conceivable that the normal activation of these genes associated with early embryonic development and/or the inactivation of somatically expressed genes may not occur readily in reconstructed embryos (Chung et al., 2003). Therefore, nuclear reprogramming is often abnormal or incomplete in cloned embryos.
There are many studies pointing out the different constraints seen in such type of embryos which disturb their development, such as aberrant DNA methylation patterns, incomplete chromatin reorganization and irregular nuclear compartmentalization, as well as abnormal histone modification patterns. Several studies have been published about irregular reprogramming of epigenetic markers, such as DNA methylation and histone acetylation and methylation (Wang et al., 2007; Zhang et al., 2009; Kang et al., 2011).
For instance, the signals for H3K4me2 which are supposed to increase at the 2-cell stage concomitant to the genome activation in mouse embryos, have been observed in lower levels during the same stage in cloned mouse embryos (Shao et al., 2008). Another epigenetic marker found abnormal in cloned embryos is H3K9 acetylation. This modification is detected in lower levels in cloned embryos when compared to their in vivo counterparts (Wang et al., 2007). As mentioned before the DNA methylation pattern is also disturbed in cloned embryos. One of the enzymes related to DNA methylation, the methyltransferase Dnmt1, is precociously detected in cloned mouse embryos at the 8-cell stage, whereas in normal embryos this protein should only be expressed in later stages (Chung et al., 2003). Moreover, studies carried out by Yamagata et al. (2007) have shown altered DNA methylation in reconstructed embryos by SCNT as well. They have found by live-cell imaging experiments that the centromeric and pericentromeric heterochromatin domains of cloned embryos were highly methylated when compared to their IVF counterparts. These results clearly illustrate the heterogeneity of epigenetic alterations found in cloned embryos. Taken all together, further research needs to be done to unveil the basic mechanisms regulating the development of SCNT embryos, as well as to acquire a better understanding of these mechanisms to deeply analyse nuclear reprogramming in order to improve this fabulous technique.

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TSA and the improvement of reprogramming after SCNT

Different methods have been employed by several research groups in attempts to improve the SCNT technique, such as inhibiting cytokinesis, changing the time of enucleation and injection of the donor nucleus, using various types of donor cells and the use of drugs like trichostatin A (TSA), an inhibitor of histone deacetylases. Histone acetylation has the greatest potential for unfolding chromatin, making it more accessible to different transcriptional factors which will lead to gene transcription (Wang et al., 2007). In fact, the treatment of the reconstructed embryos with TSA has shown a significant improvement in cloning efficiency in mouse, pig, bovine and rabbit (Maalouf et al., 2009; Bui et al., 2010). It is said that TSA improves nuclear reprogramming as a consequence of the modulation of histone modifications. As TSA is an inhibitor of deacetylases, this drug causes hyperacetylation, therefore enhancing the acetylation state of the cloned embryos which do then reach almost normal levels of acetylation as observed in normal embryos. In fact, the TSA treatment brought many benefits to the SNCT technique. It increased the time taken for chromosome condensation, enhanced the levels of H3K4me2 (a marker of transcriptionally active chromatin) and reduced the levels of a marker of inactive chromatin, H3K9me3 (Bui et al., 2010). Improved levels of H3K9 acetylation was also seen when the mouse-cloned embryos were incubated with TSA (Wang et al., 2007).

Pericentromeric Heterochromatin remodelling after SCNT

As mentioned before, the epigenetic status of the reconstructed embryo is a crucial step in normal embryonic development after nuclear transfer. As a matter of fact, different epigenetic modifications are found misregulated after this procedure including DNA methylation and histone modifications, as mentioned above. Additionally, as these epigenetic modifications are in close relation to chromatin remodelling and nuclear higher-order organization such misregulation can be one of the causes of the abnormal nuclear reprogramming seen in cloned embryos. Therefore, providing the means for the donor-cell genome to find its way to the correct reorganization after nuclear transfer is primordial for the improvement of cloning procedures.
It is clearly evident that the different nuclear arrangements seen in various types of cells and embryos result from the interaction between epigenetic markers and chromatin. In mouse, the somatic cells possess a characteristic way of organizing their chromatin which consists of an agglomeration of pericentromeric domains of different chromosomes forming blocks of constitutive heterochromatin which are called chromocenters. In embryos for example, the parental genomes display a unique distribution of centromeres and pericentromeric heterochromatin organized around the nucleolar precursor bodies (NPBs), with the chromocenters being formed later by the end of the 2-cell stage in mouse (Martin et al., 2006a) and around the 8-cell stage in bovine embryos (Santos et al., 2002). It is exactly these epigenetic markers together with chromatin that dictate the way the nucleus should be organized. Therefore, it is very interesting to look at how these heterochromatin domains behave after SCNT because the donor cell injected in an enucleated oocyte is thought to overcome its epigenetically imposed nuclear organization and higher-order chromatin structure, undergoing profound changes in its global nuclear structure in order to achieve the same epigenetic markers and embryonic nuclear configuration as a normal embryo.
Even though there is a great chromatin reshape imposed by the oocyte reprogramming factors in the donor nucleus, this switch from a somatic configuration to an embryonic one is not well achieved. It is known that with nuclear transfer, the donor cell nucleus which has blocks of heterochromatin (chromocenters) can be remodeled into a zygotic-like heterochromatin which is characterized by the arrangement of the centromeres around the NPBs (Martin et al., 2006a; Merico et al., 2007). However, it has been shown that a great number of cloned mouse embryos present a high number of centromeres not associated to the NPBs compared to normal embryos (Martin et al., 2006b). Moreover, the percentage of cloned embryos showing this abnormal heterochromatin redistribution correlates to the proportion of the cloned embryos which failed to develop to blastocyst stage (Maalouf et al., 2009).
It is evident that the somatic heterochromatin is often not well remodelled by nuclear reprogramming after SCNT. It is therefore important to further investigate how these heterochromatin domains are “reshuffled” through nuclear reprogramming so that new tools and approaches can be found to improve reorganization of these regions after SCNT.

Table of contents :

CHAPTER ONE : INTRODUCTION
1.1 EPIGENETICS OVERVIEW
1.2 INTRODUCTION ON CHROMATIN
1.2.1 Euchromatin & Heterochromatin
1.3 GLOBAL CHROMATIN MODIFICATIONS
1.3.1 Histone Post-translation Modifications
1.3.2 DNA Methylation
1.4 CENTROMERIC & PERICENTROMERIC HETEROCHROMATIN
1.5 HISTONE H3 AND ITS SERINE 10 PHOSPHORYLATION
1.5.1 H3S10P in somatic cells
1.5.2 H3S10P in mammalian gametes
1.6 THE MOUSE AS A MODEL
1.6.1 Preimplantation development
1.6.2 Mouse preimplantation development
1.6.3 Heterochromatin in embryos
1.7 H3S10P IN MAMMALIAN EMBRYOS
1.8 SOMATIC CELL NUCLEAR TRANSFER (SCNT)
1.8.1 SCNT and its issues
1.8.2 Epigenetic reprogramming after SCNT
1.8.3 TSA and the improvement of reprogramming after SCNT
1.8.4 Pericentromeric Heterochromatin remodelling after SCNT
1.9 PROJECT AIMS
CHAPTER TWO: H3S10 PHOSPHORYLATION MARKS CONSTITUTIVE HETEROCHROMATIN DURING INTERPHASE IN EARLY MOUSE EMBRYOS, UNTIL THE 4-CELL STAGE
2.1 INTRODUCTION
2.1 ORIGINAL PAPER #1
2.2 SUPPLEMENTARY FIGURES
CHAPTER THREE: NUCLEAR DYNAMICS OF HISTONE H3 TRIMETHYLATED ON LYS9 AND/OR PHOSPHORYLATED ON SER10 IN MOUSE CLONED EMBRYOS AS NEW MARKERS OF REPROGRAMMING?
3.1 INTRODUCTION
3.2 ORIGINAL PAPER #2
3.3 SUPPLEMENTARY FIGURE: IMMUNODETECTION OF H3S10P IN IVF EMBRYOS
CHAPTER FOUR: GENERAL DISCUSSION & PERSPECTIVES
BIBLIOGRAPHY

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