Periodic Reporting for period 1 - Epigenome_embryo (How epigenetic reprogramming modulates 3D chromatin organization in totipotent embryos)
Période du rapport: 2021-06-01 au 2023-05-31
One of the most intriguing questions in biology is how a single cell (zygote) has the potential to give rise to all cell types of an organism, known as totipotency. Afterwards this potential is progressively lost along cleavage divisions to allow cell specification and differentiation. Upon fertilization a single cell embryo is generated, and it has the potential to activate a series of coordinated events that lead to the achievement of the totipotent state. These events encompass an extensive epigenetic reprogramming of the parental genomes, 3D chromatin organization reorganization and zygotic genome activation (ZGA). The overall purpose of this project is to deepen our understanding of the mechanisms underlying the oocyte-to-zygote transition (OZT) in mammals. The understanding of ZGA is very promising, not only from the intellectual point of view in the developmental biology field, but also these factors will be useful for cell reprogramming in regenerative medicine to generate the first totipotent cell lines.
The first aim of the project is to address whether and how epigenetic reprogramming modulates 3D chromatin structure in zygotes. Shortly after fertilization, both parental genomes undergo genome wide demethylation. Paternal DNA becomes largely demethylated. Though, changes in maternal genome are less evident. This DNA demethylation can proceed passively by dilution through replication, or actively, mediated by several mechanisms including catalysis by the methylcytosine dioxygenase Tet3. Given the asymmetry between the parental genomes, both in terms of epigenetic landscape and the chromatin compartmentalization, it would be interesting to determine how epigenetic changes in the paternal pronuclei lead to a reorganization of the chromatin. More specifically, the aim of this project is one the one hand, to assess how DNA methylation modulates compartmentalization on paternal and maternal genome; and in the other hand, determine the effect of histone methylation on chromatin compartmentalization, due to the asymmetry between the maternal/paternal genome in the histone marks. The preliminary results using a Tet3 conditional knock-out model, point to weaker compartmentalization in the zygotic genome, suggesting a role of Tet3 in chromatin organization. Whether if this change is due to the DNA demethylation activity of Tet3 is yet to be determined.
The main aim of the second part of the project is to identify a candidate regulator of the zygotic genome activation. A key question is which proteins are essential for activation of embryonic transcription during ZGA. It is conceivable that these regulators are maternally inherited from the oocyte. Based on a previous screen of potential regulators of ZGA carried out in the laboratory, one promising candidate factor was chosen to study in detail. It will be analyzed if the candidate is essential for embryonic development and its involvement in the regulation of ZGA. The knock down of the candidate show a reduction in the major ZGA transcripts, suggesting an implication of this candidate in the regulation of ZGA in mammals.
Unfortunately, this project has to be terminated prematurely after these 5 months, as the hosting research group has moved its location from IMBA to Max Planck Institute of Biochemistry (MPIB), Germany. The proposal was submitted in the Society and Enterprise panel, as IMBA is a non-academic institution. However, the MPIB has an academic status, which makes it impossible the transfer of the fellowship. Because of that, no further conclusions have been obtained in this short period of the project.
The first aim of the project is to address whether and how epigenetic reprogramming modulates 3D chromatin structure in zygotes. Shortly after fertilization, both parental genomes undergo genome wide demethylation. Paternal DNA becomes largely demethylated. Though, changes in maternal genome are less evident. This DNA demethylation can proceed passively by dilution through replication, or actively, mediated by several mechanisms including catalysis by the methylcytosine dioxygenase Tet3. Given the asymmetry between the parental genomes, both in terms of epigenetic landscape and the chromatin compartmentalization, it would be interesting to determine how epigenetic changes in the paternal pronuclei lead to a reorganization of the chromatin. More specifically, the aim of this project is one the one hand, to assess how DNA methylation modulates compartmentalization on paternal and maternal genome; and in the other hand, determine the effect of histone methylation on chromatin compartmentalization, due to the asymmetry between the maternal/paternal genome in the histone marks. The preliminary results using a Tet3 conditional knock-out model, point to weaker compartmentalization in the zygotic genome, suggesting a role of Tet3 in chromatin organization. Whether if this change is due to the DNA demethylation activity of Tet3 is yet to be determined.
The main aim of the second part of the project is to identify a candidate regulator of the zygotic genome activation. A key question is which proteins are essential for activation of embryonic transcription during ZGA. It is conceivable that these regulators are maternally inherited from the oocyte. Based on a previous screen of potential regulators of ZGA carried out in the laboratory, one promising candidate factor was chosen to study in detail. It will be analyzed if the candidate is essential for embryonic development and its involvement in the regulation of ZGA. The knock down of the candidate show a reduction in the major ZGA transcripts, suggesting an implication of this candidate in the regulation of ZGA in mammals.
Unfortunately, this project has to be terminated prematurely after these 5 months, as the hosting research group has moved its location from IMBA to Max Planck Institute of Biochemistry (MPIB), Germany. The proposal was submitted in the Society and Enterprise panel, as IMBA is a non-academic institution. However, the MPIB has an academic status, which makes it impossible the transfer of the fellowship. Because of that, no further conclusions have been obtained in this short period of the project.
During these first months of the project, I have trained the general techniques that are essential to carry out this project (oocyte/zygote isolation, microinjection, snHi-C).
Preliminary results in the laboratory using snHi-C of zygotes treated with an inhibitor of Tet3 showed weaker compartmentalization. This suggests that Tet3 activity, presumably by DNA demethylation, contributes to the asymmetry in compartmentalization between maternal and paternal zygotic chromatin. To determine whether Tet3 is required for compartmentalization, I will use mouse genetic approaches. Considering that Tet3 is inherited from the egg, it makes it necessary to use Tet3 conditional knockout mice in order to have oocytes deficient for Tet3. We have obtained a Tet3Flox/Flox strain and I will generate conditional Tet3Flox/D maternal knockout mice using female germline-specific Zp3-Cre deleter strain. As starting point of the project, it was performed snHi-C analysis in G2 zygotes. In addition, as a contingency plan in case these mouse strains still will not be complete knock-out I have designed a knock-in auxin-inducible degron (AID)-Tet3 mouse to get rid of maternal Tet3 protein using auxin.
Regarding the study of a regulator of ZGA, the fellow has participated in the lab in a targeted knockdown screen of potential regulators of ZGA. Candidates were selected based on molecular features such as DNA binding domains and their detection during the OZT as mRNA or proteins. Based on this screen, a potential candidate has been already picked and confirmed by knock-down the decrease in the levels of ZGA transcripts during major ZGA through single-molecule FISH. In addition, given the availability of an already generated conditional knock-out mouse strain, this strain has been acquired and started the expansion of it for further analysis.
As this is a very preliminary phase of the project, the results obtained so far are not ready for dissemination. However, the project is still ongoing besides it will not be founded from this point on under MSCA fellowship, and the final results will be published once the project is completed.
Preliminary results in the laboratory using snHi-C of zygotes treated with an inhibitor of Tet3 showed weaker compartmentalization. This suggests that Tet3 activity, presumably by DNA demethylation, contributes to the asymmetry in compartmentalization between maternal and paternal zygotic chromatin. To determine whether Tet3 is required for compartmentalization, I will use mouse genetic approaches. Considering that Tet3 is inherited from the egg, it makes it necessary to use Tet3 conditional knockout mice in order to have oocytes deficient for Tet3. We have obtained a Tet3Flox/Flox strain and I will generate conditional Tet3Flox/D maternal knockout mice using female germline-specific Zp3-Cre deleter strain. As starting point of the project, it was performed snHi-C analysis in G2 zygotes. In addition, as a contingency plan in case these mouse strains still will not be complete knock-out I have designed a knock-in auxin-inducible degron (AID)-Tet3 mouse to get rid of maternal Tet3 protein using auxin.
Regarding the study of a regulator of ZGA, the fellow has participated in the lab in a targeted knockdown screen of potential regulators of ZGA. Candidates were selected based on molecular features such as DNA binding domains and their detection during the OZT as mRNA or proteins. Based on this screen, a potential candidate has been already picked and confirmed by knock-down the decrease in the levels of ZGA transcripts during major ZGA through single-molecule FISH. In addition, given the availability of an already generated conditional knock-out mouse strain, this strain has been acquired and started the expansion of it for further analysis.
As this is a very preliminary phase of the project, the results obtained so far are not ready for dissemination. However, the project is still ongoing besides it will not be founded from this point on under MSCA fellowship, and the final results will be published once the project is completed.
In the zygote, the paternal and maternal genomes are spacially separated in two different pronucleus. In this phase, paternal chromatin shows stronger compartmentalization than maternal chromatin. One hypothesis to explain this difference is that the active DNA demethylation that is mediated by Tet3 leads to a different chromatin state of the paternal genome. Consistent with this, preliminary results in the host laboratory using snHi-C of zygotes treated with an inhibitor of Tet3 showed weaker compartmentalization. This suggests that Tet3 activity, presumably by DNA demethylation, contributes to the asymmetry in compartmentalization between maternal and paternal zygotic chromatin. Similar results have been obtained with the conditional Tet3 knock-model. Given the existence of three different Tet dioxygenases, the aim is to determine the contribution of the paralogs Tet1 and Tet2, as well as if the potential chromatin changes are dependent on the catalytic activity of Tet3.
Chromatin compartmentalization can be also modified due to the histone methylation changes that takes place in the zygote. In this aspect, it will be assessed how the absence of the histone lysine demethylase JMJD2A (Kdm4a) influence the 3D chromatin structure by snHi-C.
Focusing in the ZGA regulator, to assess whether the candidate regulates genome-wide ZGA, a single cell RNAseq analysis in G2 and 2-cell embryo will be carried out in the absence of the candidate as well as CUT&Tag to determine where it binds in the genome and for motif analysis.
Chromatin compartmentalization can be also modified due to the histone methylation changes that takes place in the zygote. In this aspect, it will be assessed how the absence of the histone lysine demethylase JMJD2A (Kdm4a) influence the 3D chromatin structure by snHi-C.
Focusing in the ZGA regulator, to assess whether the candidate regulates genome-wide ZGA, a single cell RNAseq analysis in G2 and 2-cell embryo will be carried out in the absence of the candidate as well as CUT&Tag to determine where it binds in the genome and for motif analysis.