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Modulation of an essential histone methyltransferase in mouse embryonic stem cells
Modulation of an essential histone methyltransferase in mouse embryonic stem cells
The H3K9me3 is one of the major modifications characteristic of repressed chromatin. Its accumulation is linked to silencing of transcription and compaction of chromatin. Among the methyltransferases able to catalyze H3K9me3, SETDB1 leads to the earliest lethal phenotype in knockout mice embryos. In vitro studies showed that its interactor ATF7IP is capable of modulating SETDB1-dependent catalysis of H3K9me3. The implications of this interaction in a cellular system still remains an open question. Thus, to understand the modulation of SETDB1 methyltransferase activity during mouse early development was the main goal of this work. Using ChIP-Seq the genome-wide occupancy of both SETDB1 and ATF7IP was identified in FLAG knockin mouse embryonic stem cells (mESC). To investigate the epigenetic outcome of this interaction, the genome-wide enrichment for different H3K9 marks was characterized in control mESC and cells where Atf7ip was deleted by the CRISPR/Cas system. Afterwards, these data were coupled with transcriptome profiles to address whether the epigenetic changes implicated in transcriptional deregulation. In contrast to Setdb1 knockout mESC, cells lacking ATF7IP survive and grow normally. However, several families of endogenous retrovirus (ERV) belonging to classes I and II known to be controlled by SETDB1 were bound by both SETDB1 and ATF7IP and became derepressed in Atf7ip knockout mESC. This phenotype is further enhanced when cells are devoid of DNA methylation. Interestingly, while mutant cells are able to differentiate and repress repetitive sequences during differentiation as in control cells, DNA methylation-depleted mutant mESC could not properly repress those sequences. Unexpectedly, H3K9me3 levels in Atf7ip knockout mESC were reduced neither globally nor site specifically at transposable elements targets of SETDB1-ATF7IP complex. On the contrary, H3K9me3 deposition increased at those regions and was followed by increase in H3K9me2. To identify other proteins that might be involved in SETDB1-ATF7IP silencing mechanism mass spectrometry of FLAG-ATF7IP immune-complexes was performed. Then, taking advantage of an exogenous retrotransposon repression reporter system, which is impaired in Atf7ip-depleted mESC, ATF7IP partner proteins and other known repressors were screened for genetic interaction with Atf7ip. Some factors were found to act within the same pathway, while others had synergistic effects and probably belong to independent pathways. Lastly, exchanging the expression of the endogenous locus by that of different Atf7ip mutants by using a Bxb1-mediated recombination system demonstrated that nuclear localization, as well as the conserved Domains 1 and 2, are essential for proper ERV repression. Altogether, this work provides a better understanding of the molecular mechanism of repression by SETDB1 and its modulation by the co-factor ATF7IP.
Atf7ip, Setdb1, H3K9me3, epigenetics, chromatin, retrotransposon
Almeida, Gustavo Pereira de
2018
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Almeida, Gustavo Pereira de (2018): Modulation of an essential histone methyltransferase in mouse embryonic stem cells. Dissertation, LMU München: Faculty of Medicine
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Abstract

The H3K9me3 is one of the major modifications characteristic of repressed chromatin. Its accumulation is linked to silencing of transcription and compaction of chromatin. Among the methyltransferases able to catalyze H3K9me3, SETDB1 leads to the earliest lethal phenotype in knockout mice embryos. In vitro studies showed that its interactor ATF7IP is capable of modulating SETDB1-dependent catalysis of H3K9me3. The implications of this interaction in a cellular system still remains an open question. Thus, to understand the modulation of SETDB1 methyltransferase activity during mouse early development was the main goal of this work. Using ChIP-Seq the genome-wide occupancy of both SETDB1 and ATF7IP was identified in FLAG knockin mouse embryonic stem cells (mESC). To investigate the epigenetic outcome of this interaction, the genome-wide enrichment for different H3K9 marks was characterized in control mESC and cells where Atf7ip was deleted by the CRISPR/Cas system. Afterwards, these data were coupled with transcriptome profiles to address whether the epigenetic changes implicated in transcriptional deregulation. In contrast to Setdb1 knockout mESC, cells lacking ATF7IP survive and grow normally. However, several families of endogenous retrovirus (ERV) belonging to classes I and II known to be controlled by SETDB1 were bound by both SETDB1 and ATF7IP and became derepressed in Atf7ip knockout mESC. This phenotype is further enhanced when cells are devoid of DNA methylation. Interestingly, while mutant cells are able to differentiate and repress repetitive sequences during differentiation as in control cells, DNA methylation-depleted mutant mESC could not properly repress those sequences. Unexpectedly, H3K9me3 levels in Atf7ip knockout mESC were reduced neither globally nor site specifically at transposable elements targets of SETDB1-ATF7IP complex. On the contrary, H3K9me3 deposition increased at those regions and was followed by increase in H3K9me2. To identify other proteins that might be involved in SETDB1-ATF7IP silencing mechanism mass spectrometry of FLAG-ATF7IP immune-complexes was performed. Then, taking advantage of an exogenous retrotransposon repression reporter system, which is impaired in Atf7ip-depleted mESC, ATF7IP partner proteins and other known repressors were screened for genetic interaction with Atf7ip. Some factors were found to act within the same pathway, while others had synergistic effects and probably belong to independent pathways. Lastly, exchanging the expression of the endogenous locus by that of different Atf7ip mutants by using a Bxb1-mediated recombination system demonstrated that nuclear localization, as well as the conserved Domains 1 and 2, are essential for proper ERV repression. Altogether, this work provides a better understanding of the molecular mechanism of repression by SETDB1 and its modulation by the co-factor ATF7IP.