Zfp322a regulates mouse ES cell pluripotency and enhances reprogramming efficiency [ChIP-Seq]
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ABSTRACT: Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells. E14 mES cells were immunoprecipitated with Zfp322a antibody or IgG from rabbit.
Project description:Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells. Zfp322a depleted (RNAi) E14 mES cells were used for RNA extraction and hybridization on Affymetrix microarrays. We compared these microarray samples with the corresponding wild type (control RNAi cells).
Project description:Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells.
Project description:Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotentcy maintenance of ES cells. Here, we report that Zfp322a is required for ES cell identity since depletion of Zfp322a directs ES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Oct4 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of the ES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated ES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits ES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka’s factors (OSKM). It can be even used in combination with Yamanaka’s factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of ES cells.
Project description:Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of developing blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and are characterized by a restricted developmental potential. Although certain mouse strains, such as the non-obese diabetic (NOD) mice, are considered “non-permissive” for ES cell derivation, they retain the capacity to generate EpiSCs. Using the NOD strain as a model, we characterized the stability of pluripotent states in cells generated by ICM explantation or direct in vitro reprogramming. We find that ES-like NOD stem cells can be captured in both approaches by providing exogenous constitutive expression of Klf4 or c-Myc transcription factors or small molecules that can replace these factors during in vitro reprogramming. The fully pluripotent NOD ES and iPS cells appear “metastable”, as the cells acquire an alternative EpiSC-like identity after removal of the exogenous factors, while reintroduction of these factors converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by the continuous presence of defined exogenous factors. Gene expression profiling was performed in mouse ES, EpiSC and EpiSC-like cell lines.
Project description:Sall4 is a stem cell factor which is important for embryogenesis. We have genetically modified Sall4 in mouse embryonic stem cells to access the preference of Sall4 binding site. There are three different genetic modifications for the ES cells in the form of Sall4 Knockout (KO), Sall4 Zinc Finger Cluster 4 Mutation (ZFC4mut) and Sall4 Zinc Finger Cluster 1-2 Deletion (ZFC1-2Δ) respectively that we have considered for our study.
Project description:The histone H3 lysine 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using ChIP-seq analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and subsequently placental tissues. Co-immunoprecipitation and ChIP assays further demonstrates that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our result suggests that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor. Keywords: Epigenetics Examine Eset-binding sites and compare the H3K9me3 state between Eset knockdown mouse ES cells and control knockdown mouse ES cells.
Project description:CTCF and CTCFL DNA binding profile in CTCFL induced and non-induced ES cells.CTCF is a highly conserved and essential zinc finger protein that in conjunction with cohesin organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralogue of CTCF, is less clear. Here, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. Our data show that CTCFL is functionally different from CTCF and its absence in mice causes sub-fertility due to a partially penetrant testicular atrophy. Genome-wide studies reveal that CTCFL and CTCF bind similar consensus sequences. However, only ~2000 out of the ~5,700 CTCFL and ~31,000 CTCF binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Thus, nucleosome dynamics specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes ChIP-seq for CTCF (with CTCF antibody) and CTCFL (with V5 antibody) in CTCFL_V5_GFP induced and non-induced ES cells
Project description:First lineage specification in the mammalian blastocyst embryo leads to formation of the inner cell mass (ICM) and trophectoderm (TE), which respectively give rise to the embryo proper and extraembryonic tissues. We show histone methylation asymmetry on promoters in the first two developmental lineages, and highlight epigenetic skewing associated with derivation of embryonic stem (ES) cells. Comparison of histone methylation patterns on promoters in the ICM, TE and in ES cells derived from ICM. ChIP-chip experiments using anti-H3K4me3 or anti-H3K27me3 antibodies. Two or three replicates per sample.
Project description:Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of developing blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and are characterized by a restricted developmental potential. Although certain mouse strains, such as the non-obese diabetic (NOD) mice, are considered “non-permissive” for ES cell derivation, they retain the capacity to generate EpiSCs. Using the NOD strain as a model, we characterized the stability of pluripotent states in cells generated by ICM explantation or direct in vitro reprogramming. We find that ES-like NOD stem cells can be captured in both approaches by providing exogenous constitutive expression of Klf4 or c-Myc transcription factors or small molecules that can replace these factors during in vitro reprogramming. The fully pluripotent NOD ES and iPS cells appear “metastable”, as the cells acquire an alternative EpiSC-like identity after removal of the exogenous factors, while reintroduction of these factors converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by the continuous presence of defined exogenous factors.
Project description:Reverse transcription-derived sequences account for at least half of the human genome. Although these retroelements are formidable motors of evolution, they can occasionally cause disease, and accordingly are inactivated during early embryogenesis through epigenetic mechanisms. In the mouse, at least for endogenous retroviruses, important mediators of this process are the tetrapod-specific KRAB-containing zinc finger proteins (KRAB-ZFPs) and their cofactor TRIM28. The present study demonstrates that KRAB/TRIM28-mediated regulation is responsible for controlling a very broad range of human-specific endogenous retroelements (EREs) in human embryonic stem (ES) cells and that it exerts, as a consequence, a marked effect on the transcriptional dynamics of these cells. It further reveals reciprocal dependence between TRIM28 recruitment at specific families of EREs and DNA methylation. It finally points to the importance of persistent TRIM28-mediated control of ERE transcriptional impact beyond their presumed inactivation by DNA methylation. Analyses of epigentic effectors and marks in KAP1 WT and KD human embryonic stem cells