Lysine acetyltransferase Tip60 is required for hematopoietic stem cells maintenance
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ABSTRACT: Hematopoietic stem cells (HSC) have the potential to replenish the blood system for the lifetime of the organism. Their two defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Here, using conditional gene knockout models, we demonstrate a critical requirement of lysine acetyltransferase 5 (also known as Tip60) for murine HSC maintenance both in the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling revealed that Tip60 co-localizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell-cycle and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin and Tip60 deletion induced a robust reduction in the acH2A.Z / H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance through Tip60 dependent H2A.Z acetylation to activate Myc target genes.
Project description:The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to play crucial roles regulating gene expression and maintaining genome stability. Our cryo-EM studies show that within the NuA4/TIP60 complex, the EP400 subunit serves as an architectural scaffold holding the different functional modules in specific positions and giving rise to a novel arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, thereby preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome,emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
Project description:The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to play crucial roles regulating gene expression and maintaining genome stability. Our cryo-EM studies show that within the NuA4/TIP60 complex, the EP400 subunit serves as an architectural scaffold holding the different functional modules in specific positions and giving rise to a novel arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, thereby preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
Project description:The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to play crucial roles regulating gene expression and maintaining genome stability. Our cryo-EM studies show that within the NuA4/TIP60 complex, the EP400 subunit serves as an architectural scaffold holding the different functional modules in specific positions and giving rise to a novel arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, thereby preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
Project description:The human NuA4/TIP60 co-activator complex, a fusion of the yeast SWR1 and NuA4 complexes, both incorporates the histone variant H2A.Z into nucleosomes and acetylates histones H4/H2A/H2A.Z to play crucial roles regulating gene expression and maintaining genome stability. Our cryo-EM studies show that within the NuA4/TIP60 complex, the EP400 subunit serves as an architectural scaffold holding the different functional modules in specific positions and giving rise to a novel arrangement of the ARP module. EP400 interacts with the TRRAP subunit using a footprint that overlaps with that of the SAGA acetyltransferase complex, thereby preventing the formation of a hybrid complex. Loss of the TRRAP subunit leads to mislocalization of NuA4/TIP60, resulting in the redistribution of H2A.Z and its acetylation across the genome, emphasizing the dual functionality of NuA4/TIP60 as a single macromolecular assembly.
Project description:Although histone-modifying enzymes are assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here we show the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT–deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT–independent role of Tip60 in ESC maintenance. In contrast, KAT–deficient ESCs exhibited impaired differentiation into mesoderm and endoderm, demonstrating a second, KAT–dependent function in differentiation. Consistent with this phenotype, KAT–deficient mouse embryos exhibited post-implantation developmental defects. These findings establish separable KAT–dependent and KAT–independent functions of Tip60 in ESCs and during embryonic development, raising the possibility of undiscovered catalysis-independent functions of additional KATs.
Project description:Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc to regulate target genes required for neuroblast maintenance. Knockdown of members of this complex results in loss of cortical polarity, symmetric neuroblast division and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes which were identified before as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex and the molecular network controlling cell polarity and asymmetric cell division.
Project description:Background Tip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood. Results Gel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of âfreeâ Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50â60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions. Conclusions Our study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs. Genome- wide binding of Tip60 co-activator complexes
Project description:Chromosome translocations involving the MLL gene are common rearrangements in leukemia. Such translocations fuse the MLL 5’-region in frame to partner genes , and the resultant fusion proteins can cause MLL-related leukemia. MLL-fusions activate transcription of target genes such as the HoxA cluster and Meis1, but the underlying mechanisms remain elusive. We found that the MLL-AF10 fusion recruits Tip60 to the Hoxa9 locus, where it acetylates H2A.Z, thereby promoting Hoxa9 expression. Following conditional deletion of Tip60, hypoacetylation of H2A.Z was accompanied by recruitment of Ezh2, the catalytic subunit of PRC2, suggesting that nucleosomes with hypoacetylated H2A.Z are the preferential targets of Ezh2. Our findings suggest that MLL-AF10 achieves active chromatin states by recruiting Tip60, which acetylates H2A.Z to prevent gene silencing by Ezh2.
Project description:In this study we examined the effects of loss of the MYST histone acetyltransferase TIP60 (KAT5) in mouse embryonic fibroblasts (MEFs), human embryonic kidney cells 293 (HEK293), and human osteosarcoma cells (U2OS) on cell proliferation, BrdU incorporation, cell cycle progression, apoptotic and other forms of cell death, DNA damage, histone acetylation at specific lysine residues and RNA expression levels. This dataset relates to MEFs. To assess the effects of loss of TIP60 on RNA levels, RNA-seq was performed on MEFs, where the TIP60 gene was deleted using Cre/loxP technology. Nuclear translocation was induced with 4-OH-tamoxifen treatment for 3 and 5 days to induce TIP60 gene deletion in the samples also containing the loxP sited in the Tip60 locus.