Gene name - reptin
Cytological map position-76A3-76A3
Function - DNA helicase
Keywords - chromatin remodeling, histone exchange, component of TIP60 complex
Symbol - rept
FlyBase ID: FBgn0040075
Genetic map position -3L
Classification - AAA+ family of ATPases
Cellular location - nuclear
|Recent literature|| David-Morrison, G., Xu, Z., Rui, Y.N., Charng, W.L., Jaiswal, M., Yamamoto, S., Xiong, B., Zhang, K., Sandoval, H., Duraine, L., Zuo, Z., Zhang, S. and Bellen, H.J. (2016). WAC regulates mTOR activity by acting as an adaptor for the TTT and Pontin/Reptin complexes. Dev Cell 36: 139-151. PubMed ID: 26812014
The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. Under energy-sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. This study shows that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, wacky, the homolog of WAC, was identified. Loss of Wacky leads to neurodegeneration, defective mTOR activity, and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin, which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction.
|Vinayagam, A., Kulkarni, M. M., Sopko, R., Sun, X., Hu, Y., Nand, A., Villalta, C., Moghimi, A., Yang, X., Mohr, S. E., Hong, P., Asara, J. M. and Perrimon, N. (2016). An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to insulin signaling. Cell Rep 16: 3062-3074. PubMed ID: 27626673
Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and metabolism. To expand understanding of the insulin pathway, biochemical, genetic, and computational approaches were applied to build a comprehensive Drosophila InR/PI3K/Akt network. First, the dynamic protein-protein interaction network surrounding the insulin core pathway was mapped using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, it was found that 47% of interacting proteins affect pathway activity, and, using quantitative phosphoproteomics, it was demonstrates that approximately 10% of interacting proteins are regulated by insulin stimulation at the level of phosphorylation. Next, these orthogonal datasets were integrated to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate this method by identifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, this study represents a comprehensive resource for the study of the evolutionary conserved insulin network.
Histone acetyltransferase (HAT) complexes have been linked to activation of transcription. Reptin is a subunit of different chromatin-remodeling complexes, including the TIP60 HAT complex (see Tip60). In Drosophila, Reptin also copurifies with the Polycomb group (PcG) complex PRC1, which maintains genes in a transcriptionally silent state. Genetic interactions have been demonstrated between reptin mutant flies and PcG mutants, resulting in misexpression of the homeotic gene Scr. Genetic interactions are not restricted to PRC1 components, but are also observed with another PcG gene. In reptin homozygous mutant cells, a Polycomb response-element-linked reporter gene is derepressed, whereas endogenous homeotic gene expression is not. Furthermore, reptin mutants suppress position-effect variegation (PEV), a phenomenon resulting from spreading of heterochromatin. These features are shared with three other components of TIP60 complexes, namely Enhancer of Polycomb, Domino, and dMRG15. It is concluded that Drosophila Reptin participates in epigenetic processes leading to a repressive chromatin state as part of the fly TIP60 HAT complex rather than through the PRC1 complex. This shows that the TIP60 complex can promote the generation of silent chromatin (Qi, 2006).
A fundamental regulatory step in transcription and other DNA-dependent processes in eukaryotes is the control of chromatin structure, which regulates access of proteins to DNA. Histone acetylation and the protein complexes that mediate this modification have been linked to activation of transcription. It is believed that lysine acetylation of histone N termini results in less compact chromatin by neutralizing the positive charge of histones and that the acetyl groups are recognized by regulatory proteins that promote transcription. However, it is becoming clear that histone acetyltransferases (HATs) can have functions other than facilitating transcription. For example, the TIP60 HAT complex has been implicated in DNA repair in yeast, flies, and mammals (Ikura, 2000; Bird, 2002; Kusch, 2004). This study investigated the role of Drosophila Reptin and other TIP60 components in chromatin regulation in vivo (Qi, 2006).
The Reptin protein, also known as TIP48, TIP49b, or RUVBL2, is related to bacterial RuvB, an ATP-dependent DNA helicase that promotes branch migration in Holliday junctions (Kanemaki, 1999). Reptin, and the related Pontin (TIP49, TIP49a, or RUVBL1) protein, possess intrinsic ATPase and helicase activities and can heterodimerize (Kanemaki, 1999; Makino, 1999. In yeast, both Reptin and Pontin are part of the INO80 chromatin-remodeling complex (Shen, 2000), as well as the Swr1 complex that can exchange histone H2A with the variant histone H2A.Z (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). Reptin and Pontin appear to play antagonistic roles in development by regulating Wnt signaling (Bauer, 2000) and heart growth in zebrafish embryos (Rottbauer, 2002). Mammalian Reptin and Pontin are present in TIP60 HAT complexes (Wood, 2000; Fuchs, 2001; Cai, 2003; Frank, 2003; Doyon, 2004), which are involved in induction of apoptosis in response to DNA damage and which interact with the c-Myc protein to promote its oncogenic activity (Qi, 2006).
TIP60 is a HAT of the MYST family (Utley, 2003). The homologous yeast protein Esa1 is the catalytic subunit of the nucleosome acetyltransferase of H4 (NuA4) complex, which acetylates lysines in histone H4 and H2A (Doyon, 2004). In Drosophila, the TIP60 complex acetylates the phosphorylated variant histone H2Av after DNA double-strand breaks and exchanges it with unmodified H2Av (Kusch, 2004). The composition of TIP60 and NuA4 complexes has recently been determined (Doyon, 2004). TIP60 (yeast Esa1), ING3 (Yng2), and Enhancer of Polycomb (EPC1, yeast Epl1) form a core complex that is sufficient for acetylation of histones in nucleosomes (Boudreault, 2003; Doyon, 2004). Mammalian and Drosophila TIP60 complexes contain four subunits not present in yeast NuA4 (Doyon, 2004; Kusch, 2004): Brd8, Reptin, Pontin, and Domino (also known as p400), the homolog of yeast Swr1 (Qi, 2006).
Polycomb group (PcG) proteins are evolutionarily conserved chromatin regulators that maintain appropriate expression patterns of developmental control genes, such as the Hox genes. PcG proteins are generally repressors that maintain the off state of genes and exist in at least two distinct protein complexes. The EscE(z) complex is a histone methyltransferase that includes the catalytic subunit Enhancer of zeste [E(z)], as well as the Extra sex combs (Esc) and Suppressor of zeste 12 [Su(z)12] subunits. Another complex purified from Drosophila embryos, Polycomb repressive complex 1 (PRC1) has a mass of >1 MDa. In addition to genetically identified PcG proteins, it includes TFIID subunits, the Reptin protein, and other polypeptides. The PRC1 complex can block chromatin remodeling by the SWI/SNF complex in vitro. A core PRC1 complex consisting of Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), and dRING1/Sex combs extra (Sce) is sufficient for the in vitro activities of PRC1. Recently, it was shown that dRing1/Sce as well as its mammalian orthologs are E3 ubiquitin ligases that monoubiquitylate histone H2A (Qi, 2006 and references therein).
This study investigates the role of Drosophila Reptin in chromatin regulation. Reptin is shown to interact genetically with PcG gene products and suppresses position-effect variegation (PEV), properties shared by other Drosophila TIP60 complex components. It is suggested that the fly TIP60 complex regulates epigenetic processes leading to a repressive chromatin state. This is a novel activity of a HAT complex that has previously been implicated in transcription activation and DNA repair (Qi, 2006).
It is proposed that Reptin acts as a subunit of the TIP60 HAT complex to generate a repressive chromatin state. This is a novel activity of a HAT complex previously shown to promote transcription (Doyon, 2004). This study shows that Reptin copurifes with the Polycomb complex PRC1. This prompted an investigation of whether the biochemical interaction with PRC1 was accompanied by a genetic interaction. It was shown that Reptin and PRC1 components genetically interact to regulate expression of the Hox gene Scr. However, Reptin also interacts with a PcG gene product not associated with the PRC1 complex, Pcl. Although no interactions were detected between reptin heterozygous mutants and several PREs tested, a PRE from the Ubx gene is derepressed in reptin homozygous mutant cells. This shows that Reptin contributes an essential function to the activity of this PRE. However, unlike most PcG genes, reptin homozygous mutants do not derepress endogenous Hox gene expression. It appears that repression of endogenous Hox genes is more complex and not as sensitive to the loss of Reptin as the Ubx PRE. In contrast to most PcG genes, reptin mutants suppress PEV. Interestingly, derepression of the Ubx PRE also occurs in embryos mutant for other suppressors of PEV, indicating that this PRE may be highly sensitive to the chromatin environment in its vicinity. Since reptin mutants suppress PEV and fail to derepress endogenous Hox gene expression, reptin is not considered a bona fide PcG gene, and it is found unlikely that Reptin protein contributes an essential function to the PRC1 complex. In fact, the biochemical activities ascribed to PRC1 can be reconstituted either with recombinant dRing1/Sce or with four core components whose activity can be further enhanced by the DNA-binding proteins Zeste and GAGA (Qi, 2006).
Given that Reptin is present in TIP60 complexes in mammals and recently was shown to be a component of a Drosophila TIP60 complex (Ikura, 2000; Fuchs, 2001; Cai, 2003; Doyon, 2004; Kusch, 2004), the possibility is considered that the genetic interactions observed with PcG genes are due to the presence of Reptin in the fly TIP60 complex. The products of two previously characterized Drosophila genes, E(Pc) and domino, are also present in the TIP60 complex. Strikingly, E(Pc) and domino mutants share with reptin the ability to genetically interact with PcG genes and suppress PEV. E(Pc) is an unusual PcG gene that has very minor effects on Hox gene expression, and unlike most PcG genes, modifies PEV. In both yeast and humans, E(Pc) homologs form a core complex with Esa1 (TIP60) and Yng2 (ING3) that is sufficient for the nucleosomal acetylation of histones H4 and H2A by the NuA4 complex (Boudreault, 2003; Doyon, 2004). That such an integral NuA4/TIP60 complex component displays phenotypes similar to reptin mutants suggests that Reptin functions through the fly TIP60 complex (Qi, 2006).
Domino protein is similar to p400 and to SRCAP in mammals and to Swr1 in yeast (Eissenberg, 2005). Swr1 has recently been shown to exchange the variant histone H2A.Z (Htz1 in yeast) for H2A in nucleosomes (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004). Intriguingly, an involvement of Htz1 (H2A.Z) in controlling the spreading of silenced chromatin has recently been demonstrated in yeast. Exchange of variant histones may be a conserved feature of chromatin regulation since a recent report demonstrates that Drosophila H2Av behaves genetically as a PcG gene and suppresses PEV (Swaminathan, 2005). Domino exchanges phosphorylated and acetylated H2Av for unmodified H2Av after DNA damage (Kusch, 2004). However, no change was found in binding of H2Av to polytene chromosomes prepared from domino mutant larvae (Qi, 2006).
A P-element insertion was identified in the gene encoding one additional TIP60 complex component, the chromodomain-containing protein MRG15. Human MRG15 (MORF-related gene on chromosome 15) has been implicated in cellular senescence and regulation of the B-myb promoter. Both human and yeast (Eaf3/Alp13) MRG15 have been found in Sin3/HDAC complexes in addition to the TIP60 (NuA4) complex, where it directs the histone deacetylase to coding regions through interaction of its chromodomain with methylated histone H3 lysine 36. This study found that MRG15 mutant flies interact with PcG genes and suppress PEV, just as other TIP60 complex components do. This is taken as further support of the conclusion that Reptin's effects on chromatin processes are mediated through its association with the fly TIP60 complex (Qi, 2006).
What is the basis for the genetic interaction between TIP60 components and PcG genes? One possibility is that the TIP60 complex regulates PcG expression. However, no reduction was observed in Pc expression in reptin mutant embryos. Another possibility is that the enzymatic activities of the TIP60 complex cooperate with PcG genes to mediate transcriptional silencing. Since binding of Pc to polytene chromosomes is abolished in H2Av mutant animals (Swaminathan, 2005), TIP60 complex-mediated histone variant exchange might cause the genetic interaction with PRC1. However, this study found that binding of PcG proteins to polytene chromosomes is unaffected in domino mutant larvae. It is possible that PRC1-mediated H2A ubiquitylation helps to recruit the TIP60 complex, whose histone acetylation or histone exchange activity assists in transcriptional repression. Alternatively, histone acetylation or exchange facilitates binding of the PRC1 complex to PREs. A similar mechanism has been invoked for the cooperation of the EscE(z) complex and PRC1, where EscE(z) trimethylates histone H3 lysine 27, which is recognized by the chromodomain of Polycomb (Qi, 2006).
This study has shown that the Drosophila TIP60 complex plays a role in epigenetic gene silencing in vivo. A similar case has been described for the yeast HAT complex SAGA (Spt-Ada-Gen5-acetyltransferase) that is required for both activation and repression of the ARG1 gene. Two other yeast HATs, Sas2 and Sas3, also promote gene silencing. Interestingly, the Drosophila HAT Chameau suppresses PEV and cooperates with PcG genes as well. TIP60, Sas2, Sas3, and Chameau are HATs that belong to the MYST family. Therefore, MYST family HATs in both yeast and flies can control epigenetic inheritance of silent chromatin (Qi, 2006).
The Reptin protein, also known as TIP48, TIP49b, or RUVBL2, is related to bacterial RuvB, an ATP-dependent DNA helicase that promotes branch migration in Holliday junctions (Kanemaki, 1999). Reptin, and the related Pontin (TIP49, TIP49a, or RUVBL1) protein, possess intrinsic ATPase and helicase activities and can heterodimerize (Kanemaki, 1999; Makino, 1999). In yeast, both Reptin and Pontin are part of the INO80 chromatin-remodeling complex (Shen, 2000), as well as the Swr1 complex that can exchange histone H2A with the variant histone H2A.Z (Krogan, 2003; Kobor, 2004; Mizuguchi, 2004; Qi, 2006 and references therein).
date revised: 18 July 2007
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