InteractiveFly: GeneBrief

PAX transcription activation domain interacting protein: Biological Overview | References

BIOLOGICAL OVERVIEW

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).

The establishment and maintenance of gene expression patterns in development is regulated in part at the level of chromatin modification through the concerted actions of the Polycomb and trithorax family of genes (PcG/trxG). In Drosophila, Polycomb and Trithorax response elements (PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb protein complexes and maintain active or silent states, presumably in a heritable manner. In mammalian cells however, such PRE/TREs have not been conclusively identified. Polycomb and Trithorax gene products function by methylating specific histone lysine residues, yet how these complexes recognize individual loci in a temporal and tissue specific manner during development is unclear. Recently, a novel protein, PTIP (also known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins (Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax DNA-binding site. These data suggest that Pax proteins, and other similar DNA-binding proteins, can provide the locus and tissue specificity for HMT complexes during mammalian development (Fang, 2009).

In mammals, the PTIP protein is found within an HMT complex that includes the SET domain proteins ALR (GFER) and MLL3, and the accessory proteins WDR5, RBBP5 and ASH2 (Cho, 2007; Issaeva, 2007; Patel, 2007). This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip^-/- mouse embryos and conditionally inactivated Ptip^-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals, PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a time when the mesoderm becomes compartmentalized into axial, intermediate and lateral plate. These data suggest that PTIP provides a link between tissue specific DNA-binding proteins that specify cell lineages and the H3K4 methylation machinery (Fang, 2009).

To extend these finding to a non-mammalian organism and address the evolutionary conservation of Ptip, it was asked whether a Drosophila ptip homolog could be identified and if so, whether it is also an essential developmental regulator and part of the epigenetic machinery. The mammalian Ptip gene encodes a novel nuclear protein with two amino-terminal and four carboxy-terminal BRCT domains, flanking a glutamine-rich sequence (Lechner, 2000). Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning defects and developmental arrest, whereas zygotic null mutants developed to the third instar larval stage but also exhibited anterior/posterior (A/P) patterning defects. In cell culture, depletion of Polycomb-mediated repression activates developmental regulatory genes, such as the homeotic gene Ultrabithorax (Ubx). This derepression is dependent on trxG activity and also requires PTIP. Microarray analyses in cell culture of Polycomb and polyhomeotic target genes indicate that many, but not all, require PTIP for activation once repression is removed. The activation of PcG target genes is coincident with PTIP binding to promoter sequences and increased H3K4 trimethylation. These data argue for a conserved role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).

Embryonic development requires epigenetic imprinting of active and inactive chromatin in a spatially and temporally regulated manner, such that correct gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic development. In larval development, ptip coordinately regulates the methylation of histone H3K4 and demethylation of H3K27, consistent with the reports that mammalian PTIP complexes with HMT proteins ALR and MLL3, and the histone demethylase UTX (Cho, 2007; Issaeva, 2007). In wing discs, ptip is required for appropriate A/P patterning by affecting morphogenesis determinant genes, such as en and ci. These data demonstrate in vivo that dynamic histone modifications play crucial roles in animal development and PTIP might be necessary for coherent histone coding. In addition, ptip is required for the activation of a broad array of PcG target genes in response to derepression in cultured fly cells. These data are consistent with a role for ptip in trxG-mediated activation of gene expression patterns (Fang, 2009).

Early development requires ptip for the appropriate expression of the pair rule genes eve and ftz. The characteristic seven-stripe eve expression pattern is regulated by separate enhancer sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The characteristic 14 stripes of en expression depends on the correct expression of pair rule genes, which are clearly affected in ptip mutants. However, the maintenance of en expression at later stages and in imaginal discs is regulated by PREs and PcG proteins. If ptip functions as a trxG cofactor, then expression of en along the entire A/P axis in the imaginal discs of ptip mutants might be due to the absence of a repressor. This might explain the surprising presence of ectopic en in the anterior halves of imaginal discs from zygotic ptip mutants. This ectopic en expression is likely to result in suppression of ci through a PcG-mediated mechanism. Yet, it is not clear how en is normally repressed in the anterior half, nor which genes are responsible for derepression of en in the ptip mutant wing and leg discs (Fang, 2009).

The direct interaction of PTIP protein with developmental regulatory genes is supported by ChIP studies in cell culture. Given the structural and functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG target genes that are activated upon loss of PC and PH activity. Consistent with the interpretation that a PTIP trxG complex is necessary for activation of repressed genes, mPTIP only bound to DNA upon loss of Pc and ph function. In the Kc cells, suppression of both Pc and ph results in the activation of many important developmental regulators, including homeotic genes. A recent report details the genome-wide binding of PcG complexes at different developmental stages in Drosophila and reveals hundreds of PREs located near transcription start sites. Strikingly, most of the genes found to be activated in the Kc cells after PcG knockdown also contain PRE elements near the transcription start site (Fang, 2009).

In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to promote assembly of an H3K4 methyltransferase complex. The tissue and locus specificity for assembly may be mediated by DNA-binding proteins such as PAX2 (Patel, 2007) or SMAD2 (Shimizu, 2001), which regulate cell fate and cell lineages in response to positional information in the embryo. In flies, recruitment of PcG or trxG complexes to specific sites also can require DNA-binding proteins such as Zeste, DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG complexes. Purification of a trxG complex capable of histone acetylation (TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By contrast, the mammalian MLL/ALL proteins are components of large multi-protein complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).

Mammalian PTIP is also thought to play a role in the DNA damage response based on its ability to bind to phosphorylated p53BP1 (Munoz, 2007). PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases (Manke, 2003). Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex (Cho, 2007; Patel, 2007). Both budding and fission yeast contain multiple BRCT domain proteins that are involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All of these yeast proteins have mammalian counterparts. However, neither the fission nor budding yeast genomes encodes a protein with six BRCT domains and a glutamine-rich region between domains two and three, whereas such characteristic PTIP proteins are found in Drosophila, the honey bee, C. elegans and all vertebrate genomes. These comparative genome analyses suggest that ptip evolved in metazoans, consistent with an important role in development and differentiation (Fang, 2009).

In summary, Drosophila ptip is an essential gene for early embryonic development and pattern formation. Maternal ptip null embryos show early patterning defects including altered and reduced levels of pair rule gene expression prior to gastrulation. In cultured cells PTIP activity is required for the activation of Polycomb target genes upon derepression, suggesting an important role for the PTIP protein in trxG-mediated activation of developmental regulatory genes. The conservation of gene structure and function, from flies to mammals, suggests an essential epigenetic role for ptip in metazoans that has remained unchanged (Fang, 2009).

Search PubMed for articles about Drosophila Ptip

Cho, Y. W., Hong, T., Hong, S., Guo, H., Yu, H., Kim, D., Guszczynski, T., Dressler, G. R., Copeland, T. D., Kalkum, M. et al. (2007). PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. J. Biol. Chem. 282: 20395-20406. PubMed ID: 17500065

Fang, M., Ren, H., Liu, J., Cadigan, K. M., Patel, S. R. and Dressler, G. R. (2009). Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways. Development 136(11): 1929-38. PubMed ID: 19429789

Issaeva, I., Zonis, Y., Rozovskaia, T., Orlovsky, K., Croce, C. M., Nakamura, T., Mazo, A., Eisenbach, L. and Canaani, E. (2007). Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol. Cell. Biol. 27: 1889-1903. PubMed ID: 17178841

Lechner, M. S., Levitan, I. and Dressler, G. R. (2000). PTIP, a novel BRCT domain-containing protein interacts with Pax2 and is associated with active chromatin. Nucleic Acids Res. 28: 2741-2751. PubMed ID: 10908331

Manke, I. A., Lowery, D. M., Nguyen, A. and Yaffe, M. B. (2003). BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302: 636-639. PubMed ID: 14576432

Munoz, I. M., Jowsey, P. A., Toth, R. and Rouse, J. (2007). Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage. Nucleic Acids Res. 35: 5312-5322. PubMed ID: 17690115

Patel, S. R., Kim, D., Levitan, I. and Dressler, G. R. (2007). The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex. Dev. Cell 13: 580-592. PubMed ID: 17925232

Shimizu, K., Bourillot, P. Y., Nielsen, S. J., Zorn, A. M. and Gurdon, J. B. (2001). Swift is a novel BRCT domain coactivator of Smad2 in transforming growth factor beta signaling. Mol. Cell. Biol. 21: 3901-3912. PubMed ID: 11359898

date revised: 22 November 2022

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