48 hours after transfection with plasmids, cells were harvested for protein expression and ChIP experiments as described below. Chromatin Immunoprecipitation and Real-time PCR HEK 293 cells with an integrated PRS4EGFP reporter were transfected with 1 ug CMV-Pax2 (Cai et al, 2002) and harvested 48 hour after transfection. a novel component of a histone methyltransferase complex that links DNA binding developmental regulators to epigenetic imprinting. Trithorax, including MLL/ALL (Milne et al., 2002; Nakamura et al., 2002), ALR (Goo et al., 2003), and MLL2 (Glaser et al., 2006). How does methylation at specific residues alter chromatin structure? The modified histone tails may interact with specific proteins such as chromatin remodeling factors that recognize methylated lysine residues (Jacobs and Khorasanizadeh, 2002; Min et al., 2003; Nielsen Lox et al., 2002). The WDR5 protein facilitates tri-methylation of H3K4 (Wysocka et al., 2005) and subsequent interaction with the PhD finger of the BPTF subunit of a nucleosome remodeling factor (Wysocka et al., 2006). Thus, histone methylation may provide docking sites for chromatin remodeling proteins to establish and maintain open or closed Canagliflozin hemihydrate configurations. Methylation of histones must be regulated in a spatial and temporal manner as specific loci are differentially marked during development. Yet, how the histone methylation complexes recognize genes in a tissue specific manner is not clear. In this report, we show that the modular BRCT-domain protein PTIP (Pax Transcription activation domain Interacting Protein) links the developmental regulator Pax2 to the H3K4 methylation machinery. Pax2 specifies a region of mesoderm fated to become kidney and urogenital epithelium and also demarcates the midbrain-hindbrain junction (Bouchard et al., 2002; Torres et al., 1995). The Pax family of DNA binding proteins determine cell fate or patterns in the post-gastrulation embryo. PTIP was discovered because of its interactions with Pax2 (Lechner et al., 2000) but also interacts with other transcription factors (Shimizu et al., 2001). PTIP is part of a histone methyltransferase complex that includes the SET domain proteins ALR and MLL3 and other homologues of the yeast Canagliflozin hemihydrate Compass complex (Miller et al., 2001). The PTIP complex localizes to a Pax2 DNA binding sequence, in a Pax2 dependent manner, and recruits the ALR methyltransferase complex to the Pax2 binding site. In PTIP siRNA knockdown cells, Pax2 still binds an integrated DNA target site, but the ALR complex fails to form and no histone H3K4 methylation occurs. Furthermore, PTIP null embryos are severely disorganize from the time of gastrulation and show a clear reduction of H3K4 staining, suggesting that PTIP is required for H3K4 methylation at many loci. Similar reductions of global H3K4 levels are observed in the developing spinal cord of conditional PTIP mutants, whereas methylation at histone H4K20 appears unaffected. Thus, PTIP links DNA binding proteins that specify cell lineages to an H3K4 histone methyltransferase complex. Results Biochemical Purification of a PTIP complex In order to better understand the function of PTIP, we purified PTIP containing nuclear complexes. HEK293 cells were transfected with flag-tagged PTIP or control vectors. Some PTIP expressing cells were subject to Canagliflozin hemihydrate 10 Gy gamma irradiation because PTIP had been shown to interact with proteins in the DNA damage response pathway. Nuclear extracts were prepared from 200 plates of cells as outlined (Fig. 1A). After initial purification over P11 phosphocellulose and DEAE Sephacel columns, proteins were bound to M2 flag agarose and eluted with 3x flag peptide. Elutions were tested for PTIP using anti-flag and anti-PTIP antibodies and pooled for further analysis. Concentrated elutions were separated on 12% SDS/PAGE gels and stained with colloidal coomasie (Fig. 1B). Proteins co-purifying with PTIP were analyzed by mass spectrometry and peptide sequences determined. The identities of specific proteins and the number of peptides sequenced for each protein are listed (Table 1). Open in a separate window Figure 1 PTIP Co-purifies with a Histone Methylatransferase ComplexA) The biochemical purification of PTIP and associated proteins is shown. B) The final 3x FLAG elutions were concentrated, run on a 12% SDS/PAGE gel, and stained with colloidal coomasie blue. The proteins identified by MS/MS are indicated. The abundant protein at 60 kD in the PTIP lane was a Keratin contaminant. C) Chicken antibodies against PTIP or rabbit anti-Ash2L were used to immunoprecipitate (IP) from nuclear extracts directly. Co-precipitated proteins were detected by western blotting as Canagliflozin hemihydrate indicated. Controls antibodies for the IP are chicken (Ch) IgY and rabbit (Rb) IgG. D) WDR5 co-immunoprecipitates with anti-PTIP but not with control antibodies, as indicated. E) Rbbp5 co-immunoprecipitates with anti-PTIP antibodies. Similarly, PTIP is co-immunoprecipitated with antibodies against Rbbp5. F) Antibodies against human ALR were.