was identified as a member of the TRF2-DREF complex that is involved in core promoter selection. TATA-box-binding-protein-related element 2 (TRF2) and the DNA-replication related element (DRE) binding element DREF (Hochheimer mutant cis-Urocanic acid background (Kugler and Nagel 2007 ). The TRF2-DREF Ppia complex consists of more than a dozen of proteins and the biochemical function of most of them remains still elusive. Interestingly it also consists of three members of the nucleosome redesigning element (NURF) imitation switch (ISWI) Nurf 55 and Nurf 38 (Hochheimer is required cis-Urocanic acid for Notch target gene manifestation which is definitely impaired in mutant cell clones. Consistent with this mutants enhance the mutant wing phenotype strongly arguing for an involvement of the NURF complex in Pzg-mediated epigenetic Notch target gene activation. MATERIALS AND METHODS Genetics Take flight Strains and Paperwork of Phenotypes To generate mutant clones using the Flp/FRT system the null mutant allele was recombined with and selected relating to its gentamicin resistance and failure of complementation with additional alleles. Flies of the genotype (Janody (Neufeld and Edgar 1998 ) (Lecuit (Kugler and Nagel cis-Urocanic acid 2007 ) (Roy (VDRC 24740) (VDRC 26455) (VDRC 3200) all from Vienna Stock Center (Kugler and Nagel 2007 ); mutants: (gift of A. Preiss University or college of Hohenheim Germany) (Deuring ((Badenhorst (Lecourtois and Schweisguth 1995 ) and (Kim induction assays which were performed at 25 and at 29°C. Adult wings were inlayed in Euparal (Roth Karlsruhe Germany) and at least 30 wings of each genotype were measured. Wing size was identified using ImageJ software for pixel measurements (http://rsb.info.nih.gov/ij/). The wing area was cis-Urocanic acid encircled with the Polygon tool. The Segmented Collection tool was used to reconstruct the space of wing notches. To test statistical significance p ideals were calculated relating to Student’s test (http://www.physics.csbsju.edu/stats/t-test.html). Photos were taken with Normarski optics on a Zeiss Axiophot (Carl Zeiss Jena Germany). Immunhistochemistry Immunoprecipitation and Chromatin Immunoprecipitation Wing imaginal discs were prepared from crawling third instar larvae of the respective genotype. The following antibodies were used: mouse anti-β-galactosidase (1:100; JIE7) mouse anti-Cut (1:20; 2B10) and mouse anti-Wg (1:25; 4D4) all from the Developmental Studies Hybridoma Bank University or college of Iowa (NICHD contract NO1-HD-7-3262) rabbit anti-cleaved caspase 3 (1:200 NEB Cell Signaling Technology Beverly MA) rat anti-Ci 2A1 at 1:2 dilutions (Motzny and Holmgren 1995 ). Photos of stained discs were taken having a Bio-Rad MRC1024 confocal microscope (Hercules CA) on a Zeiss Axiophot. Immunoprecipitations were performed relating to Nagel (2005) by using protein components from 100 1st instar larvae. For precipitations we used guinea pig anti-Pzg antibodies (1:250; Kugler and Nagel 2007 ) and rabbit anti-MOF antibodies (Matyunina [(encodes a Zn-finger protein we investigated at first whether Pzg binds directly to Notch target gene promoters. We performed electromobility shift analyses with the sequences precipitated by Pzg in the XChIP however found no suggestions for a direct DNA binding of Pzg to these sites (data not shown). From this we conclude that Pzg requires at least 1 further partner for binding to Notch target gene promoters. Pzg’s Presence at Notch Target Genes Depends on the Presence of the NURF Complex Pzg depletion is definitely correlated with a reduction of open chromatin structure at different Notch target genes assuming an important part of Pzg in chromatin activation at these Notch target sites (Kugler and Nagel 2007 ). Because mutant cells do not display this effect Pzg must influence Notch signaling inside a TRF2-DREF-independent manner (Kugler and Nagel 2007 ). Three proteins of the TRF2-DREF complex are also components of the nucleosome redesigning factor NURF that has been involved in chromatin activation (examined in Bouazoune and Brehm 2006 ). Hence Pzg might take action together with NURF in the context of Notch target gene activation. To investigate a possible association of Pzg and NURF in more detail we 1st performed XChIPs with anti-Pzg antibodies in mutants of all four components of the NURF complex. This includes the NURF-specific subunit Nurf 301 which is not part of the TRF2-DREF complex (Xiao and in the absence of ISWI Nurf 38 Nurf 55 or Nurf 301 whereas it was present in the crazy type.