Anti-PHF1 monoclonal antibody, clone 3H8

The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste (E(Z)) is a Drosophila PcG protein that previously has been shown to bind directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, we show that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL-E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. EM was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with EM and ESC in Drosophila embryos. We discuss the implications of PCL association with ESC.E(Z) complexes and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes.

Alzheimer's disease is characterized by the accumulation of amyloid-beta (A beta) and Tau in the brain. In mature neurons, Tau is concentrated in the axon and found at lower levels in the dendrite where it is required for targeting Fyn to the spines. Here Fyn mediates A beta toxicity, which is vastly abrogated when Tau is either deleted or a truncated form of Tau (Tau(1-255)) is co-expressed. Interestingly, MAP2, a microtubule-binding protein with mainly dendritic localization that shares Fyn-binding motifs with Tau, does not mediate A beta's synaptic toxicity in the absence of Tau. Here we show in hippocampal neurons that endogenous Tau enters the entire spine, albeit at low levels, whereas MAP2 only enters its neck or is restricted to the dendritic shaft. Based on an extensive mutagenesis study, we also reveal that the spine localization of Tau is facilitated by deletion of the microtubule-binding repeat domain. When distinct phosphorylation sites (AT180-T231/S235, 12E8-S262/S356, PHF1-S396/S404) were pseudophosphorylated (with glutamic acid, using alanine replacements as controls), Tau targeting to spines was markedly increased, whereas the pseudophosphorylation of the late phospho-epitope S422 had no effect. In determining the role physiological Fyn has in the spine localization of Tau, we found that neither were endogenous Tau levels reduced in Fyn knockout compared with wild-type synaptosomal brain fractions nor was the spine localization of over-expressed pseudophosphorylated or P301L Tau. This demonstrates that although Fyn targeting to the spine is Tau dependent, elevated levels of phosphotylated Tau or P301L Tau can enter the spine in a Fyn-independent manner. (C) 2015 The Authors. Published by Elsevier B.V.