News
11.05.2012
Pichler G, Jack A, Wolf P, Hake SB (2012) Versatile Toolbox for High Throughput Biochemical and Functional Studies with Fluorescent Fusion Proteins. PLoS ONE 7(5): e36967. doi:10.1371/journal.pone.0036967
Fluorescent fusion proteins are widely used to study protein localization and interaction dynamics in living cells. However,
to fully characterize proteins and to understand their function it is crucial to determine biochemical characteristics such as
enzymatic activity and binding specificity. Here we demonstrate an easy, reliable and versatile medium/high-throughput
method to study biochemical and functional characteristics of fluorescent fusion proteins. Using a new system based on 96-
well micro plates comprising an immobilized GFP-binding protein (GFP-mulitTrap), we performed fast and efficient one-step
purification of different GFP- and YFP-fusion proteins from crude cell lysate. After immobilization we determined highly
reproducible binding ratios of cellular expressed GFP-fusion proteins to histone-tail peptides, DNA or selected RFP-fusion
proteins. In particular, we found Cbx1 preferentially binding to di-and trimethylated H3K9 that is abolished by
phosphorylation of the adjacent serine. DNA binding assays showed, that the MBD domain of MeCP2 discriminates between
fully methylated over unmethylated DNA and protein-protein interactions studies demonstrate, that the PBD domain of
Dnmt1 is essential for binding to PCNA. Moreover, using an ELISA-based approach, we detected endogenous PCNA and
histone H3 bound at GFP-fusions. In addition, we quantified the level of H3K4me2 on nucleosomes containing different
histone variants. In summary, we present an innovative medium/high-throughput approach to analyse binding specificities
of fluroescently labeled fusion proteins and to detect endogenous interacting factors in a fast and reliable manner in vitro.
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01.04.2012
Bönisch C, Schneider K, Pünzeler S, Wiedemann SM, Bielmeier C, Bocola M, Eberl HC, Kuegel W, Neumann J, Kremmer E, Leonhardt H, Mann M, Michaelis J, Schermelleh L, Hake SB. H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes sever
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
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07.03.2012
Ratnakumar K, Duarte LF, Leroy G, Hasson D, Smeets D, Vardabasso C, Bönisch C, Zeng T, Xiang B, Zhang DY, Li H, Wang X, Hake SB, Schermelleh L, Garcia BA, Bernstein E. ATRX-mediated chromatin association of histone variant macroH2A1 regulates α-globi
The histone variant macroH2A generally associates with transcriptionally inert chromatin; however, the factors that regulate its chromatin incorporation remain elusive. Here, we identify the SWI/SNF helicase ATRX (α-thalassemia/MR, X-linked) as a novel macroH2A-interacting protein. Unlike its role in assisting H3.3 chromatin deposition, ATRX acts as a negative regulator of macroH2A's chromatin association. In human erythroleukemic cells deficient for ATRX, macroH2A accumulates at the HBA gene cluster on the subtelomere of chromosome 16, coinciding with the loss of α-globin expression. Collectively, our results implicate deregulation of macroH2A's distribution as a contributing factor to the α-thalassemia phenotype of ATRX syndrome.
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