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First published online 22 November 2005
doi: 10.1242/jcs.02688

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Histone H2AZ dimerizes with a novel variant H2B and is enriched at repetitive DNA in Trypanosoma brucei

Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA

View larger version (29K): [in a new window]   Fig. 1. T. brucei H2AZ localizes to distinct foci within the nucleus. (A) Sequence conservation of H2AZ. For each H2AZ, the polypeptide length is denoted as well as the percent sequence identity/similarity relative to T. brucei H2AZ. The amino terminal extension of T. brucei H2AZ (red) is unique and was excluded from these calculations. (B) A polyclonal antibody to a 14 amino-acid region of the amino-terminal tail of H2AZ (marked by a black bar in A) recognizes endogenous H2AZ in wild-type procyclic (PF) and bloodstream-form (BF) cell lines as well as those in which ectopically expressed GFP- or TY1-tagged H2AZ has replaced the endogenous H2AZ. (C) A comparison of the localization of H2AZ (red) and TY1-H2A (green) by indirect immunofluorescence in a bloodstream-form cell line over the course of the cell cycle reveals that H2AZ is not uniformly distributed. Both nuclear (n) and mitochondrial kinetoplast (k) DNA (blue) were detected with DAPI. Owing to its compact state, the kinetoplast DNA signal appears stronger than the nuclear DNA at this focal plane. Top, an interphase cell (1n 1k); middle, an early mitotic cell (1n 2k); bottom, cytokinesis (2n 2k). Bar, 2 µm.

View larger version (32K): [in a new window]   Fig. 2. A novel histone H2B variant colocalizes with H2AZ. (A) The sequence of H2BV aligned with T. brucei H2B (Tb10.406.0330). Amino acids conserved between H2BV and H2B are highlighted. (B) A bloodstream-form cell line expressing TY1-tagged H2BV was examined using antibodies that recognize TY1 (green) and H2AZ (red). DNA (blue) was detected with DAPI. The H2BVand H2AZ signals are completely coincident (yellow) at all stages of the cell cycle. Top, two interphase cells (1n 1k); bottom, a late mitotic cell (2n 2k). Bar, 2 µm.

View larger version (35K): [in a new window]   Fig. 3. H2AZ and H2BV show similar genomic distributions. (A) Chromatin immunoprecipitations were carried out on cell lines expressing TY1-H2A, TY1-H2AZ, TY1-H2BV, or parental `wild-type' cells. For each cell line, chromatin was immunoprecipitated using nonspecific (FLAG) or specific (TY1) antibodies. Input chromatin (10% of total) and immunoprecipitated material were analyzed by slot blot using radiolabeled probes to a variety of sequences: transcriptionally active genes [-tubulin (TUB), ß-tubulin (ßTUB), histone H3 (HHT), 5SDNA and VSG221]; silenced genes [procyclin EP1 (EP1), VSG224 and VSGVO2]; a retrotransposon-like element (INGI); and repetitive, noncoding sequences [rDNA spacer region (rDNA spacer), expression-site promoter (ES pro), telomeric repeat (TEL), mini-chromosome 177 bp repeat (MC177) and 50 bp repeat (50 bp)]. (B) Quantification of the data in A. Using a phosphorimager, each signal was quantified and the amount of material immunoprecipitated relative to the input was calculated. Each ChIP was performed 2-4 times and the values averaged. Error bars are indicated.

View larger version (27K): [in a new window]   Fig. 4. H2AZ does not colocalize with sites of transcription. (A) Nascent RNA transcripts from bloodstream-form cells were labeled with BrUTP and examined by indirect immunofluorescence. H2AZ (red) and BrUTP (green) occupy distinct sites within the nucleus. (B) In the presence of 100 µg/ml -amanitin, incorporation of BrUTP (green) is limited to sites of RNA polymerase I transcription, which do not overlap with H2AZ (red). For both A and B, DNA (blue) was detected with DAPI. Bar, 2 µm. BrUTP incorporation into the kinetoplasts is detected in the presence and, less obviously, in the absence of -amanitin, probably reflecting the massive uridine incorporation that occurs during RNA editing, including in the bloodstream form (Schnaufer et al., 2001).

View larger version (46K): [in a new window]   Fig. 5. H2AZ and H2BV co-immunoprecipitate. Mononucleosomes were prepared from cell lines expressing TY1-H2BV, FLAG-H2B, TY1-H2A or TY1-H3V, and were subjected to immunoprecipitations using antibodies to TY1 or FLAG. Immunoprecipitated material was then examined by western blotting for the presence of the TY1- or FLAG-tagged histone along with H2AZ and H3. For each experiment, a lane corresponding to whole-cell extract (wce), input material (input) and immunoprecipitated material (IP) is shown. (A) Both H2AZ and histone H3 co-immunoprecipitate with TY1-H2BV. (B) Histone H3, but not H2AZ, co-immunoprecipitates with FLAG-H2B. (C) Histone H3, but not H2AZ, co-immunoprecipitates with TY-H2A. (D) Neither H2AZ nor histone H3 co-immunoprecipitate with TY1-H3V.

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