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First published online 2 November 2004
doi: 10.1242/jcs.01515

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A variant histone H3 is enriched at telomeres in Trypanosoma brucei

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

View larger version (90K): [in a new window]   Fig. 1. HTV encodes a variant of histone H3. (A) The sequence of H3V aligned with variant forms of histone H3 from T. cruzi (http://TcruziDB.org) and L. major (Q9U196) and major histone H3 from T. brucei (Q810K5), S. cerevisiae (P02303), S. pombe (P09988), Arabidopsis thaliana (P59226), C. elegans (P08898), Drosophila melanogaster (P02299) and Homo sapiens (P16106). Amino acids conserved between H3V and other proteins are highlighted in black; boxed regions reveal identity among all proteins except H3V. An asterisk marks the site of a glutamine residue that is conserved in histone H3 but is often absent in CenH3s. (B) The primary structure of the histone-fold domain of H3V compared to Cse4p from S. cerevisiae (P36012), Cnp1 from S. pombe (Q9Y812), HTR12 from A. thaliana (AAL86775), Hcp-3 from C. elegans (CAA80160), Cid from D. melanogaster (AF259371) and Cenp-A from H. sapiens (P49450). Amino acids conserved between H3V and CenH3s are highlighted in black whereas those only conserved among CenH3s are boxed. Regions identified as corresponding to the helices N, 1 and 2, and loop 1 and 2 are based on the crystal structure of histone H3 (Luger et al., 1997).

View larger version (45K): [in a new window]   Fig. 2. The localization of H3V shifts dramatically over the cell cycle. A procyclic-form cell line (PFJEL12) expressing TY1-H3V (green) from its endogenous locus was subjected to indirect immunofluorescence, stained with DAPI to detect DNA (blue) and examined by deconvolution microscopy. (A) In interphase cells, TY1-H3V is often present as discrete foci at the nuclear periphery. (B) As the cell enters mitosis, TY1-H3V moves to the center of the nucleus. (C) As mitosis proceeds, TY1-H3V heads towards the poles. (D) At the end of mitosis, TY1-H3V remains at the poles before dispersing along the nuclear periphery. The region within the nucleus that is not stained by DAPI corresponds to the nucleolus as verified previously (Das et al., 1998). n, nucleus; k, kinetoplast. Bar, 2 µm.

View larger version (73K): [in a new window]   Fig. 3. H3V associates with the spindle during mitosis. (A-D) PFJEL4, a cell line expressing H3V-YFP (green), was fixed and stained with an antibody that recognizes ß-tubulin (red). This antibody detects both the cytoskeleton and the mitotic spindle, and thus the outline of each cell is seen. DNA (blue) was visualized with DAPI. (A) At interphase, no spindle is observed and H3V-YFP is predominantly at the nuclear periphery. (B) In early mitosis, the spindle is visualized and H3V-YFP appears arrayed across it. (C,D) H3V-YFP continues its association with the spindle as it is pulled toward the poles. Bar, 2 µm.

View larger version (57K): [in a new window]   Fig. 4. H3V associates with mini-chromosomes. (A-D) Indirect-immunofluorescence of TY1-H3V (green) was combined with detection of mini-chromosomes by FISH using a probe specific for the 177-bp repeats (MC177, red). DNA (blue) was detected with DAPI. All cells are procyclics (PFJEL12) at (A) interphase, (B,C) mitosis and (D) late mitosis. Arrowheads indicate trailing TY1-H3V foci that do not overlap with 177-bp repeats. Bar, 2 µm.

View larger version (45K): [in a new window]   Fig. 5. H3V colocalizes with telomeres. (A-E) Indirect immunofluorescence of TY1-H3V (green) was combined with FISH using a probe specific for the telomeric TTAGGG repeats (TEL, red) found at the ends of all chromosomes. DNA (blue) was detected with DAPI stain. All cells are procyclics (PFJEL12) at (A) interphase, (B,C) mitosis, (D) late mitosis and (E) karyokinesis. Arrowheads indicate TY1-H3V foci that are coincident with telomeric foci that may correspond to MBCs. Asterisk indicates a kinetoplast of an adjacent cell. Bar, 2 µm.

View larger version (21K): [in a new window]   Fig. 6. H3V is enriched at telomeres. (A) Chromatin immunoprecipitation was carried out on procyclic wild-type cells (29.13) or cells expressing H3V-YFP (PFJEL4) or H3-YFP (PFJEL7). In each series, chromatin was immunoprecipitated with either a non-specific (117) or a specific antibody (GFP). Immunoprecipitated DNA was analyzed by slot blots hybridized with probes specific for telomeric repeats (TEL), mini-chromosome 177-bp repeats (MC177), or 5SDNA. Note that the input control corresponds to 10% of total. ChIPs were performed two to four times and a representative slot blot is shown for each set. (B) A quantitative presentation of the data shown in A and from one to three other independent experiments reveals the average percentage of input material present in control (117) and test (GFP) ChIPs. (C) Relative to major H3, H3V is enriched at telomeres and is found at reduced levels at two other genomic loci. To determine these ratios, the background signal value (i.e. the material immunoprecipitated by the non-specific antibody) was subtracted from GFP signal value to generate a specific signal value; the specific signal value of H3V-YFP divided by the specific signal value of H3-YFP equals the relative enrichment.

View larger version (24K): [in a new window]   Fig. 7. Silencing of an expression site upon differentiation to the procyclic form is unaffected by loss of H3V. (A) Schematic diagram of telomere (zig-zag) proximal portion of the 221 expression site and the 221 expression site in which either GFP-BLE cassette or T7 promoter driven GFP-BLE was inserted. (B) Comparison of the GFP fluorescence intensity from the wild type (single marker), wild type + GFP (BFJEL36), wild type + T7-GFP (BFJEL37) and htv/htv (BFJEL25), htv/htv + GFP (BFJEL38) and htv/htv + T7-GFP (BFJEL39) bloodstream-form cell lines and the same cell lines following two weeks differentiation to the procyclic form.

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