The H2ABbd nucleosome contains 127 bp of DNA and its unusual

By Vanessa Watson,2014-11-29 17:17
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The H2ABbd nucleosome contains 127 bp of DNA and its unusual

Dissection of the unusual structural and functional properties of the variant

    H2A.Bbd nucleosome

    1,22,312,45Cécile-Marie Doyen, Fabien Montel, Thierry Gautier, Hervé Ménoni, Cyril Claudet,

    12,465Marlène Delacour-Larose, Dimitri Angelov, Ali Hamiche, Jan Bednar, Cendrine Favre-

    2,32,4*1, 2*Moskalenko, Philippe Bouvet and Stefan Dimitrov .

    1Institut Albert Bonniot, INSERM U309, 38706 La Tronche cedex, France 234Laboratoire Joliot-Curie, Laboratoire de Physique, CNRS UMR 5672, Laboratoire de Biologie

    Moléculaire de la Cellule, CNRS-UMR 5161/INRA 1237/IFR128 Biosciences, Ecole Normale

     Supérieure de Lyon, 46 Allée d'Italie, 69007 Lyon, France

    5 CNRS, Laboratoire de Spectrometrie Physique, UMR 5588, BP87, 140 Av. de la Physique , 38402 St. Martin d'Heres Cedex, France

    6Institut André Lwoff, CNRS UPR 9079, 7 rue Guy Moquet, 94800 Villejuif, France

*Corresponding authors

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    Fax: (33) 4 76 54 95 95 Tel: (33) 4 76 54 94 73


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    The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (EC-M) showed that the H2A.Bbd histone octamer organizes only 130 bp of DNA, suggesting that 10 bp of each end of

    nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180? and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, EC-M, FRAP and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its properties to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.



    DNA is packaged into chromatin in the cell nucleus. The chromatin exhibits repeating structure and the repeating unit, the nucleosome, consists of an octamer of the core histones (two each of H2A, H2B, H3 and H4) around which two superhelical turns of DNA are wrapped (van Holde, 1988). The nucleosome is an obstacle for the protein factors to bind to their cognate DNA sequences and it interferes with several vital cellular processes (Beato and Eisfeld, 1997). Histone modifications, ATP-remodeling machines and the incorporation of histone variants within chromatin are used by the cell to overcome the nucleosomal obstacle (Becker, 2002; Henikoff and Ahmad, 2005; Henikoff et al., 2004; Strahl and Allis, 2000).

    Histone variants are nonallelic isoforms of the conventional histones, which mRNA, in contrast to this of conventional histones, is polyadenylated (Tsanev et al., 1993; van Holde, 1988). The function of the different histone variants is poorly understood, but the emerging general picture suggests that the incorporation of histone variants within the nucleosome results in a particle with novel structural and functional properties (Abbott et al., 2001; Angelov et al., 2003; Bao et al., 2004; Gautier et al., 2004 Fan, 2004 #231; Suto et al., 2000). The presence of histone variants in the nucleosome has serious impacts on several processes, including transcription, repair, cell division and meiosis and may result in important epigenetic consequences (Ahmad and Henikoff, 2002a; Ausio and Abbott, 2002; Kamakaka and Biggins, 2005; Sarma and Reinberg, 2005). CENP-A, a histone H3 variant, is specifically associated with the centromeric DNA and it is essential for mitosis. Its presence is crucial for the assembly and maintenance of the kinetochores (for a recent review see (Henikoff and Dalal, 2005)). H3.3, another variant belonging to the H3 family, marks active chromatin (Ahmad and Henikoff, 2002b). Genome-


    scale distribution of H3.3 showed that transcription units, from upstream to downstream, are enriched in this histone variant (Mito et al., 2005). The recently identified variant of H2B, H2BFWT, seemed to be localized specifically on the telomeric sequences and might be involved in mitotic chromosome condensation (Boulard et al., 2006; Churikov et al., 2004).

    The histone H2A has the largest family of identified variants (Redon et al., 2002; Sarma and Reinberg, 2005). This could reflect both the more labile interaction of the H2A-H2B dimer with the remaining histones and DNA (van Holde, 1988) and its strategic position within the core particle (Luger et al., 1997). The variants of the H2A family include H2A.X, H2A.Z, macroH2A and H2A.Bbd. H2A.Z and H2A.X are the best studied H2A histone variants to date. H2A.Z is highly conserved, suggesting an important function of this protein (Redon et al., 2002). Indeed, the mammalian H2A.Z is encoded by an essential gene, since its knockout results in embryonal lethality (Faast et al., 2001). H2A.Z is involved in both gene activation (Santisteban et al., 2000) and gene silencing (Dhillon and Kamakaka, 2000). In yeast, H2A.Z is found at nearly all promoters (Guillemette et al., 2005; Li et al., 2005; Raisner et al., 2005; Zhang et al., 2005), while hyperacetylated H2A.Z is concentrated at the 5‟ ends of active genes in higher eukaryotes (Bruce et al., 2005). It was also reported that H2A.Z is important for chromosome segregation (Rangasamy et al., 2004).

    H2AX is intimately related to repair. Double strands breaks (DSB) induce the phosphorylation of H2AX at its C-terminus and this is mediated by members of the PIKK protein

    -/- deficient mice showed that H2AX family (Rogakou et al., 1998). Experiments with H2AX

    assists the prevention of aberrant repair of DSB and functions as suppressor of genomic instability and tumors (Bassing et al., 2003; Celeste et al., 2003). H2AX is specifically


    recognized by the mammalian MDC1 and the interaction of MDC1 with H2AX is crucial for the cell response to DNA damage (Stucki et al., 2005).

    MacroH2A (mH2A) is an unusual histone variant with a size threefold this of the conventional H2A (Pehrson and Fried, 1992). mH2A consists of a histone H2A-like domain fused to a large non-histone region, termed the macro-domain (Allen et al., 2003; Ladurner, 2003; Pehrson and Fried, 1992). Immunofluorescence data suggested that the inactive chromosome X is enriched of mH2A (Chadwick et al., 2001; Costanzi and Pehrson, 1998; Costanzi and Pehrson, 2001; Mermoud et al., 1999), but this view was, however, challenged and it was proposed that the higher concentrations of mH2A in the inactive X chromosome reflected higher nucleosome density (Perche et al., 2000). The incorporation of mH2A within the nucleosome interfered with both transcription factor binding and nucleosome remodeling (Angelov et al., 2003). Both in

    vitro and transient transfection experiments showed that mH2A inhibited initiation of transcription and histone acetylation (Doyen et al., 2006; Perche et al., 2000). The available data suggest that mH2A is a major stopper of transcription (Doyen et al., 2006). It was recently reported that the macro-domain is an ADP-ribose binding module, suggesting that mH2A might be also involved in the biology of ADP-ribose (Karras et al., 2005; Kustatscher et al., 2005).

    H2A.Bbd (Barr body deficient) is the least studied histone variant and microscopy data showed that it is largely excluded from the inactive X chromosome (Chadwick and Willard, 2001). This histone variant is quite divergent and its primary sequence showed only 48% identity compared to its conventional H2A counterpart (Chadwick and Willard, 2001). The N-terminus of H2A.Bbd exhibits a row of six arginines, which could be important for its function. In addition, H2A.Bbd is relatively shorter and lacks both C-terminus and the very last sequence of the


    docking domain (Chadwick and Willard, 2001). Microccocal nuclease digestion suggested that the H2A.Bbd octamer organized only 118 bp of DNA (Bao et al., 2004). FRAP, FRET and sedimentation measurements point to a less stable structure of the variant H2A.Bbd nucleosome (Angelov et al., 2004b; Bao et al., 2004; Gautier et al., 2004). The current view is that H2A.Bbd is enriched in nucleosomes associated with transcriptionally active regions of the genome (Chadwick and Willard, 2001) and in vitro experiments demonstrated that nucleosomal arrays

    containing H2A.Bbd are easier transcribed and their histones easier acetylated (Angelov et al., 2004b). The role of the different domains of H2A.Bbd in these processes is not known.

    In this work we report a detailed analysis of the properties of the H2A.Bbd nucleosomes in solution. We have dissected the role of the different domains of H2A.Bbd in the structure and function of H2A.Bbd nucleosomes using a number of physical methods including Atomic Force Microscopy (AFM), Electron cryomicroscopy (EC-M), optical tweezers and FRET combined with molecular biology approaches. We showed that the H2A.Bbd octamer organizes 130 bp of

    DNA and that its structural and functional properties are determined by the whole histone fold domain of H2A.Bbd.



    130 base pairs of DNA are wrapped around the histone variant H2A.Bbd octamer

    First we analyzed the structure in solution of the H2A.Bbd particle using microccocal nuclease. Microccocal nuclease digestion measures the accessibility of nucleosomal DNA that is not protected by the histone octamer. To better characterize this accessibility, we performed kinetics studies of the microccocal nuclease digestion of conventional H2A and H2A.Bbd particles reconstituted on the 601 positioning sequence (Figure 1 A). The digestion pattern of the conventional particle showed discrete kinetics intermediates at about 208, 166, 146, and 128 bp, while the kinetics intermediates of the H2A.Bbd particle were observed mainly at 146, 128, and around 118 bp. The band at 166 bp, generated upon digestion of the conventional nucleosome, was attributed to the interaction of the N-terminal histone tails with nucleosomal DNA (Van Holde et al., 1980), the 146 bp band represents the core particle, while the 128 bp band reflects a subnucleosomal digestion (Van Holde et al., 1980). The origin of the 208 bp band is unknown, but it might be associated with the presence of histones on DNA, since upon digestion of naked 601 DNA, no such band was observed (data not shown). The presence of very faint 166 bp band in the digestion pattern of the H2A.Bbd nucleosome suggests that the interaction of the histone N-termini with DNA were perturbed. The relatively fast disappearance of the 128 bp band in the kinetics of microccocal nuclease digestion and the presence of a stable 118 bp intermediate (Figure 1A, see the scans of the microccocal nuclease digestion pattern) indicates that the protection against microccocal nuclease digestion is weaker in the variant H2A.Bbd particle, suggesting a more relaxed structure.

    Next we used microscopy techniques to measure the length of DNA, which is wrapped around the histone octamer. Briefly, using purified recombinant histones we have reconstituted both conventional and variant H2A.Bbd centrally positioned nucleosomes on a 255 bp DNA


    fragment containing the 601 positioning sequence. Initially, AFM was used to visualize the reconstituted particles (Figure 1B-D). The large free DNA sequences present at each end of the nucleosomes allowed the precise measurement of the length of DNA which is non-wrapped around the histone octamer, and therefore the determination of the length of the DNA organized by the histone octamer. The measurements were carried out on a large number of particles (458 conventional and 290 H2A.Bbd particles), which made the experiment statistically relevant. The mean of distribution of the length of DNA, organized by conventional octamer (Figure 1D) peaked at 146 +/- 1.3 bp, in perfect agreement with the crystal structure of the nucleosome (Luger et al., 1997). In contrast, the mean length of DNA organized by the octamer containing H2A.Bbd (Figure 1D) peaked at 127 +/- 2.2 bp.

     The AFM experiment could be affected by the deposition of the material on the functionalized mica surface and by the fact that the measurements were carried out in air. To overcome these potential problems the same types of experiments were repeated, but using electron cryomicroscopy (EC-M). Indeed, EC-M measurements carried out in solution provide high resolution images of the conformation of the studied samples and were successfully used to study both nucleosome and 30 nm chromatin fiber structures (Bednar et al., 1995; Bednar and Woodcock, 1999). The electron cryomicrographs clearly showed that the conventional and the variant H2A.Bbd particles exhibit different conformations (Figure 2A and B). The majority of the entry and exit DNA ends of the conventional nucleosomes formed a V-type structure with the nucleosome particle located at the middle of the structure (Figure 2A). In contrast, only a small number of the H2A.Bbd particles exhibited such structure, while the majority of the DNA nucleosomal ends run close to parallel to each other (Figure 2B). This suggests that the H2A.Bbd octamer interacts weakly with the entry/exit nucleosomal DNA and it is unable to generate a stable V-type orientation to the free DNA ends. The length of the DNA wrapped around the


conventional histone octamer was 148 1.9 bp and 132 2.6 bp for the variant H2A.Bbd

    octamer (Figure 2C), which is in complete agreement with the AFM measurements. Thus, using two different microscopy techniques we found that H2A.Bbd octamer organizes 130 bp of DNA.

    Taken together, these results point to a distinct structure of the H2A.Bbd nucleosome with altered interactions of the variant octamer with the nucleosomal DNA.

Force-extension measurements of a single H2A.Bbd nucleosomal array

    These as well as previous experiments (Bao et al., 2004; Gautier et al., 2004) suggest that the H2A.Bbd nucleosome exhibits lower stability compared to the conventional nucleosomes, but no direct measurements of the forces maintaining the structure of the H2A.Bbd particle were reported. We have addressed this problem using optical tweezers and measured the force necessary for the unfolding of a single H2A.Bbd nucleosome. A fragment of DNA, containing twelve 208 bp repeats comprising the 5S rRNA sea urchin gene nucleosome positioning sequence, was used to reconstitute H2A.Bbd nucleosome arrays. Upon digestion of the reconstituted arrays a clear 200 bp repeat was observed evidencing for a proper organization of the reconstituted samples (data not shown). A single array was then attached between two polysterene beads and subjected to traction using optical tweezers (for detail see Materials and Methods and ref. (Claudet et al., 2005)). Each peak in the “saw tooth” profile of the force-extension curve (Figure

    3A) reflects the unfolding of a single H2A.Bbd particle (Claudet et al., 2005). The same experiment was carried out, but using conventional H2A arrays (results not shown and (Claudet et al., 2005)). The measurements showed that the force necessary for the disruption of a single H2A.Bbd nucleosome was 16.1 4.8 pN, which was very close to the force required for the

    disruption of a single conventional H2A particle (17.8 5.3 pN) (Figure 3B). The two sets of


    values of threshold disruption forces were compared using standard t-test for two independent populations. The resulting values of p and t factors were 0.00243 and -3.04722, respectively, indicating that both means cannot be viewed as different.

     We have also studied the stability of the H2A.Bbd nucleosome variant at very low particle concentrations. Under these conditions, a selective release of the H2A-H2B dimer occurred,

     tetramer which reflects the disruption of the H2A-H2B dimer interactions with both the (H3-H4)2

    and DNA (Claudet et al., 2005). If the H2A.Bbd-H2B variant dimer exhibited lower stability relative to the conventional one, a release of H2A.Bbd-H2B dimer at higher nucleosome concentration would be expected. Figure 3C showed that this is indeed the case. At the lowest particle concentration studied (3.8 nM) the vast majority of the H2A.Bbd-H2B dimer was released from the variant H2A.Bbd particle (Figure 3C, the last two panels and the scan nuc Bbd-H3*) in contrast to conventional particle (Figure 3C, the first panel and the nuc H2A-H3* scan). This showed that the interactions between the H2A.Bbd-H2B dimer with both the (H3-H4) 2

    tetramer and DNA within the H2A.Bbd particle were altered, which conferred lower stability of the H2A.Bbd variant nucleosomal particle.

    SWI/SNF is unable to remodel swapped tail H2A-H2A.Bbd mutants containing the H2A.Bbd histone fold domain

    The H2A.Bbd particle exhibits distinct structural and functional properties, including an inability to be remodeled by SWI/SNF (Angelov et al., 2004b). The mechanism and role of the different H2A.Bbd domains in this interference in the remodeling are not known. We approached this problem by studying the properties of nucleosomes containing H2A.Bbd-H2A swapped domain mutants. Initially we have concentrated on the N-ter and C-ter swapped-tail mutants (schematically depicted in Figure 4A). The different mutant proteins were purified (Figure 4B)


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