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The incorporation of the novel histone variant H2AL2 confers unusual structural and functional properties of the nucleosome.

Syed SH, Boulard M, Shukla MS, Gautier T, Travers A, Bednar J, Faivre-Moskalenko C, Dimitrov S, Angelov D - Nucleic Acids Res. (2009)

Bottom Line: AFM imaging showed that the H2AL2 histone octamer was complexed with only approximately 130 bp of DNA.H2AL2 reconstituted trinucleosomes exhibited a type of a 'beads on a string' structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes.The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles.

View Article: PubMed Central - PubMed

Affiliation: Université Joseph Fourier - Grenoble 1, INSERM Institut Albert Bonniot, U823, Site Santé-BP 170, 38042 Grenoble Cedex 9, France.

ABSTRACT
In this work we have studied the properties of the novel mouse histone variant H2AL2. H2AL2 was used to reconstitute nucleosomes and the structural and functional properties of these particles were studied by a combination of biochemical approaches, atomic force microscopy (AFM) and electron cryo-microscopy. DNase I and hydroxyl radical footprinting as well as micrococcal and exonuclease III digestion demonstrated an altered structure of the H2AL2 nucleosomes all over the nucleosomal DNA length. Restriction nuclease accessibility experiments revealed that the interactions of the H2AL2 histone octamer with the ends of the nucleosomal DNA are highly perturbed. AFM imaging showed that the H2AL2 histone octamer was complexed with only approximately 130 bp of DNA. H2AL2 reconstituted trinucleosomes exhibited a type of a 'beads on a string' structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes. The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles. These unusual properties of the H2AL2 nucleosomes suggest a specific role of H2AL2 during mouse spermiogenesis.

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The histone variant H2AL2 can substitute for conventional H2A in the nucleosome. (A) Sequence alignment of mouse H2A.1 and H2AL2 and human H2A.Bbd. The N- and C-termini, the histone-fold domain as well as the docking domain (in bold) are indicated. (B) SDS PAGE of the purified recombinant histones used for nucleosome reconstitution. (C) EMSA of reconstituted nucleosome core particles. 32P-end labeled 147 bp 601.2 DNA sequence was used to reconstitute conventional and histone variant H2AL2 core particles. The reconstituted particles were run on 5% PAGE under native conditions. The positions of the core particles and of free DNA are indicated. Note that under the conditions of reconstitution essentially no free DNA was observed. (D) Preparative EMSA of reconstituted nucleosomes. Conventional and H2AL2 nucleosomes, reconstituted on 255 bp 601 DNA sequence, were run on 5% native PAGE, the bands corresponding to the nucleosomes were excised and then the nucleosomes were eluted from the gel. The gel-purified nucleosomes were run on SDS electrophoresis. (E) SDS PAGE of both conventional and H2AL2 histone octamers (lanes 3 and 4) and gel-purified reconstituted nucleosomes (lanes 1 and 2).
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Figure 1: The histone variant H2AL2 can substitute for conventional H2A in the nucleosome. (A) Sequence alignment of mouse H2A.1 and H2AL2 and human H2A.Bbd. The N- and C-termini, the histone-fold domain as well as the docking domain (in bold) are indicated. (B) SDS PAGE of the purified recombinant histones used for nucleosome reconstitution. (C) EMSA of reconstituted nucleosome core particles. 32P-end labeled 147 bp 601.2 DNA sequence was used to reconstitute conventional and histone variant H2AL2 core particles. The reconstituted particles were run on 5% PAGE under native conditions. The positions of the core particles and of free DNA are indicated. Note that under the conditions of reconstitution essentially no free DNA was observed. (D) Preparative EMSA of reconstituted nucleosomes. Conventional and H2AL2 nucleosomes, reconstituted on 255 bp 601 DNA sequence, were run on 5% native PAGE, the bands corresponding to the nucleosomes were excised and then the nucleosomes were eluted from the gel. The gel-purified nucleosomes were run on SDS electrophoresis. (E) SDS PAGE of both conventional and H2AL2 histone octamers (lanes 3 and 4) and gel-purified reconstituted nucleosomes (lanes 1 and 2).

Mentions: H2AL2 is a recently identified mouse H2A histone variant (23). H2AL2 shows only 41% identity with conventional H2A (23). The protein exhibits similar primary sequence to that of H2A.Bbd, a human variant of H2A (see Figure 1A). Indeed, both H2A.Bbd and H2AL2 show an arginine tract at their N-termini and shorter docking domain than that of H2A (Figure 1A). H2A.Bbd is mainly expressed in testis, but it was also found in other tissues (39,40). RT-PCR data have suggested that H2AL2, similarly to H2A.Bbd, was expressed in different tissues, but mainly in testis (23). Since PCR is, however, very sensitive to small contaminations; we have studied the expression of H2AL2 in different mouse tissues by using Northern blot analysis. The data show that, in general agreement with the reported data, H2AL2 is expressed only in testis (see Supplementary Figure 1), suggesting that H2AL2 is a mouse testis-specific histone variant.


The incorporation of the novel histone variant H2AL2 confers unusual structural and functional properties of the nucleosome.

Syed SH, Boulard M, Shukla MS, Gautier T, Travers A, Bednar J, Faivre-Moskalenko C, Dimitrov S, Angelov D - Nucleic Acids Res. (2009)

The histone variant H2AL2 can substitute for conventional H2A in the nucleosome. (A) Sequence alignment of mouse H2A.1 and H2AL2 and human H2A.Bbd. The N- and C-termini, the histone-fold domain as well as the docking domain (in bold) are indicated. (B) SDS PAGE of the purified recombinant histones used for nucleosome reconstitution. (C) EMSA of reconstituted nucleosome core particles. 32P-end labeled 147 bp 601.2 DNA sequence was used to reconstitute conventional and histone variant H2AL2 core particles. The reconstituted particles were run on 5% PAGE under native conditions. The positions of the core particles and of free DNA are indicated. Note that under the conditions of reconstitution essentially no free DNA was observed. (D) Preparative EMSA of reconstituted nucleosomes. Conventional and H2AL2 nucleosomes, reconstituted on 255 bp 601 DNA sequence, were run on 5% native PAGE, the bands corresponding to the nucleosomes were excised and then the nucleosomes were eluted from the gel. The gel-purified nucleosomes were run on SDS electrophoresis. (E) SDS PAGE of both conventional and H2AL2 histone octamers (lanes 3 and 4) and gel-purified reconstituted nucleosomes (lanes 1 and 2).
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Figure 1: The histone variant H2AL2 can substitute for conventional H2A in the nucleosome. (A) Sequence alignment of mouse H2A.1 and H2AL2 and human H2A.Bbd. The N- and C-termini, the histone-fold domain as well as the docking domain (in bold) are indicated. (B) SDS PAGE of the purified recombinant histones used for nucleosome reconstitution. (C) EMSA of reconstituted nucleosome core particles. 32P-end labeled 147 bp 601.2 DNA sequence was used to reconstitute conventional and histone variant H2AL2 core particles. The reconstituted particles were run on 5% PAGE under native conditions. The positions of the core particles and of free DNA are indicated. Note that under the conditions of reconstitution essentially no free DNA was observed. (D) Preparative EMSA of reconstituted nucleosomes. Conventional and H2AL2 nucleosomes, reconstituted on 255 bp 601 DNA sequence, were run on 5% native PAGE, the bands corresponding to the nucleosomes were excised and then the nucleosomes were eluted from the gel. The gel-purified nucleosomes were run on SDS electrophoresis. (E) SDS PAGE of both conventional and H2AL2 histone octamers (lanes 3 and 4) and gel-purified reconstituted nucleosomes (lanes 1 and 2).
Mentions: H2AL2 is a recently identified mouse H2A histone variant (23). H2AL2 shows only 41% identity with conventional H2A (23). The protein exhibits similar primary sequence to that of H2A.Bbd, a human variant of H2A (see Figure 1A). Indeed, both H2A.Bbd and H2AL2 show an arginine tract at their N-termini and shorter docking domain than that of H2A (Figure 1A). H2A.Bbd is mainly expressed in testis, but it was also found in other tissues (39,40). RT-PCR data have suggested that H2AL2, similarly to H2A.Bbd, was expressed in different tissues, but mainly in testis (23). Since PCR is, however, very sensitive to small contaminations; we have studied the expression of H2AL2 in different mouse tissues by using Northern blot analysis. The data show that, in general agreement with the reported data, H2AL2 is expressed only in testis (see Supplementary Figure 1), suggesting that H2AL2 is a mouse testis-specific histone variant.

Bottom Line: AFM imaging showed that the H2AL2 histone octamer was complexed with only approximately 130 bp of DNA.H2AL2 reconstituted trinucleosomes exhibited a type of a 'beads on a string' structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes.The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles.

View Article: PubMed Central - PubMed

Affiliation: Université Joseph Fourier - Grenoble 1, INSERM Institut Albert Bonniot, U823, Site Santé-BP 170, 38042 Grenoble Cedex 9, France.

ABSTRACT
In this work we have studied the properties of the novel mouse histone variant H2AL2. H2AL2 was used to reconstitute nucleosomes and the structural and functional properties of these particles were studied by a combination of biochemical approaches, atomic force microscopy (AFM) and electron cryo-microscopy. DNase I and hydroxyl radical footprinting as well as micrococcal and exonuclease III digestion demonstrated an altered structure of the H2AL2 nucleosomes all over the nucleosomal DNA length. Restriction nuclease accessibility experiments revealed that the interactions of the H2AL2 histone octamer with the ends of the nucleosomal DNA are highly perturbed. AFM imaging showed that the H2AL2 histone octamer was complexed with only approximately 130 bp of DNA. H2AL2 reconstituted trinucleosomes exhibited a type of a 'beads on a string' structure, which was quite different from the equilateral triangle 3D organization of conventional H2A trinucleosomes. The presence of H2AL2 affected both the RSC and SWI/SNF remodeling and mobilization of the variant particles. These unusual properties of the H2AL2 nucleosomes suggest a specific role of H2AL2 during mouse spermiogenesis.

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