The Histone H3 Variant Nucleosomes

The two major histone variants of H3, CENP-A (centromeric protein A) and H3.3, are the object of several studies. CENP-A is present in all eukaryotes and it has different names (Kamakaka and Biggins, 2005), but here for simplicity we will use the name CenH3 (centromeric H3) for this protein. CenH3 is specifically localized to the centromeres and it replaces the canonical H3 in the centromeric chromatin (Palmer et al, 1989, 1990). The CenH3 gene is essential and its disruption resulted in very early embryonic lethality (Howman et al, 2000). The structured region of CenH3 is well evolutionary conserved, while its N-terminal tail exhibits high divergence between the different organisms (Malik and Henikoff, 2003). In vitro studies using reconstituted nucleosome, where conventional H3 was substituted with CenH3 (CenH3 nucleosome), showed that the overall structure of this particle was very similar to the conventional nucleosome (Yoda et al, 2000).

Within the centromere the conventional and CenH3 nucleosomes are interspersed (Blower et al, 2002). It is widely accepted that CenH3 is absolutely required for the assembly of the kinetochores (Blower and Karpen, 2001; Howman et al, 2000). The depletion of CenH3 in worm, fly and mouse resulted in massive mislocalization of the kinetochore proteins (Blower and Karpen, 2001; Howman et al, 2000; Oegema et al, 2001). The reported data indicated that CenH3 interacts directly or indirectly with several kinetochore proteins (Van Hooser et al, 2001). When CenH3 was mistargeted to non-centromeric regions of chromatin, kinetochore proteins were also mistargeted to these regions by a mechanism, which required the N-terminal tail of CenH3 (Van Hooser et al, 2001).

CenH3 is selectively and quantitatively retained in the mature spermatozoa and it forms discrete foci in the nucleus (Palmer et al, 1990). This suggests that information on the centromeric organization of chromatin is transmitted through generations and thus, CenH3 could act as a specific epigenetic marker.

Three recent reports addressed the mechanism of deposition of CenH3 and the assembly of CenH3 nucleosomes (Foltz et al, 2006; Furuyama et al, 2006; Okada et al, 2006). In Drosophila cells, a CenH3 soluble assembly complex was isolated. This complex consists of CenH3, histone H4 and the protein RbAp48 (Furuyama et al, 2006). The single protein RbAp48 exhibited histone chaperone properties and it has the ability to promote the assembly of CenH3 nucleosomes in vitro. This observation made the chaperone (which is active on its own) distinct from the other chromatin assembly complexes, which consists of several subunits (Furuyama et al, 2006). By using tandem affinity purification, a human multiprotein complex (CenH3 NAC) directly recruited by CenH3 nucleosome, was identified (Foltz et al, 2006). CenH3 NAC comprised three new centromere proteins (CENP-M, CENP-N and CENP-T) and the assembly of CenH3 NAC depended on these proteins (Foltz et al, 2006). In addition, the transcription implicated protein FACT and the histone chaperone nucleophosmin were found associated with CenH3 independent of CenH3 NAC (Foltz et al, 2006). Another study claimed that the CenH3 NAC consists of three subcomplexes and each one of them was found to be essential for faithfull chromosome segregation (Fukagawa et al, 2004).

H3.3, the other major histone variant of H3, differs from H3 by only four aminoacids, three of which are in the histone fold (globular) domain of the protein. These three residues are crucial for the distinctive deposition of H3.3 during the cell cycle. The epigenetic markers of H3.3, such as di- and tri-methylation of lysine4 (K4), acetylation of lysine9, 18, and -23 and methylation at K79 suggest its important role in the transcriptional activation. H3.3 could be deposited at all stages of cell cycle, unlike histone H3 which assembles only in the S-phase. In Drosophila, a good correlation between the transcriptional gene activity and genome-wide localization of H3.3 was demonstrated (Mito et al, 2005). In contrast to H3, it is deposited exclusively by the histone chaperone HIRA in a replication-independent manner (Bosch and Suau, 1995; Tagami et al, 2004). However, involvement of histone chaperone(s) in the deposition as well as removal of H3.3 during transcriptional activation and repair could not be ruled out.

5. THE H2B VARIANT NUCLEOSOMES

Very few variants of H2B, have been identified so far. The identified H2B variants play some yet not well understood role during gametogenesis (Doenecke et al, 1997; Poccia and Green, 1992; Tanphaichitr et al, 1978). Recently, H2BFWT, a novel H2B variant, was cloned (Churikov et al, 2004). This variant, has very low homology (45% identity) with the conventional H2B but could replace H2B efficiently in the nucleosome (Boulard et al, 2006). The H2BFWT nucleosome showed identical DNase I footprinting pattern of the conventional nucleosome, indicating that the variant nucleosome also may have very similar solution structure (Boulard et al, 2006). Intriguingly, the highly divergent N-terminal tail of H2AFWT, was unable to recruit chromosome assembly factors and to participate in the formation of condensed mitotic chromosomes (Boulard et al, 2006).

The involvement of a testis specific H2B variant in specialized higher order chromosomal domain such as telomeric heterochromatin has been documented (Gineitis et al, 2000). Indeed, an uncharacterized H2B variant was identified as a component of the telomere binding complex, which is implicated in telomere membrane attachment in human spermatozoa (Gineitis et al, 2000). Presumably, H2BFWT is a sperm specific histone variant associated with telomeric sequences (Churikov et al, 2004). Nevertheless, it has been also suggested that H2BFWT could be a component of the large telomeric chromatin of some somatic cells (Churikov et al, 2004). Taken together these data suggest that H2BFWT, as CenH3, could be an epigenetic marker required for the transmission of distinct chromatin structure through generations.

6. HISTONE VARIANTS AND DISEASE 6.1. Histone Variants and Cancer

The involvement of histone variants in disease is just beginning to emerge. Some causal relationships between H2AX and tumorigenesis are already well established in a series of experiments with H2AX knockout mice (Celeste et al, 2002). H2AX-/-mice are immune deficient, radiosensitive, exhibited repair defects, chromosomal instability and enhanced susceptibility to cancer in the absence of p53 (Bassing et al, 2003; Celeste et al, 2003a). In particular, mice deficient for both H2AX and p53, developed rapidly immature T and B lymphomas, sarcomas, leukemia and solid tumors (Bassing et al, 2003; Celeste et al, 2003a). The lymphomas in these mice exhibited an increased frequency of clonal nonreciprocal translocations and amplifications (Celeste et al, 2003a). Rescue with H2AX restores both genomic stability and radiation resistance (Celeste et al, 2003a). Importantly, if replacement of the H2AX null allele was carried out with a non-phosphorylable H2AX (a single

H2AX aminoacid mutant bearing a serine within the SQ motif substituted to either alanine or glutamic acid residues), no rescue of genome stability and radiation resistance was achieved (Celeste et al, 2003a). These data support the hypothesis that H2AX functions as a genome caretaker and tumor suppressor. This is consistent with the cytogenetic location of the H2AX gene in the region 11q23, which is altered in numerous cancers (Monni and Knuutila, 2001).

A recent study claimed that H2AX could be used as a potential target for radiotherapy (Taneja et al, 2004). When a peptide of the H2AX C-terminal tail, containing the SQ motif (containing the phosphorylable serine), was used to compete for phophorylation with the endogenous H2AX and to affect the efficiency of DNA repair, the radiotherapy effect on resistant tumor cell lines was clearly increased (Taneja et al, 2004). Of note is that in contrast to the cytotoxic agents commonly used in chemotherapy, the targeting of H2AX would not affect the non-irradiated tissue (Taneja et al, 2004).

A well characterized feature of human cancers is aneuploidy. CenH3 is, as described above, a key player in kinetochore assembly, maintenance and function. CenH3 was overexpressed in primary human colorectal cancer tissues and this occurred at the transcriptional level (Tomonaga et al, 2003). Immunofluores-cence studies showed that the tumor cells in these tissues contained the overex-pressed CenH3, which in addition was mistargetted to noncentromeric chromatin (Tomonaga et al, 2003). In Drosophila, mistargetting of CenH3 resulted in the promotion of the formation of ectopic centromeres and multicentric chromosomes, which in turn led to aneuploidy and growth defects (Heun et al, 2006). These results strongly suggest that CenH3 could be involved in genomic instability during cancer progression and, in particular, it could play an important role in aneuploidy in colorectal cancers (Tomonaga et al, 2003).

6.2. Histone Variants and Infertility

Since, H2AX deficient males, but not female mice, are infertile, H2AX could be involved in male infertility (Celeste et al, 2002). The size of the testes of male H2AX-/- mice was twofold reduced, the diameter of the seminuferous tubules was smaller and at early pachytene stage the H2AX-/- cells showed apoptotic features, pointing that the H2AX spermatocytes were arrested in the pachytene stage of meiosis I (Celeste et al, 2002). An accumulation of -y-H2AX in the sex body (a condensed structure observed in the nuclei of the mammalian spermatocytes at meotic prophase I, which contains the highly condensed X-Y chromosome pair) was also observed (Fernandez-Capetillo et al, 2003). This accumulation did not depend on the meiotic recombination-associated DSB (Fernandez-Capetillo et al, 2003). Importantly, the loss of H2AX resulted in the absence of sex-body and no initiation of meiotic sex chromosome inactivation was detected (Fernandez-Capetillo et al, 2003). In addition, the X and Y chromosomes in H2AX-/- mice exhibited severe defects in chromosome pairing. All these data evidence for participation of H2AX in remodeling of chromatin and silencing in male meiosis (Fernandez-Capetillo et al, 2003).

To date the identified and characterized H2B variants are specifically express in the testes at the early (for instance in spermatogonia) or later stages of spermatogenesis (Moss et al, 1989; Unni et al, 1995). During spermatogenesis somatic histones are initially replaced by the testis specific histone variants, then by transitional proteins and finally by protamines. It was found that spermatozoa from infertile males contained more histones than these of fertile males, indicating that insufficient removal of histone or the lacking of one variant might be involved in infertility (Chevaillier et al, 1987; Foresta et al, 1992; Hofmann and Hilscher, 1991). Thus, the human testis H2B variant TH2B has been observed in variable amount among several subfertile males witch are characterized by abnormality of spermatozoa morphology (van Roijen et al, 1998). This example points an association between an altered regulation in a testis specific histone variant and infertility.

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