Interestingly, we also noticed that few U2OS cells showed a strong nuclear -H3.X/Y staining, colocalizing with DNA (Fig. DNA and octamers of AZ 23 the core histones H2A, H2B, H3, and H4 (van Holde, 1988). To allow changes in chromatin structure, which are necessary to promote different biological functions, several interconnected mechanisms have evolved (for review see B?nisch et al., 2008). Among others, these include the sliding or eviction of nucleosomes by ATP-dependent chromatin remodeling machines (for review see Clapier AZ 23 and Cairns, 2009), posttranslational AZ 23 modifications (PTMs) of histone proteins (Strahl and Allis, 2000), and the exchange of canonical histones with specialized histone variants (for reviews see Pusarla and Bhargava, 2005; Bernstein and Hake, 2006). Histone variants differ in sequence and expression timing from their canonical counterparts and are enriched in chromatin of specific functional states, ranging from DNA repair and centromere determination to the regulation of gene expression. In mammals, variants of the H3, H2A, and H2B families of histones have been identified whose incorporation results in nucleosomes with novel functional and structural properties (Suto et al., 2000; Abbott et al., 2001; Angelov et al., 2003; Bao et al., 2004; Gautier et al., 2004). To date, five different H3 variants have been found in mammals: H3.1, H3.2, H3.3, H3.1t (tH3), and CENP-A. The centromeric H3 variant CENP-A causes changes to the nucleosomal structure (Black et al., 2004) and is crucial for proper chromosome segregation (for review see Allshire and Karpen, 2008). tH3 is usually a testis-specific histone variant with a putative function in chromatin reorganization during spermatogenesis (Witt et al., 1996). H3.1 and H3.2 sequences are distinguishable by just one amino acid. Although expression of both is usually replication dependent (Ahmad and Henikoff, 2002a), they differ in their cell type expression levels as well as their enrichment of PTMs (Hake et al., 2006). Furthermore, H3.1 has been implicated in DNA damage response pathways (Polo et al., 2006) and is deposited AZ 23 into chromatin by the chaperone complex CAF-1 (Tagami et al., 2004), whereas H3.3 is expressed and incorporated into chromatin in a replication-independent manner by HIRA (Tagami et al., 2004). The latter variant is highly decorated with modifications associated with gene transcription (McKittrick et al., 2004; Hake et al., 2006) and is thought to be involved in activating gene expression (Ahmad and Henikoff, 2002a) and epigenetic reprogramming (for review see Santenard and Torres-Padilla, 2009). Here, we describe the identification of two novel primate-specific histone H3 variants (and (HIST1H3I; available from GenBank/EMBL/DDBJ under accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003533″,”term_id”:”1780002153″NM_003533), we searched the public database (National Center for Biotechnology Information) and identified two highly comparable genes initially annotated as pseudogenes (Fig. S1 A). These two intron-free genes, which we named (GenBank/EMBL/DDBJ accession no. LOC340096) and (GenBank/EMBL/DDBJ accession no. LOC391769), are located on human chromosome 5 (5p15.1). Further database searches revealed the presence of comparable genes in primate genomes (H3.X, GenBank/EMBL/DDBJ accession no. LOC471464; and H3.Y, GenBank/EMBL/DDBJ accession no. LOC471473; H3.X, GenBank/EMBL/DDBJ accession no. LOC718189; and H3.Y, GenBank/EMBL/DDBJ accession no. Nt5e LOC718280; Fig. S1 B). Searches for these genes in other mammalian genomes yielded no positive hits, which suggests that they evolved in evolutionarily younger terms and might constitute primate-specific histones. Both human genes contain a sequence matching the translation initiation start site consensus (underline) for vertebrates (GCCGCCACCAUGGCG; Kozak, 1991; Nakagawa et al., 2008), and depending on the search program used (polyadq or PolyA_SVM program), and 3 genomic sequences of human and primate origins were predicted to include a conserved poly-A site (Tabaska and Zhang, 1999; Cheng et al., 2006). Alignment of human and primate and coding sequences with respective human variant sequences revealed a higher similarity to than to and (Fig. S1 C). and AZ 23 genes are predicted to encode proteins of 146 and 135 amino acids, respectively (Fig. 1 A). Both putative variant proteins are highly comparable to each other, with differences of only four amino acids in their overlapping region (89.7% identity). H3.X has an unusual long C-terminal tail with no sequence homology to other proteins (Fig. 1 A). H3.X and H3.Y display interesting changes in amino acids that are known to be.