Anti-Heterochromatin Protein 1, alpha (HP1a, Antigen p25, Chromobox Homolog 5 (Drosophila), Cbx5, HP

Heterochromatin protein-1 (HP1) is a methyl-lysine binding protein localized at heterochromatin sites, where it mediates gene silencing. It has been shown that mammalian methyltransferases that selectively methylate histone H3 on lysine-9 generate a binding site for HP1 proteins, a family of heterochromatic adaptor molecules implicated in both gene silencing and supranucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromodomain. Thus, the HP1 chromodomain is a specific interaction motif for the methyl epitope on lysine-9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after reintroduction of a catalytically active SUV39H1 HMTase. A molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin has been defined. It has been demonstrated that HP1 can bind with high affinity to histone H3 methylated at lysine-9 but not at lysine-4. The chromodomain of HP1 as its methyl-lysine-binding domain has been identified. A point mutation in the chromodomain, which destroys the gene silencing activity of HP1 in Drosophila, abolished methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe showed that the methylase activity of Clr4 (the SUV39H1 homolog) is necessary for the correct localization of Swi6 (the HP1 equivalent) at centromeric heterochromatin and for gene silencing. A stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a methyl marker on histone H3, which is then recognized by HP1 through its chromodomain has been suggested. This model may also explain the stable inheritance of the heterochromatic state. SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39H1, the activity of the cyclin E and cyclin A2 genes are specifically elevated. The SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other corepressor proteins. Applications: Suitable for use in ELISA, Immunohistochemistry and Western Blot. Other Applications not tested. Recommended Dilutions: ELISA: 1:128,000 Immunohistochemistry (paraffin): 2-3ug/ml, Western Blot: 0.03-0.1ug/ml, Optimal dilutions to be determined by the researcher, Positive Control: Human fibroblast A431 and HeLa cell lysates. Storage and Stability: May be stored at 4°C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and store at -20°C. Aliquots are stable for at least 12 monthsat -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer. Peptide Blocking: Corresponding peptide is available for Peptide Blocking studies. See H2034-50D-P. Antibody is typically 0.5mg/ml and peptide is supplied as a 100ul pellet. When peptide is reconstituted in 200ul water, the concentration would also be 0.5mg/ml. To start, the best ratio would be 1:1 (molar excess of peptide relative to antibody when identical volumes are mixed). Mix equal volumes of peptide and antibody at the required dilution and leave at ambient temperature. It is best is to have two identical blots to be incubated with equal amount of antibody, but one with the antibody pre-adsorbed to the peptide for 20min. Then incubate and develop the two blots in parallel.