Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1

Abstract

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1␣ or HP1␤ proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1␣, HP1␤, and HP1␥ in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1␣, HP1␤, and HP1␥ from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1␣, HP1␤, and HP1␥ at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains. INTRODUCTIONThe HP1 (heterochromatin protein 1) gene was first discovered in Drosophila melanogaster as a mutant suppressing position effect variegation, a phenomenon in which a gene adjacent to a heterochromatin domain shows a mosaic expression pattern (James and Elgin, 1986;Eissenberg et al., 1990;Festenstein et al., 1999). Since then, a variety of HP1 homologues has been found in many eukaryotes (Li et al., 2002). There are at least three HP1 proteins in mammals. HP1␣ and HP1␤ are present in heterochromatic regions, whereas HP1␥ has been found either exclusively in euchromatin or in both euchromatin and heterochromatin (Wreggett et al., 1994;Horsley et al., 1996;Minc et al., 1999Minc et al., , 2000Cheutin et al., 2003). HP1 proteins are thought to have a role in gene regulation in a broad range of eukaryotes: from fission yeast to mammals (Hiragami and Festenstein, 2005;Hediger and Gasser, 2006).The HP1 gene products are proteins of ϳ180 amino acids, containing two protein-binding domains linked by a flexible hinge region. The chromodomain (CD) located at the Nterminal side of the hinge region specifically binds histone H3 methylated at lysine 9 (H3K9me; Bannister et al., 2001;Lachner et al., 2001;Jacobs and Khorasanizadeh, 2002), which is a marker for heterochromatin and is related to gene silencing (Cowell et al., 2002;Sims et al., 2003). HP1 is able to repress gene activity when artificially targeted to a site near a reporter gene (van der Vlag et al., 2000). The C-terminal part of HP1 contains the chromoshadow domain (CSD), which is partially homologous to the CD (Aasland and Stewart, 1995;Sims et al., 2003). The CSD binds to a consensus peptide that is present in a number of proteins, including the CSD of HP1 itself, thereby targeting proteins to heterochromatin and forming HP1 dimers (Brasher et al., 2000;Smothers and Henikoff, 2...

HP1α is not necessary for the structural maintenance of centromeric heterochromatin

“…Most important, the compact packaging of centromeric heterochromatin seemed preserved after HP1α removal. The results of this study, along with previously published results, 45 strongly suggest that HP1α is not necessary to maintain a compact state of pericentromeric heterochromatin. It should be noted that Drosophila HP1 (a homolog of mammalian HP1α) was identified as an essential component of heterochromatin, based on the studies of suppression of position effect variegation.…”

The PHD Domain of Np95 (mUHRF1) Is Involved in Large-Scale Reorganization of Pericentromeric Heterochromatin

“…This, however, is not surprising because several studies show that the H3:K9 methylation pathway is not involved in chromocenter dynamics. Accumulation of HP1 in PH regions does not cause large-scale chromocenter modifications (Mateos-Langerak et al, 2007). The number and size of chromocenters in Suv39h1/2dn cells are comparable to control cells (Peters et al, 2001).…”

Heterochromatin and histone modifications in the germline-restricted chromosome of the zebra finch undergoing elimination during spermatogenesis

“…This latter possibility seems most plausible in view that the release of HP1 proteins from chromatin without major changes in the levels of histone H3K9 methylation has already been demonstrated in different systems. For instance, in mouse fibroblasts endogenous HP1 is lost from DAPI-stained pericentric heterochromatin without the occurrence of alterations in the levels of methylated H3K9 (Mateos-Langerak et al 2007). Moreover, during terminal differentiation in nucleated chicken erythrocytes, HP1 proteins are totally removed from and replaced by specific condensing factors whereas methylated H3K9 levels experiment only a slight reduction (Gilbert et al 2003).…”