Heterochromatin protein 1 (HP1) family members are chromatin-associated proteins involved in transcription, replication, and chromatin organization. We show that HP1 isoforms HP1-α, HP1-β, and HP1-γ are recruited to ultraviolet (UV)-induced DNA damage and double-strand breaks (DSBs) in human cells. This response to DNA damage requires the chromo shadow domain of HP1 and is independent of H3K9 trimethylation and proteins that detect UV damage and DSBs. Loss of HP1 results in high sensitivity to UV light and ionizing radiation in the nematode Caenorhabditis elegans, indicating that HP1 proteins are essential components of DNA damage response (DDR) systems. Analysis of single and double HP1 mutants in nematodes suggests that HP1 homologues have both unique and overlapping functions in the DDR. Our results show that HP1 proteins are important for DNA repair and may function to reorganize chromatin in response to damage.

Steroid receptors regulate gene expression in a ligand-dependent manner by binding specific DNA sequences. Ligand binding also changes the conformation of the ligand binding domain (LBD), allowing interaction with coregulators via LxxLL motifs. Androgen receptors (ARs) preferentially interact with coregulators containing LxxLL-related FxxLF motifs. The AR is regulated at an extra level by interaction of an FQNLF motif in the N-terminal domain with the C-terminal LBD (N/C interaction). Although it is generally recognized that AR coregulator and N/C interactions are essential for transcription regulation, their spatiotemporal organization is largely unknown. We performed simultaneous fluorescence resonance energy transfer and fluorescence redistribution after photobleaching measurements in living cells expressing ARs double tagged with yellow and cyan fluorescent proteins. We provide evidence that AR N/C interactions occur predominantly when ARs are mobile, possibly to prevent unfavorable or untimely cofactor interactions. N/C interactions are largely lost when AR transiently binds to DNA, predominantly in foci partly overlapping transcription sites. AR coregulator interactions occur preferentially when ARs are bound to DNA.

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.

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...