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.

Heterochromatin protein 1 (HP1) was originally identified as a constitutive component of heterochromatin. However it is recognized now that it plays an important role in a number of dynamic processes in the cell nucleus, including transcriptional repression and regulation of euchromatic genes. Recent reports demonstrate that HP1 may be involved in the DNA damage response. Two seemingly contradictory phenomena have been observedÀ ÀHP1 detachment from chromatin and HP1 recruitment to damaged DNA foci. Based on quantitative FRAP and FLIP studies carefully designed to minimize phototoxicity, we demonstrate that HP1 is recruited to the damaged regions in heteroas well as euchromatin within a few minutes after damage. ' 2009 International Society for Advancement of Cytometry Key termsBER; base excision repair; DNA breaks; DNA damage; DNA repair; chromatin; epigenetic information; heterochromatic protein 1; HP1; oxidative damage HETEROCHROMATIN protein 1 (HP1) is a dominant suppressor of position-effect variegation, and a major component of heterochromatin in Drosophila (1), found in centromeres and telomeres (2). It is a conservative nonhistone chromatin protein. The HP1 molecule consists of an amino-terminal, chromatin organization modifier domain (chromodomain, CD), and a carboxy-terminal, chromoshadow domain (CSD). CD binds to tri-and di-methylated lysine 9 on histone H3; binding to a modified histone H3 can induce transcriptional repression (3,4). The CSD is highly conserved and can dimerize to form both homodimers and heterodimers with a nonpolar groove that acts as a docking site for proteins containing the consensus sequence P 3 V 3 L (5,6). Thus, the members of the HP1 family can interact with transcriptional regulators and chromatin-modifying proteins (e.g., SUV39H1, Polycomb, TAFIII30), DNA replication and repair proteins (e.g., CAF-Ip150, Ku70, ORC1-6), chromosome-associated proteins (e.g., INCENP, SP100), and structural nuclear proteins (e.g., lamin B and its receptor, LAP2b [reviewed in (7)]). Various functions of HP1 and interaction with protein partners are regulated by post-translational modifications, such as phosphorylation (8À10). HP1 is dynamic and exchanges with the mobile pool (11À13).Recent reports demonstrate that HP1 regulates the higher-order structure and activity not only in heterochromatin but in euchromatin as well (14). HP1 was found in transcriptionally-active (i.e., euchromatic) bands in polytene chromosomes (15) and can recruit transcriptional activators (16).Recently, HP1 has been shown to be involved in a yet unknown mechanism of DNA damage response. Two seemingly contradictory phenomena have been reported: recruitment to sites of oxidative damage (17,18) and detachment from DNA in the regions of double strand breaks (caused by radiation and etoposide) (10). We hypothesize that these two phenomena could occur in succession, or

In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neuropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oligodendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus following amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only participates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluorescent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi apparatus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lysosomes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin-coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles.