A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin

Wreggett, K A; Hill, Fergal; James, P S; Hutchings, Amanda; Butcher, Geoffrey W.; Singh, Prim B.

doi:10.1159/000133676

Cited by 193 publications

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“…Expression of all transfected proteins (full-length and truncated HP1) was detectable 12 h after transfection using an anti-flag antibody (data not shown). After 24 h, cells with high expression levels were analyzed for HP1 localization and DAPI-stained heterochromatin domains.Immunofluorescent labeling, using mouse-anti-flag antibodies ( Figure 1) and rabbit-anti-flag (data not shown), showed that the transfected wild-type HP1␣-flag and HP1␤-flag proteins are targeted to the DAPI-intense stained heterochromatin domains in the nucleus, as shown also by others (Wreggett et al, 1994;Horsley et al, 1996;Minc et al, 1999;Cheutin et al, 2003). The same anti-flag antibodies showed that HP1␣-⌬(2-39)-flag and HP1␤-⌬(2-40)-flag proteins are localized in the nucleus.…”

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confidence: 67%

Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1

Mateos‐Langerak

Brink

Luijsterburg

et al. 2007

MBoC

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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...

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supporting

confidence: 67%

Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1

Mateos‐Langerak

Brink

Luijsterburg

et al. 2007

MBoC

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“…We further proceeded to investigate whether Udu is localized in the replicating pericentromeric heterochromatin by staining the cells for Udu or PAH-L and SANT-L domains and HP1␤, a marker for pericentromeric heterochromatin (Wreggett et al, 1994). Indeed, Udu (and PAH-LϩSANT-L) foci were colocalized with HP1␤ foci (Figure 5, T-W; data not shown).…”

Section: Udu Binds MCM Proteins and Localizes In Chromatin During Dnamentioning

confidence: 99%

Udu Deficiency Activates DNA Damage Checkpoint

Lim

Chong

Jiang

2009

MBoC

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Udu has been shown to play an essential role during blood cell development; however, its roles in other cellular processes remain largely unexplored. In addition, ugly duckling (udu) mutants exhibited somite and myotome boundary defects. Our fluorescence-activated cell sorting analysis also showed that the loss of udu function resulted in defective cell cycle progression and comet assay indicated the presence of increased DNA damage in udu tu24 mutants. We further showed that the extensive p53-dependent apoptosis in udu tu24 mutants is a consequence of activation in the Atm-Chk2 pathway. Udu seems not to be required for DNA repair, because both wild-type and udu embryos similarly respond to and recover from UV treatment. Yeast two-hybrid and coimmunoprecipitation data demonstrated that PAH-L repeats and SANT-L domain of Udu interacts with MCM3 and MCM4. Furthermore, Udu is colocalized with 5-bromo-2 -deoxyuridine and heterochromatin during DNA replication, suggesting a role in maintaining genome integrity. INTRODUCTIONugly duckling (udu tu24 ) mutant was first isolated from the 1996 Tü bingen screen and has been shown to exhibit a short body-axis with massive cell death (Hammerschmidt et al., 1996). Another udu allele, udu sq1 , was isolated in a genetic screen aiming at mutants with defects in hematopoiesis (Liu et al., 2007). Positional cloning revealed that Udu protein encodes a novel nuclear factor consisting of three conserved regions (CR-1, CR-2, and CR-3) that do not share similarity with any known domains, two paired amphipathic ␣-helix like (PAH-L) repeats, and one putative SW13, ADA2, N-Cor and TFIIIB like (SANT-L) domain. The C-terminal PAH-L and SANT-L domains have been shown to be essential in primitive erythroid cell development (Liu et al., 2007). The PAH domain, first identified in yeast SIN3 (Wang et al., 1990), has been demonstrated to mediate protein-protein interactions (Spronk et al., 2000). SIN3 has no intrinsic DNA binding ability and has to be targeted to gene promoters by interacting with DNA binding proteins, thereafter positively and negatively regulating genes involved in diverse cellular functions (Silverstein and Ekwall, 2005). The SANT domain is a highly conserved motif with similarity to Myb DNA binding domain (Aasland et al., 1996), which has been shown to be critical in regulating chromatin accessibility (Boyer et al., 2002(Boyer et al., , 2004.The DNA damage response pathway is a cellular surveillance system that senses the presence of damaged DNA and elicits checkpoint activation and subsequent lesion repair in preventing amplification or loss of genes or chromosomes (Zhou and Elledge, 2000). The critical components of DNA damage checkpoint are two phosphatidyl inositol 3-kinaselike kinase family proteins: Ataxia telangiectasia mutated (ATM) and ATM-and Rad3-related (ATR) (Abraham, 2003;Shiloh, 2003). ATR functions as a sensor and transducer in response to UV light and presumably to genotoxic agents that give rise to stalled replication forks, and subsequently activates Checkp...

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“…1a, panel J), suggesting that GH promotes the relocalization of C/EBP␤ to areas of heterochromatin. To examine further the relocalization of C/EBP␤ to pericentromeric heterochromatin, heterochromatin protein 1-␣ (HP-1␣), a constitutive non-histone heterochromatin protein (33,34) was labeled with anti-HP-1␣ antibodies. HP-1␣ exhibits the same pattern of distribution as C/EBP␤ in cells treated with GH (Fig.…”

Section: Merging Of Immunofluorescense For C/ebp␤ (Panel A) and Dapmentioning

confidence: 99%

Subnuclear Localization of C/EBPβ Is Regulated by Growth Hormone and Dependent on MAPK

Pilipuk

Galigniana

Schwartz

2003

Journal of Biological Chemistry

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Localization of transcription regulatory proteins in the nucleus is dynamically regulated, and may alter nucleoplasmic concentrations and/or assembly of multimolecular transcription regulatory complexes, which ultimately regulate gene expression. Since growth hormone (GH) regulates multiple transcription factors including C/EBP␤, the effect of GH on the subcellular localization of C/EBP␤ was examined in 3T3-F442A preadipocytes. Indirect immunofluorescence shows that C/EBP␤ is diffusely distributed in nuclei of quiescent cells. . Indirect immunofluorescence using antibodies specific for C/EBP␤ phosphorylated on the conserved MAPK site shows that GH also rapidly induces a punctate pattern of staining for the phosphorylated C/EBP␤. In addition, phosphorylated C/EBP␤ colocalizes to pericentromeric heterochromatin. The satellite DNA present in heterochromatin contains multiple C/EBP binding sites. DNA binding analysis shows that C/EBP␤, C/EBP␦, and C/EBP␣ (p42 and p30 forms) can bind to satellite DNA as homo-or heterocomplexes in vitro. Importantly, GH rapidly and transiently increases binding of endogenous C/EBP␤ from 3T3-F442A cells to satellite DNA. Further, the GH-promoted nuclear relocalization of C/EBP␤ to pericentromeric heterochromatin was prevented by the MEK inhibitor U0126. This observation suggests that GH-dependent MAPK activation plays a role in the regulation of nuclear relocalization of C/EBP␤. Nuclear redistribution introduces a new level of transcriptional regulation in GH action, since GHmediated phosphorylation and nuclear redistribution of C/EBP␤ may be coordinated to achieve spatial-temporal control of gene expression.Chromatin is organized into higher order fibers in interphase chromosomes, which localize into discrete territories, providing an excellent scenario for establishing spatio-temporal regulation of gene transcription (1, 2). Transcriptional regulatory proteins are generally restricted to several hundred discrete foci throughout the nucleoplasm (3, 4). Interestingly, foci rich in some transcription factors such as E2F-1 or Oct1 show only minor overlap with sites of transcription or RNA polymerase II (3, 4), whereas steroid hormone receptors and RNA polymerase II show significant colocalization (5, 6). Thus, the function of the transcription factor-rich foci in the nucleus that do not contain RNA polymerase II or nascent RNA is unclear. A simple explanation is that foci represent nonchromatin structures that function in the regulation of nucleoplasmic concentrations of gene regulatory factors and/or the assembly of multimolecular transcription factor complexes. This subnuclear

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A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin

Cited by 193 publications

References 0 publications

Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1

Pericentromeric Heterochromatin Domains Are Maintained without Accumulation of HP1

Udu Deficiency Activates DNA Damage Checkpoint

Subnuclear Localization of C/EBPβ Is Regulated by Growth Hormone and Dependent on MAPK

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