Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, Vrt||ICC, IHC, IP, WB, ChIP||M||Purified||Monoclonal Antibody|
|Presentation||Purified mouse monoclonal IgG in buffer containing 0.02 M phosphate buffer, pH 7.6, 0.25 M NaCl, and 0.1% Sodium Azide before the addition of glycerol to 30%.|
|Safety Information according to GHS|
|Material Size||200 µg|
|Anti-HP1#945;, clone15.19s2 - 2153146||2153146|
|Anti-HP1#945;, clone15.19s2 - 2021757||2021757|
|Anti-HP1#945;, clone15.19s2 - DAM1770284||DAM1770284|
|Anti-HP1#945;, clone15.19s2 - DAM1810805||DAM1810805|
|Anti-HP1, clone15.19s2 - 1964540||1964540|
|Anti-HP1, clone15.19s2 - 2043519||2043519|
|Anti-HP1, clone15.19s2 - 2297222||2297222|
|Anti-HP1, clone15.19s2 - DAM1412887||DAM1412887|
|Anti-HP1, clone15.19s2 - DAM1650326||DAM1650326|
|Anti-HP1, clone15.19s2 - DAM1695859||DAM1695859|
|Reference overview||Application||Species||Pub Med ID|
|Hierarchical clustering of breast cancer methylomes revealed differentially methylated and expressed breast cancer genes.|
Lin, IH; Chen, DT; Chang, YF; Lee, YL; Su, CH; Cheng, C; Tsai, YC; Ng, SC; Chen, HT; Lee, MC; Chen, HW; Suen, SH; Chen, YC; Liu, TT; Chang, CH; Hsu, MT
PloS one 10 e0118453 2015
Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.
|Epigenetic silencing of Oct4 by a complex containing SUV39H1 and Oct4 pseudogene lncRNA.|
Scarola, M; Comisso, E; Pascolo, R; Chiaradia, R; Maria Marion, R; Schneider, C; Blasco, MA; Schoeftner, S; Benetti, R
Nature communications 6 7631 2015
Pseudogene-derived, long non-coding RNAs (lncRNAs) act as epigenetic regulators of gene expression. Here we present a panel of new mouse Oct4 pseudogenes and demonstrate that the X-linked Oct4 pseudogene Oct4P4 critically impacts mouse embryonic stem cells (mESCs) self-renewal. Sense Oct4P4 transcription produces a spliced, nuclear-restricted lncRNA that is efficiently upregulated during mESC differentiation. Oct4P4 lncRNA forms a complex with the SUV39H1 HMTase to direct the imposition of H3K9me3 and HP1α to the promoter of the ancestral Oct4 gene, located on chromosome 17, leading to gene silencing and reduced mESC self-renewal. Targeting Oct4P4 expression in primary mouse embryonic fibroblasts causes the re-acquisition of self-renewing features of mESC. We demonstrate that Oct4P4 lncRNA plays an important role in inducing and maintaining silencing of the ancestral Oct4 gene in differentiating mESCs. Our data introduces a sense pseudogene-lncRNA-based mechanism of epigenetic gene regulation that controls the cross-talk between pseudogenes and their ancestral genes.
|Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin.|
Boros, J; Arnoult, N; Stroobant, V; Collet, JF; Decottignies, A
Molecular and cellular biology 34 3662-74 2014
Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.
|A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.|
Smith, CL; Matheson, TD; Trombly, DJ; Sun, X; Campeau, E; Han, X; Yates, JR; Kaufman, PD
Molecular biology of the cell 25 2866-81 2014
Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.
|Dynamics of the two heterochromatin types during imprinted X chromosome inactivation in vole Microtus levis.|
Vaskova, EA; Dementyeva, EV; Shevchenko, AI; Pavlova, SV; Grigor'eva, EV; Zhelezova, AI; Vandeberg, JL; Zakian, SM
PloS one 9 e88256 2014
In rodent female mammals, there are two forms of X-inactivation - imprinted and random which take place in extraembryonic and embryonic tissues, respectively. The inactive X-chromosome during random X-inactivation was shown to contain two types of facultative heterochromatin that alternate and do not overlap. However, chromatin structure of the inactive X-chromosome during imprinted X-inactivation, especially at early stages, is still not well understood. In this work, we studied chromatin modifications associated with the inactive X-chromosome at different stages of imprinted X-inactivation in a rodent, Microtus levis. It has been found that imprinted X-inactivation in vole occurs in a species-specific manner in two steps. The inactive X-chromosome at early stages of imprinted X-inactivation is characterized by accumulation of H3K9me3, HP1, H4K20me3, and uH2A, resembling to some extent the pattern of repressive chromatin modifications of meiotic sex chromatin. Later, the inactive X-chromosome recruits trimethylated H3K27 and acquires the two types of heterochromatin associated with random X-inactivation.
|HP1α mediates defective heterochromatin repair and accelerates senescence in Zmpste24-deficient cells.|
Liu, J; Yin, X; Liu, B; Zheng, H; Zhou, G; Gong, L; Li, M; Li, X; Wang, Y; Hu, J; Krishnan, V; Zhou, Z; Wang, Z
Cell cycle (Georgetown, Tex.) 13 1237-47 2014
Heterochromatin protein 1 (HP1) interacts with various proteins, including lamins, to play versatile functions within nuclei, such as chromatin remodeling and DNA repair. Accumulation of prelamin A leads to misshapen nuclei, heterochromatin disorganization, genomic instability, and premature aging in Zmpste24-null mice. Here, we investigated the effects of prelamin A on HP1α homeostasis, subcellular distribution, phosphorylation, and their contribution to accelerated senescence in mouse embryonic fibroblasts (MEFs) derived from Zmpste24(-/-) mice. The results showed that the level of HP1α was significantly increased in Zmpste24(-/-) cells. Although prelamin A interacted with HP1α in a manner similar to lamin A, HP1α associated with the nuclease-resistant nuclear matrix fraction was remarkably increased in Zmpste24(-/-) MEFs compared with that in wild-type littermate controls. In wild-type cells, HP1α was phosphorylated at Thr50, and the phosphorylation was maximized around 30 min, gradually dispersed 2 h after DNA damage induced by camptothecin. However, the peak of HP1α phosphorylation was significantly compromised and appeared until 2 h, which is correlated with the delayed maximal formation of γ-H2AX foci in Zmpste24(-/-) MEFs. Furthermore, knocking down HP1α by siRNA alleviated the delayed DNA damage response and accelerated senescence in Zmpste24(-/-) MEFs, evidenced by the rescue of the delayed γ-H2AX foci formation, downregulation of p16, and reduction of senescence-associated β-galactosidase activity. Taken together, these findings establish a functional link between prelamin A, HP1α, chromatin remodeling, DNA repair, and early senescence in Zmpste24-deficient mice, suggesting a potential therapeutic strategy for laminopathy-based premature aging via the intervention of HP1α.
|Developmentally regulated linker histone H1c promotes heterochromatin condensation and mediates structural integrity of rod photoreceptors in mouse retina.|
Popova, EY; Grigoryev, SA; Fan, Y; Skoultchi, AI; Zhang, SS; Barnstable, CJ
The Journal of biological chemistry 288 17895-907 2013
Mature rod photoreceptor cells contain very small nuclei with tightly condensed heterochromatin. We observed that during mouse rod maturation, the nucleosomal repeat length increases from 190 bp at postnatal day 1 to 206 bp in the adult retina. At the same time, the total level of linker histone H1 increased reaching the ratio of 1.3 molecules of total H1 per nucleosome, mostly via a dramatic increase in H1c. Genetic elimination of the histone H1c gene is functionally compensated by other histone variants. However, retinas in H1c/H1e/H1(0) triple knock-outs have photoreceptors with bigger nuclei, decreased heterochromatin area, and notable morphological changes suggesting that the process of chromatin condensation and rod cell structural integrity are partly impaired. In triple knock-outs, nuclear chromatin exposed several epigenetic histone modification marks masked in the wild type chromatin. Dramatic changes in exposure of a repressive chromatin mark, H3K9me2, indicate that during development linker histone plays a role in establishing the facultative heterochromatin territory and architecture in the nucleus. During retina development, the H1c gene and its promoter acquired epigenetic patterns typical of rod-specific genes. Our data suggest that histone H1c gene expression is developmentally up-regulated to promote facultative heterochromatin in mature rod photoreceptors.
|Phosphorylation of KRAB-associated protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by nuclear tyrosine kinases inhibits the association of KAP1 and heterochromatin protein 1α (HP1α) with heterochromatin.|
Kubota, S; Fukumoto, Y; Aoyama, K; Ishibashi, K; Yuki, R; Morinaga, T; Honda, T; Yamaguchi, N; Kuga, T; Tomonaga, T; Yamaguchi, N
The Journal of biological chemistry 288 17871-83 2013
Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1β/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1-3YF). In DNA damage, KAP1-3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.
|DNA replication fading as proliferating cells advance in their commitment to terminal differentiation.|
Estefanía, MM; Ganier, O; Hernández, P; Schvartzman, JB; Mechali, M; Krimer, DB
Scientific reports 2 279 2012
Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation.
|Exploiting the MDM2-CK1α protein-protein interface to develop novel biologics that induce UBL-kinase-modification and inhibit cell growth.|
Huart, AS; MacLaine, NJ; Narayan, V; Hupp, TR
PloS one 7 e43391 2012
Protein-protein interactions forming dominant signalling events are providing ever-growing platforms for the development of novel Biologic tools for controlling cell growth. Casein Kinase 1 α (CK1α) forms a genetic and physical interaction with the murine double minute chromosome 2 (MDM2) oncoprotein resulting in degradation of the p53 tumour suppressor. Pharmacological inhibition of CK1 increases p53 protein level and induces cell death, whilst small interfering RNA-mediated depletion of CK1α stabilizes p53 and induces growth arrest. We mapped the dominant protein-protein interface that stabilizes the MDM2 and CK1α complex in order to determine whether a peptide derived from the core CK1α-MDM2 interface form novel Biologics that can be used to probe the contribution of the CK1-MDM2 protein-protein interaction to p53 activation and cell viability. Overlapping peptides derived from CK1α were screened for dominant MDM2 binding sites using (i) ELISA with recombinant MDM2; (ii) cell lysate pull-down towards endogenous MDM2; (iii) MDM2-CK1α complex-based competition ELISA; and (iv) MDM2-mediated ubiquitination. One dominant peptide, peptide 35 was bioactive in all four assays and its transfection induced cell death/growth arrest in a p53-independent manner. Ectopic expression of flag-tagged peptide 35 induced a novel ubiquitin and NEDD8 modification of CK1α, providing one of the first examples whereby NEDDylation of a protein kinase can be induced. These data identify an MDM2 binding motif in CK1α which when isolated as a small peptide can (i) function as a dominant negative inhibitor of the CK1α-MDM2 interface, (ii) be used as a tool to study NEDDylation of CK1α, and (iii) reduce cell growth. Further, this approach provides a technological blueprint, complementing siRNA and chemical biology approaches, by exploiting protein-protein interactions in order to develop Biologics to manipulate novel types of signalling pathways such as cross-talk between NEDDylation, protein kinase signalling, and cell survival.
|I would like to use this monoclonal antibody on mouse tissue, are there any special procedures I should follow?||To reduce the background interaction between the monoclonal antibody and the mouse tissue, you will need to perform antigen retrieval and use a special secondary antibody. For antigen retrieval, prepare a citrate buffer by mixing 19ml of 0.1M citric acid and 82ml of 0.1M sodium citrate to make 1000ml of buffer. Adjust the pH to 6.0 before each use. Add buffer to your slide. Using an 800 watt microwave oven with rotation, microwave for 7.5 min at high, 5 min at 50% (3 cycles), checking the slide at the end of each cycle to see if the solution needs to be replaced, adding water if needed. For the secondary antibody, use a biotinylated isotype specific secondary IGG (IGG-1) and a 3% donkey serum block. This will help reduce the background issues using mouse monoclonals.|