An assessment of histone-modification antibody quality

Egelhofer, Thea A.; Minoda, Aki; Klugman, Sarit; Lee, Kyungjoon; Kolasinska-Zwierz, Paulina; Alekseyenko, Artyom A.; Cheung, Ming Sin; Day, Daniel S.; Gadel, Sarah; Gorchakov, Andrey A.; Gu, Tingting; Kharchenko, Peter V.; Kuan, Samantha; Latorre, Isabel; Linder-Basso, Daniela; Luu, Ying; Ngo, Queminh; Perry, Marc D.; Rechtsteiner, Andreas; Riddle, Nicole C.; Schwartz, Yuri B.; Shanower, Gregory; Vielle, Anne; Ahringer, Julie; Elgin, Sarah C. R.; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Ren, Bing; Strome, Susan; Park, Peter J.; Karpen, Gary H.; Hawkins, R. David; Lieb, Jason D.

doi:10.1038/nsmb.1972

Cited by 375 publications

(358 citation statements)

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“…We obtained data from cells at two time points during male 63 germ cell development: prophase of meiosis I (pachytene spermatocytes), and following 64 completion of meiosis (round spermatids). These cell types are unique in that they can be 65 identified and reliably collected from whole testes in multiple mammalian species without the 66 use of transgenes or genetic markers (see Methods). In addition, they differ substantially from 67 each other in their place in the cell cycle and in the physical state of their chromatin: pachytenes 68 are tetraploid cells in meiotic prophase, with large nuclei and synapsed pairs of homologous 69 chromosomes, while round spermatids are haploid cells that have completed meiosis and have 70 small, compact nuclei.…”

mentioning

confidence: 99%

Parallel evolution of male germline epigenetic poising and somatic development in animals

et al. 2016

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mentioning

confidence: 99%

Parallel evolution of male germline epigenetic poising and somatic development in animals

et al. 2016

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“…The antibodies used were HP1a W191, Abcam 1220 H3K9me2, and Millipore 07-030 H3K4me2. Antibodies were validated by us and by others: see the Antibody Validation Database (http://compbio.med.harvard.edu/antibodies/ about) (43). Quantitaive PCR (see Table S1 for the list of primers) was performed using Bio-Rad iQ SYBR Green Supermix in a Cepheid SmartCycler.…”

Section: Methodsmentioning

confidence: 99%

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Sentmanat

Elgin

2012

Proc. Natl. Acad. Sci. U.S.A.

104

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A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landingpad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.epigenetics | position-effect variegation T ransposable elements (TEs) are major structural features of nearly all eukaryotic genomes, and can play a role as cisacting regulatory features with profound influence over the gene regulatory networks of their host (1). However, if left unchecked, the deleterious effects of TE mobilization can become insurmountable for the system, damaging genome integrity. Thus, silencing mechanisms that prevent TE mobilization are fundamental to the faithful propagation of genetic information. Position-effect variegation (PEV), a mosaic expression pattern resulting from silencing in some cells that normally express a gene, has frequently been used as an indicator of heterochromatic environments (2, 3). We reported previously that the DNA transposon 1360 can enhance reporter silencing in repeat-rich heterochromatic regions (4). To gain insight into the mechanisms involved, we sought to identify sequence elements present in 1360 that contribute to 1360-sensitive PEV, using an adjacent heat-shock protein 70 (hsp70)-driven white gene as the reporter.We used the phiC31 landing pad system, where phiC31 integrase mediates recombination between a landing pad (inserted in the genome) containing phage attachment (attP) sites and a donor construct containing bacterial attachment (attB) sites (5). This system allows us to maintain a constant genomic cont...

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“…To inhibit RNA polymerase II, actinomycin D (Sigma-Aldrich) was added to the medium at a concentration of 1 μg/ml for 4 h. To ensure the signal specificity of the primary antibodies, we have carefully selected the antibodies that have been validated previously on dot blots and western blots (anti-H4K5Ac, anti-H4K20me1, anti-H3K4me2, anti-H3K9me2, anti-H3K27me2 and anti-H3K36me3; data available at Antibody Validation Database, http://compbio.med.harvard.edu/antibodies/) (Egelhofer et al, 2011) or that have been successfully used for immunofluorescence by others. We have also performed western blots to further test the antibody specificity (Fig.…”

Section: Cell Line Inhibitors and Antibodiesmentioning

confidence: 99%

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

Fišerová

Efenberková

Sieger

et al. 2017

Journal of Cell Science

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The nuclear periphery (NP) plays a substantial role in chromatin organization. Heterochromatin at the NP is interspersed with active chromatin surrounding nuclear pore complexes (NPCs); however, details of the peripheral chromatin organization are missing. To discern the distribution of epigenetic marks at the NP of HeLa nuclei, we used structured illumination microscopy combined with a new MATLAB software tool for automatic NP and NPC detection, measurements of fluorescent intensity and statistical analysis of measured data. Our results show that marks for both active and nonactive chromatin associate differentially with NPCs. The incidence of heterochromatin marks, such as H3K27me2 and H3K9me2, was significantly lower around NPCs. In contrast, the presence of marks of active chromatin such as H3K4me2 was only decreased very slightly around the NPCs or not at all (H3K9Ac). Interestingly, the histone demethylases LSD1 (also known as KDM1A) and KDM2A were enriched within the NPCs, suggesting that there was a chromatinmodifying mechanism at the NPCs. Inhibition of transcription resulted in a larger drop in the distribution of H1, H3K9me2 and H3K23me2, which implies that transcription has a role in the organization of heterochromatin at the NP.

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An assessment of histone-modification antibody quality

Cited by 375 publications

References 10 publications

Parallel evolution of male germline epigenetic poising and somatic development in animals

Parallel evolution of male germline epigenetic poising and somatic development in animals

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Chromatin organization at the nuclear periphery as revealed by image analysis of structured illumination microscopy data

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