Extraction, purification and analysis of histones

Shechter, David; Dormann, Holger L.; Allis, C. David; Hake, Sandra B.

doi:10.1038/nprot.2007.202

Cited by 918 publications

(858 citation statements)

References 33 publications

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“…4A,B. As expected [22,23], H2B had the shortest retention time in the RP-C8 matrix (Fig. 4A,B, upper panels, fractions 91-95 and 93-96 respectively) and the autoradiogram shows no detectable phosphate incorporation in either untreated or cAMPtreated cells (Fig.…”

Section: Hplc Analysis Of Histone Phosphorylation In Metabolically-lasupporting

confidence: 77%

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines

Rodriguez-Collazo

Snyder

Chiffer

et al. 2008

Experimental Cell Research

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The phosphorylation of histone H3 is known to play a role in regulation of transcription as well as preparation of chromosomes for mitosis. Various signaling cascades induce H3 phosphorylation, particularly at genes activated by these pathways. In this study we show that signaling can also have the opposite effect. Activators of cAMP signaling induce a rapid and potent loss of H3 phosphorylation. This effect is not mediated through a cAMP metabolite since a membranepermeable form of AMP had no effect on H3 phosphorylation and a phosphodiesterase-resistant cAMP analog efficiently reduced it. cAMP is also the likely regulator of H3 phosphorylation under physiological conditions since only supra-pharmacological doses of cGMP induce the loss of H3 phosphorylation. The loss of phosphorylation is specific for histone H3 since we do not observe drastic losses in total phosphorylation of other histones. In addition, other H3 modifications are unaffected with the exception of lysine 9 methylation, which is elevated. Analysis of cell growth and cell cycle show that cAMP signaling inhibits cell growth and arrests cells at both G1 and G2/M. Similar effects of cAMP signaling on H3 phosphorylation are observed in a variety of mammary adenocarcinoma-derived cell lines. In syngeneic human breast derived cell lines, one diploid and non-transformed, the other derived from a ductal carcinoma, the loss of H3 phosphorylation is significantly more sensitive to cAMP concentration in the transformed cell line.

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Section: Hplc Analysis Of Histone Phosphorylation In Metabolically-lasupporting

confidence: 77%

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines

Rodriguez-Collazo

Snyder

Chiffer

et al. 2008

Experimental Cell Research

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“…Histones were purified in acidic condition as previously described 45 . Samples were subjected to SDS-PAGE and immunoblotting using standard procedures.…”

Section: Western Blottingmentioning

confidence: 99%

Regulation of histone modification and chromatin structure by the p53–PADI4 pathway

et al. 2012

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Histone proteins are modified in response to various external signals; however, their mechanisms are still not fully understood. Citrullination is a post-transcriptional modification that converts arginine in proteins into citrulline. Here we show in vivo and in vitro citrullination of the arginine 3 residue of histone H4 (cit-H4R3) in response to DnA damage through the p53-PADI4 pathway. We also show DnA damage-induced citrullination of Lamin C. Cit-H4R3 and citrullinated Lamin C localize around fragmented nuclei in apoptotic cells. Ectopic expression of PADI4 leads to chromatin decondensation and promotes DnA cleavage, whereas Padi4 − / − mice exhibit resistance to radiation-induced apoptosis in the thymus. Furthermore, the level of cit-H4R3 is negatively correlated with p53 protein expression and with tumour size in non-small cell lung cancer tissues. our findings reveal that cit-H4R3 may be an 'apoptotic histone code' to detect damaged cells and induce nuclear fragmentation, which has a crucial role in carcinogenesis.

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“…The nuclear histones from HeLa cells were isolated using a modified protocol of Shechter et al 27 and established the deacetylation assay using rhHDAC1/rhHDAC6 as controls along with rHos2. In these assays it was found that 0.3 μg of rhHDAC1 was able to deacetylate the nuclear acetylated histones as detected by an anti-H3-K9 histone antibody.…”

Section: Resultsmentioning

confidence: 99%

“…Acetylated histones were isolated from HeLa cells treated with the HDAC inhibitor SAHA as per published protocol 27 . The histone pellet was then resuspended in ultra pure water, stored in 50 μl aliquots at -70°C and the protein concentration was determined using a BCA kit (Pierce).…”

Section: Methodsmentioning

confidence: 99%

Functional characterization of Candida albicans Hos2 histone deacetylase

et al. 2013

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Candida albicans is a mucosal commensal organism in normal individuals, but is a major pathogen causing systemic and mucosal infections in immunocompromised individuals. Azoles have been very effective anti-fungal agents and the mainstay in treating opportunistic mold and yeast infections. Azole resistant strains have emerged compromising the utility of this class of drugs. It has been shown that azole resistance can be reversed by the co-administration of a histone deacetylase (HDAC) inhibitor, suggesting that resistance is mediated by epigenetic mechanisms possibly involving Hos2, a fungal deacetylase. We report here the cloning and functional characterization of HOS2 (High Osmolarity Sensitive) , a gene coding for fungal histone deacetylase from C. albicans. Inhibition studies showed that Hos2 is susceptible to pan inhibitors such as trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), but is not inhibited by class I inhibitors such MS-275. Purified Hos2 protein consistently deacetylated tubulins, rather than histones from TSA-treated cells. This in vitro enzymatic assay, which is amenable to high throughput could be used for screening potent fungal Hos2 inhibitors that could be a potential anti-fungal adjuvant. Hos2 has been reported to be a putative NAD+ dependent histone deacetylase, a feature of sirtuins. We assayed for sirtuin activation with resveratrol and purified Hos2 protein and did not find any sirtuin activity.

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Extraction, purification and analysis of histones

Cited by 918 publications

References 33 publications

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines

cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines

Regulation of histone modification and chromatin structure by the p53–PADI4 pathway

Functional characterization of Candida albicans Hos2 histone deacetylase

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