Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase

Minshull, Thomas C.; Dockrell, David H.; Read, Robert C.; Dickman, Mark J.

doi:10.1016/j.chroma.2016.05.025

Cited by 6 publications

(7 citation statements)

References 41 publications

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“…• High-performance liquid chromatography (HPLC) separates protein molecules by molecular weight and conformation. The method is usually combined with mass spectrometry (MS) (Minshull, Cole, Dockrell, Read, & Dickman, 2016). After the physical separation of histones by HPLC, MS allows the detection of histone post-translational modifications.…”

Section: Box 1: Techniques Used To Assess the Contribution Of Epigenementioning

confidence: 99%

Synthesizing the role of epigenetics in the response and adaptation of species to climate change in freshwater ecosystems

et al. 2018

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Freshwater ecosystems are amongst the most threatened ecosystems on Earth. Currently, climate change is one of the most important drivers of freshwater transformation and its effects include changes in the composition, biodiversity and functioning of freshwater ecosystems. Understanding the capacity of freshwater species to tolerate the environmental fluctuations induced by climate change is critical to the development of effective conservation strategies. In the last few years, epigenetic mechanisms were increasingly put forward in this context because of their pivotal role in gene-environment interactions. In addition, the evolutionary role of epigenetically inherited phenotypes is a relatively recent but promising field. Here, we examine and synthesize the impacts of climate change on freshwater ecosystems, exploring the potential role of epigenetic mechanisms in both short- and long-term adaptation of species. Following this wrapping-up of current evidence, we particularly focused on bringing together the most promising future research avenues towards a better understanding of the effects of climate change on freshwater biodiversity, specifically highlighting potential molecular targets and the most suitable freshwater species for future epigenetic studies in this context.

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Section: Box 1: Techniques Used To Assess the Contribution Of Epigenementioning

confidence: 99%

Synthesizing the role of epigenetics in the response and adaptation of species to climate change in freshwater ecosystems

et al. 2018

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“…Bacteria were washed in PBS and re-suspended in RPMI 1640 supplemented with 10% pooled human immune serum (from previously vaccinated volunteers with demonstrable antibody levels to serotype 2 pneumococci) ( 27 ). MDM were challenged with either opsonised S. pneumoniae , Δ ply or PBS, at a MOI of 10, rested on ice for 1 h and incubated at 37°C in 5% CO 2 for a further 3 h ( 28 ). For certain experiments cells were treated with 3 μM vorinostat (SAHA, Sigma) or 0.5% DMSO (vehicle control) for 30 min prior to bacterial challenge and vorinostat reintroduced after bacterial challenge.…”

Section: Methodsmentioning

confidence: 99%

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages

et al. 2021

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Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae, and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF-α production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci.

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“…Histones were extracted following the protocol previously described in Minshull et al Briefly, cell pellets underwent hypotonic lysis followed by acid extraction . Histones were re‐suspended in 100 mM of ammonium bicarbonate pH 8.0 before two rounds of chemical derivatization using propionic anhydride in isopropanol (1:3 ratio) for 15 min at 37°C, followed by trypsin digestion overnight and a further two rounds of derivatization .…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a plethora of different approaches have been described for the study of histone PTMs . These include top‐down, middle‐down and bottom‐up approaches . The top‐down approach provides information at the protein level, enabling the study of histone protein proteoforms and their associated combination of PTMs.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of data‐acquisition methods for the identification and quantification of histone post‐translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer

Hanson

James

Dockrell

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Histone post‐translational modifications (PTMs) play key roles in regulating eukaryotic gene expression. Mass spectrometry (MS) has emerged as a powerful method to characterize and quantify histone PTMs as it allows unbiased identification and quantification of multiple histone PTMs including combinations of the modifications present. Methods In this study we compared a range of data‐acquisition methods for the identification and quantification of the histone PTMs using a Q Exactive HF Orbitrap. We compared three different data‐dependent analysis (DDA) methods with MS2 resolutions of 120K, 60K, 30K. We also compared a range of data‐independent analysis (DIA) methods using MS2 isolation windows of 20 m/z and DIAvw to identify and quantify histone PTMs in Chinese hamster ovary (CHO) cells. Results The increased number of MS2 scans afforded by the lower resolution methods resulted in a higher number of queries, peptide sequence matches (PSMs) and a higher number of peptide proteoforms identified with a Mascot Ion score greater than 46. No difference in the proportion of peptide proteoforms with Delta scores >17 was observed. Lower coefficients of variation (CVs) were obtained in the DIA MS1 60 K MS2 30 K 20 m/z isolation windows compared with the other data‐acquisition methods. Conclusions We observed that DIA which offers advantages in flexibility and identification of isobaric peptide proteoforms performs as well as DDA in the analysis of histone PTMs. We were able to identify 71 modified histone peptides for histone H3 and H4 and quantified 64 across each of the different acquisition methods.

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Analysis of histone post translational modifications in primary monocyte derived macrophages using reverse phase×reverse phase chromatography in conjunction with porous graphitic carbon stationary phase

Cited by 6 publications

References 41 publications

Synthesizing the role of epigenetics in the response and adaptation of species to climate change in freshwater ecosystems

Synthesizing the role of epigenetics in the response and adaptation of species to climate change in freshwater ecosystems

Pneumolysin Is Responsible for Differential Gene Expression and Modifications in the Epigenetic Landscape of Primary Monocyte Derived Macrophages

Comparison of data‐acquisition methods for the identification and quantification of histone post‐translational modifications on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer

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