Global turnover of histone post-translational modifications and variants in human cells

<p>Abstract</p> <p>Background</p> <p>Post-translational modifications (PTMs) on the N-terminal tails of histones and histone variants regulate distinct transcriptional states and nuclear events. Whereas the functional effects of specific PTMs are the current subject of intense investigation, most studies characterize histone PTMs/variants in a non-temporal fashion and very few studies have reported kinetic information about these histone forms. Previous studies have used radiolabeling, fluorescence microscopy and chromatin immunoprecipitation to determine rates of histone turnover, and have found interesting correlations between increased turnover and increased gene expression. Therefore, histone turnover is an understudied yet potentially important parameter that may contribute to epigenetic regulation. Understanding turnover in the context of histone modifications and sequence variants could provide valuable additional insight into the function of histone replacement.</p> <p>Results</p> <p>In this study, we measured the metabolic rate of labeled isotope incorporation into the histone proteins of HeLa cells by combining stable isotope labeling of amino acids in cell culture (SILAC) pulse experiments with quantitative mass spectrometry-based proteomics. In general, we found that most core histones have similar turnover rates, with the exception of the H2A variants, which exhibit a wider range of rates, potentially consistent with their epigenetic function. In addition, acetylated histones have a significantly faster turnover compared with general histone protein and methylated histones, although these rates vary considerably, depending on the site and overall degree of methylation. Histones containing transcriptionally active marks have been consistently found to have faster turnover rates than histones containing silent marks. Interestingly, the presence of both active and silent marks on the same peptide resulted in a slower turnover rate than either mark alone on that same peptide. Lastly, we observed little difference in the turnover between nearly all modified forms of the H3.1, H3.2 and H3.3 variants, with the notable exception that H3.2K36me2 has a faster turnover than this mark on the other H3 variants.</p> <p>Conclusions</p> <p>Quantitative proteomics provides complementary insight to previous work aimed at quantitatively measuring histone turnover, and our results suggest that turnover rates are dependent upon site-specific post-translational modifications and sequence variants.</p

Histone H1 variant-specific lysine methylation by G9a/KMT1C and Glp1/KMT1D

BACKGROUND: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown. RESULTS: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle. CONCLUSIONS: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions

The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state

Towards Minimal S4 Lepton Flavor Model

We study lepton flavor models with the $S_4$ flavor symmetry. We construct simple models with smaller numbers of flavon fields and free parameters, such that we have predictions among lepton masses and mixing angles. The model with a $S_4$ triplet flavon is not realistic, but we can construct realistic models with two triplet flavons, or one triplet and one doublet flavons.Comment: 18 pages, 4 figures, references are adde

Low and intermediate mass star yields.II: The evolution of nitrogen abundances

We analyze the impact on the Galactic nitrogen abundances of using a new set of low and intermediate mass star yields. These yields have a significant yield of primary nitrogen from intermediate mass stars. We use these yields as an input to a Galactic Chemical Evolution model and study the nitrogen abundances in the halo and in the disc, and compare them with models obtained using other yield sets and with a large amount of observational data. We find that, using these new yields, our model adequately reproduce the observed trends. In particular, these yields solve the historical problem of the evolution of nitrogen, giving the right level of relative abundance N/O by the production of a primary component in intermediate mass stars. Moreover, using different evolutionary rates in each radial region of the Galaxy, we may explain the observed N dispersion.Comment: 13 pages, 13 figure

Low and intermediate mass star yields: The evolution of carbon abundances

We present a set of low and intermediate mass star yields based on a modeling of the TP--AGB phase which affects the production of nitrogen and carbon. These yields are evaluated by using them in a Galaxy Chemical Evolution model, with which we analyze the evolution of carbon abundances. By comparing the results with those obtained with other yield sets, and with a large amount of observational data, we conclude that the model using these yields combined with those from Woosley & Weaver (1995) for massive stars properly reproduce all the data. The model reproduces well the increase of C/O with increasing O/H abundances. Since these massive star yields do not include winds, it implies that these stellar winds might have a smoother dependence on metallicity than usually assumed and that a significant quantity of carbon proceeds from LIM stars.Comment: 21 pages, 11 figures. To be published in Astronomy and Astrophysic

Proteomic Interrogation of Human Chromatin

Chromatin proteins provide a scaffold for DNA packaging and a basis for epigenetic regulation and genomic maintenance. Despite understanding its functional roles, mapping the chromatin proteome (i.e. the “Chromatome”) is still a continuing process. Here, we assess the biological specificity and proteomic extent of three distinct chromatin preparations by identifying proteins in selected chromatin-enriched fractions using mass spectrometry-based proteomics. These experiments allowed us to produce a chromatin catalog, including several proteins ranging from highly abundant histone proteins to less abundant members of different chromatin machinery complexes. Using a Normalized Spectral Abundance Factor approach, we quantified relative abundances of the proteins across the chromatin enriched fractions giving a glimpse into their chromosomal abundance. The large-scale data sets also allowed for the discovery of a variety of novel post-translational modifications on the identified chromatin proteins. With these comparisons, we find one of the probed methods to be qualitatively superior in specificity for chromatin proteins, but inferior in proteomic extent, evidencing a compromise that must be made between biological specificity and broadness of characterization. Additionally, we attempt to identify proteins in eu- and heterochromatin, verifying the enrichments by characterizing the post-translational modifications detected on histone proteins from these chromatin regions. In summary, our results provide insights into the value of different methods to extract chromatin-associated proteins and provide starting points to study the factors that may be involved in directing gene expression and other chromatin-related processes

A grid of chemical evolution models as a tool to interpret spiral and irregular galaxies data

We present a generalization of the multiphase chemical evolution model applied to a wide set of theoretical galaxies with different masses and evolutionary rates. This generalized set of models has been computed using the so-called Universal Rotation Curve from Persic et al (1996) to calculate the radial mass distribution of 44 theoretical protogalaxies. This distribution is a fundamental input which, besides its own effect on the galaxy evolution, defines the characteristic collapse time-scale or gas infall rate onto the disc.We have adopted 10 sets of values, between 0 and 1, for the molecular cloud and star formation efficiencies, as corresponding to their probability nature, for each one of the radial distributions of total mass. Thus, we have constructed a bi-parametric grid of models, depending on those efficiency sets and on the rotation velocity, whose results are valid in principle for any spiral or irregular galaxy. The model results provide the time evolution of different regions of the disc and the halo along galactocentric distance, measured by the gas (atomic and molecular) and stellar masses, the star formation rate and chemical abundances of 14 elements, for a total of 440 models. This grid may be used to estimate the evolution of a given galaxy for which only present time information -- such as radial distributions of elemental abundances, gas densities and/or star formation, which are the usual observational constraints of chemical evolution models -- is available.Comment: 27 pag., 20 fig, to be published in MNRA

Outcome measures for the evaluation of treatment response in hidradenitis suppurativa for clinical practice

Importance Although several clinician- and patient-reported outcome measures have been developed for trials in hidradenitis suppurativa (HS), there is currently no consensus on which measures are best suited for use in clinical practice. Identifying validated and feasible measures applicable to the practice setting has the potential to optimize treatment strategies and generate generalizable evidence that may inform treatment guidelines. Objective To establish consensus on a core set of clinician- and patient-reported outcome measures recommended for use in clinical practice and to establish the appropriate interval within which these measures should be applied. Evidence Review Clinician- and patient-reported HS measures and studies describing their psychometric properties were identified through literature reviews. Identified measures comprised an item reduction survey and subsequent electronic Delphi (e-Delphi) consensus rounds. In each consensus round, a summary of outcome measure components and scoring methods was provided to participants. Experts were provided with feasibility characteristics of clinician measures to aid selection. Consensus was achieved if at least 67% of respondents agreed with use of a measure in clinical practice. Findings Among HS experts, response rates for item reduction, e-Delphi round 1, and e-Delphi round 2 surveys were 76.4% (42 of 55), 90.5% (38 of 42), and 92.9% (39 of 42), respectively; among patient research partners (PRPs), response rates were 70.8% (17 of 24), 100% (17 of 17), and 82.4% (14 of 17), respectively. The majority of experts across rounds were practicing dermatologists with 18 to 19 years of clinical experience. In the final e-Delphi round, most PRPs were female (12 [85.7%] vs 2 males [11.8%]) and aged 30 to 49 years. In the final e-Delphi round, HS experts and PRPs agreed with the use of the HS Investigator Global Assessment (28 [71.8%]) and HS Quality of Life score (13 [92.9%]), respectively. The most expert-preferred assessment interval in which to apply these measures was 3 months (27 [69.2%]). Conclusions and Relevance An international group of HS experts and PRPs achieved consensus on a core set of HS measures suitable for use in clinical practice. Consistent use of these measures may lead to more accurate assessments of HS disease activity and life outcomes, facilitating shared treatment decision-making in the practice setting