Distinctive Core Histone Post-Translational Modification Patterns in Arabidopsis thaliana

Post-translational modifications of histones play crucial roles in the genetic and epigenetic regulation of gene expression from chromatin. Studies in mammals and yeast have found conserved modifications at some residues of histones as well as non-conserved modifications at some other sites. Although plants have been excellent systems to study epigenetic regulation, and histone modifications are known to play critical roles, the histone modification sites and patterns in plants are poorly defined. In the present study we have used mass spectrometry in combination with high performance liquid chromatography (HPLC) separation and phospho-peptide enrichment to identify histone modification sites in the reference plant, Arabidopsis thaliana. We found not only modifications at many sites that are conserved in mammalian and yeast cells, but also modifications at many sites that are unique to plants. These unique modifications include H4 K20 acetylation (in contrast to H4 K20 methylation in non-plant systems), H2B K6, K11, K27 and K32 acetylation, S15 phosphorylation and K143 ubiquitination, and H2A K144 acetylation and S129, S141 and S145 phosphorylation, and H2A.X S138 phosphorylation. In addition, we found that lysine 79 of H3 which is highly conserved and modified by methylation and plays important roles in telomeric silencing in non-plant systems, is not modified in Arabidopsis. These results suggest distinctive histone modification patterns in plants and provide an invaluable foundation for future studies on histone modifications in plants

Multi-OMICs analysis reveals metabolic and epigenetic changes associated with macrophage polarization

Macrophages (MФ) are an essential immune cell for defense and repair that travel to different tissues and adapt based on local stimuli. A critical factor that may govern their polarization is the cross-talk between metabolism and epigenetics. However, simultaneous measurements of metabolites, epigenetics, and proteins (phenotype) has been a major technical challenge. To address this, we have developed a novel triomics approach using mass spectrometry to comprehensively analyze metabolites, proteins, and histone modifications, in a single sample. To demonstrate this technique, we investigated the metabolic-epigenetic-phenotype axis following polarization of human blood-derived monocytes into either \u27pro-inflammatory M1\u27- or \u27anti-inflammatory M2-\u27 MФs. We report here a complex relationship between arginine, tryptophan, glucose, and the citric acid cycle (TCA) metabolism, protein and histone post-translational modifications, and human macrophage polarization that was previously not described. Surprisingly, M1-MФs had globally reduced histone acetylation levels but high levels of acetylated amino acids. This suggests acetyl-CoA was diverted, in part, towards acetylated amino acids. Consistent with this, stable isotope tracing of glucose revealed reduced usage of acetyl-CoA for histone acetylation in M1-MФs. Furthermore, isotope tracing also revealed MФs uncoupled glycolysis from the TCA cycle, as evidenced by poor isotope enrichment of succinate. M2-MФs had high levels of kynurenine and serotonin which are reported to have immune-suppressive effects. Kynurenine is upstream of de novo NAD+ metabolism which is a necessary cofactor for Sirtuin-type histone deacetylases. Taken together, we demonstrate a complex interplay between metabolism and epigenetics that may ultimately influence cell phenotype

Human M1 macrophages express unique innate immune response genes after mycobacterial infection to defend against tuberculosis

Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.5 million deaths each year. Though 10% of patients develop tuberculosis (TB) after infection, 90% of these infections are latent. Further, mice are nearly uniformly susceptible to Mtb but their M1-polarized macrophages (M1-MΦs) can inhibit Mtb in vitro, suggesting that M1-MΦs may be able to regulate anti-TB immunity. We sought to determine whether human MΦ heterogeneity contributes to TB immunity. Here we show that IFN-γ-programmed M1-MΦs degrade Mtb through increased expression of innate immunity regulatory genes (Inregs). In contrast, IL-4-programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation and exhibit reduced Inregs expression. M1-MΦs and M2-MΦs express pro- and anti-inflammatory cytokine-chemokines, respectively, and M1-MΦs show nitric oxide and autophagy-dependent degradation of Mtb, leading to increased antigen presentation to T cells through an ATG-RAB7-cathepsin pathway. Despite Mtb infection, M1-MΦs show increased histone acetylation at the ATG5 promoter and pro-autophagy phenotypes, while increased histone deacetylases lead to decreased autophagy in M2-MΦs. Finally, Mtb-infected neonatal macaques express human Inregs in their lymph nodes and macrophages, suggesting that M1 and M2 phenotypes can mediate immunity to TB in both humans and macaques. We conclude that human MФ subsets show unique patterns of gene expression that enable differential control of TB after infection. These genes could serve as targets for diagnosis and immunotherapy of TB

Error Behaviour In Optical Networks

Optical fibre communications are now widely used in many applications, including local area computer networks. I postulate that many future optical LANs will be required to operate with limited optical power budgets for a variety of reasons, including increased system complexity and link speed, low cost components and minimal increases in transmit power. Some developers will wish to run links with reduced power budget margins, and the received data in these systems will be more susceptible to errors than has been the case previously. The errors observed in optical systems are investigated using the particular case of Gigabit Ethernet on fibre as an example. Gigabit Ethernet is one of three popular optical local area interconnects which use 8B/10B line coding, along with Fibre Channel and Infiniband, and is widely deployed. This line encoding is also used by packet switched optical LANs currently under development. A probabilistic analysis follows the effects of a single channel error in a frame, through the line coding scheme and the MAC layer frame error detection mechanisms. Empirical data is used to enhance this original analysis, making it directly relevant to deployed systems. Experiments using Gigabit Ethernet on fibre with reduced power levels at the receiver to simulate the effect of limited power margins are described. It is found that channel bit error rate and packet loss rate have only a weakly deterministic relationship, due to interactions between a number of non-uniform error characteristics at various network sub-layers. Some data payloads suffer from high bit error rates and low packet loss rates, compared to others with lower bit error rates and yet higher packet losses. Experiments using real Internet traffic contribute to the development of a novel model linking packet loss, the payload damage rate, and channel bit error rate. The observed error behaviours at various points in the physical and data link layers are detailed. These include data-dependent channel errors; this error hot- spotting is in contrast to the failure modes observed in a copper-based system. It is also found that both multiple channel errors within a single code-group, and multiple error instances within a frame, occur more frequently than might be expected. The overall effects of these error characteristics on the ability of cyclic redundancy checks (CRCs) to detect errors, and on the performance of higher layers in the network, is considered. This dissertation contributes to the discussion of layer interactions, which may lead to un-foreseen performance issues at higher levels of the network stack, and extends it by considering the physical and data link layers for a common form of optical link. The increased risk of errors in future optical networks, and my findings for 8B/10B encoded optical links, demonstrate the need for a cross-layer understanding of error characteristics in such systems. The development of these new networks should take error performance into account in light of the particular requirements of the application in question.The UK Engineering and Physical Sciences Research Council and Marconi Corporation supported my work financially through an Industrial CASE studentship

Comorbid depressive symptoms can aggravate the functional changes of the pain matrix in patients with chronic back pain: A resting-state fMRI study

ObjectiveThe purposes of this study are to explore (1) whether comorbid depressive symptoms in patients with chronic back pain (CBP) affect the pain matrix. And (2) whether the interaction of depression and CBP exacerbates impaired brain function.MethodsThirty-two patients with CBP without comorbid depressive symptoms and thirty patients with CBP with comorbid depressive symptoms were recruited. All subjects underwent functional magnetic resonance imaging (fMRI) scans. The graph theory analysis, mediation analysis, and functional connectivity (FC) analysis were included in this study. All subjects received the detection of clinical depressive symptoms and pain-related manifestations.ResultCompared with the CBP group, subjects in the CBP with comorbid depressive symptoms (CBP-D) group had significantly increased FC in the left medial prefrontal cortex and several parietal cortical regions. The results of the graph theory analyses showed that the area under the curve of small-world property (t = −2.175, p = 0.034), gamma (t = −2.332, p = 0.023), and local efficiency (t = −2.461, p = 0.017) in the CBP-D group were significantly lower. The nodal efficiency in the ventral posterior insula (VPI) (t = −3.581, p = 0.0007), and the network efficiency values (t = −2.758, p = 0.008) in the pain matrix were significantly lower in the CBP-D group. Both the topological properties and the FC values of these brain regions were significantly correlated with self-rating depression scale (SDS) scores (all FDR corrected) but not with pain intensity. Further mediation analyses demonstrated that pain intensity had a mediating effect on the relationship between SDS scores and Pain Disability Index scores. Likewise, the SDS scores mediated the relationship between pain intensity and PDI scores.ConclusionOur study found that comorbid depressive symptoms can aggravate the impairment of pain matrix function of CBP, but this impairment cannot directly lead to the increase of pain intensity, which may be because some brain regions of the pain matrix are the common neural basis of depression and CBP

Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation

TSLP released from human breast cancer cells promotes OX40L expression on DCs, and these OX40L-expressing DCs drive development of inflammatory Th2 cells which promote breast tumor development

Determination of acetylation and methylation sites of histones by mass spectrometry

This dissertation describes the development of a rapid method for determining the acetylation and methylation sites of core histories (H2B, H2A, H3 and H4) that gives the relative abundance of the acetylation isoforms by a method superior to the traditional micro-chemical sequencing method. The method created in this dissertation emphasizes speed, sensitivity and general applicability to all sorts of histone acetylation (methylation) scenarios. In this study, mass spectrometry method was first used to identify histone acetylation and methylation sites. Although there are 15 possible acetylation sites, only four acetylated peptide sequences were observed. The tetra-acetylated form is acetylated at lysines 5, 8, 12 and 16, the tri-acetylated form is mostly acetylated at lysines 8, 12 and 16, and the diacetylated form is acetylated at lysine 12 and 16. The only significant amount of mono-acetylated form is modified at lysine 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction from Lys16 to Lys5 and deacetylation goes in the reverse direction. This acetylation pattern is conserved in mammal species including HeLa cells, colon carcinoma cells and chicken erythrocyte. Core histones from chicken erythrocyte were isolated by the acid precipitation method. LC/MS simultaneously identified each subclass of histories and its potential acetylated or methylated isoforms. Acetylation and methylation sites of chicken core histories were identified. Histone H4 is acetylated at lysine residues 16, 12, 8 and 5 in the direction from lysine 16 to 5 as observed in butyrate treated HeLa cells. Lysine 20 in all isoforms of histone H4 is predominately di-methylated. About 17% of H2A is acetylated with a roughly even distribution of mono-acetylated at lysine 9 and di-acetylated at lysines 5 and 9. Lysine residues 18 and 23 in H3 are acetylated in the direction from lysine 23 to lysine 18 based on the observation that if lysine 18 is acetylated, lysine 23 also acetylated. Lysine 14 was determined to be partially acetylated (two thirds), partially methylated (one third) by high accuracy mass measurement. Lysine 4 of H3 is un- and mono-methylated. Lysine residues 9, 27 and 36 in H3 are un-, mono-, di- and trimethylated while lysine 79, a newly found methylation site for H3, is un, mono- and dimethylated. H2B is partially methylated (about 50%) at lysine 30 with a rough distribution of 1 : 0.4 : 0.3 : 0.2 for un-, mono-, di- and tri-methylated forms. (Abstract shortened by UMI.

Determination of acetylation and methylation sites of histones by mass spectrometry

This dissertation describes the development of a rapid method for determining the acetylation and methylation sites of core histories (H2B, H2A, H3 and H4) that gives the relative abundance of the acetylation isoforms by a method superior to the traditional micro-chemical sequencing method. The method created in this dissertation emphasizes speed, sensitivity and general applicability to all sorts of histone acetylation (methylation) scenarios. In this study, mass spectrometry method was first used to identify histone acetylation and methylation sites. Although there are 15 possible acetylation sites, only four acetylated peptide sequences were observed. The tetra-acetylated form is acetylated at lysines 5, 8, 12 and 16, the tri-acetylated form is mostly acetylated at lysines 8, 12 and 16, and the diacetylated form is acetylated at lysine 12 and 16. The only significant amount of mono-acetylated form is modified at lysine 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction from Lys16 to Lys5 and deacetylation goes in the reverse direction. This acetylation pattern is conserved in mammal species including HeLa cells, colon carcinoma cells and chicken erythrocyte. Core histones from chicken erythrocyte were isolated by the acid precipitation method. LC/MS simultaneously identified each subclass of histories and its potential acetylated or methylated isoforms. Acetylation and methylation sites of chicken core histories were identified. Histone H4 is acetylated at lysine residues 16, 12, 8 and 5 in the direction from lysine 16 to 5 as observed in butyrate treated HeLa cells. Lysine 20 in all isoforms of histone H4 is predominately di-methylated. About 17% of H2A is acetylated with a roughly even distribution of mono-acetylated at lysine 9 and di-acetylated at lysines 5 and 9. Lysine residues 18 and 23 in H3 are acetylated in the direction from lysine 23 to lysine 18 based on the observation that if lysine 18 is acetylated, lysine 23 also acetylated. Lysine 14 was determined to be partially acetylated (two thirds), partially methylated (one third) by high accuracy mass measurement. Lysine 4 of H3 is un- and mono-methylated. Lysine residues 9, 27 and 36 in H3 are un-, mono-, di- and trimethylated while lysine 79, a newly found methylation site for H3, is un, mono- and dimethylated. H2B is partially methylated (about 50%) at lysine 30 with a rough distribution of 1 : 0.4 : 0.3 : 0.2 for un-, mono-, di- and tri-methylated forms. (Abstract shortened by UMI.

Acetylation of H2AZ Lys 14 is associated with genome-wide gene activity in yeast

Histone variants and their post-translational modifications help regulate chromosomal functions. Htz1 is an evolutionarily conserved H2A variant found at several promoters in the yeast Saccharomyces cerevisiae. In this study, we undertook a genome-wide analysis of Htz1 and its modifications in yeast. Using mass spectrometric analysis, we determined that Htz1 is acetylated at Lys 3, Lys 8, Lys 10, and Lys 14 within its N-terminal tail, with K14 being the most abundant acetylated site. ChIP and microarray analysis were then used to compare the location of Htz1-K14 acetylation to that of Htz1 genome-wide. The data presented here demonstrate that while Htz1 is associated preferentially with the promoters of repressed genes, K14 acetylation is enriched at the promoters of active genes, and requires two known histone acetyltransferases, Gcn5 and Esa1. In support of our genome-wide analysis, we found that the acetylatable lysines of Htz1 are required for its full deposition during nucleosome reassembly upon repression of PHO5. Since the majority of Htz1 acetylation is seen at active promoters, where nucleosomes are known to be disassembled, our data argue for a dynamic process in which reassembly of Htz1 is regulated by its acetylation at promoters during transcription

Exogenous Free Ubiquitin Enhances Lily Pollen Tube Adhesion to an in Vitro Stylar Matrix and May Facilitate Endocytosis of SCA

Pollen tube adhesion and guidance on extracellular matrices within the pistil are essential processes that convey the pollen tube cell and the sperm cells to the ovule. In this study, we purified an additional molecule from the pistil that enhances pollen tube adhesion when combined with the SCA (stigma/stylar cysteine-rich adhesin)/pectin matrix in our in vitro assay. The enhancer of adhesion was identified as free ubiquitin (Ub). This was confirmed by use of bovine Ub as a substitute for lily (Lilium longiflorum Thunb.) stigma Ub. To study the interaction of SCA and Ub with the lily pollen tube, we labeled both proteins with biotin. We observed uptake of biotin-labeled SCA and Ub into the pollen tube cells in vitro using confocal microscopy. For SCA, a strong signal occurred first at the tip of the pollen tube, suggestive of an endocytosis event, and then progressively throughout the tube cytoplasm. SCA was also localized inside the in vivo pollen tube using immunogold electron microscopy and found to be present in endosomes, multivesicular bodies, and vacuoles, all known to be endocytic compartments. It was also confirmed that SCA is endocytosed in the in vitro adhesion assay. Internalization of SCA was increased in pollen tubes treated with exogenous Ub compared to those without Ub, suggesting that Ub may facilitate SCA endocytosis. These results show that Ub can act as an enhancer of pollen tube adhesion in vitro and that it is taken up into the pollen tube as is SCA. The Ub machinery may play a role in pollen tube adhesion and guidance in lily