Although histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here we describe an efficient single-step method to ...specifically purify histones located around an origin of replication from Saccharomyces cerevisiae. Using high-resolution MS, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that acetylation of histone H3 and H4 is dynamically regulated around an origin of replication, at the level of multiply acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S phase.
Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes ...enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for ≈25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.
DOT1 was originally identified as a gene affecting telomeric silencing in
S. cerevisiae. We now find that Dot1p methylates histone H3 on lysine 79, which maps to the top and bottom of the nucleosome ...core. Methylation occurs only when histone H3 is assembled in chromatin. In vivo, Dot1p is solely responsible for this methylation and methylates ∼90% of histone H3. In
dot1Δ cells, silencing is compromised and silencing proteins become redistributed at the expense of normally silenced loci. We suggest that methylation of histone H3 lysine 79 limits silencing to discrete loci by preventing the binding of Sir proteins elsewhere along the genome. Because Dot1p and histone H3 are conserved, similar mechanisms are likely at work in other eukaryotes.
High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de ...novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.
BACKGROUNDChronic graft-versus-host disease (cGVHD) is a serious complication of allogeneic hematopoietic cell transplantation (HCT). More accurate information regarding the risk of developing cGVHD ...is required. Bone marrow (BM) grafts contribute to lower cGVHD, which creates a dispute over whether risk biomarker scores should be used for peripheral blood (PB) and BM.METHODSDay 90 plasma proteomics from PB and BM recipients developing cGVHD revealed 5 risk markers that were added to 8 previous cGVHD markers to screen 982 HCT samples of 2 multicenter Blood and Marrow Transplant Clinical Trials Network (BMTCTN) cohorts. Each marker was tested for its association with cause-specific hazard ratios (HRs) of cGVHD using Cox-proportional-hazards models. We paired these clinical studies with biomarker measurements in a mouse model of cGVHD.RESULTSSpearman correlations between DKK3 and MMP3 were significant in both cohorts. In BMTCTN 0201 multivariate analyses, PB recipients with 1-log increase in CXCL9 and DKK3 were 1.3 times (95% CI: 1.1-1.4, P = 0.001) and 1.9 times (95%CI: 1.1-3.2, P = 0.019) and BM recipients with 1-log increase in CXCL10 and MMP3 were 1.3 times (95%CI: 1.0-1.6, P = 0.018 and P = 0.023) more likely to develop cGVHD. In BMTCTN 1202, PB patients with high CXCL9 and MMP3 were 1.1 times (95%CI: 1.0-1.2, P = 0.037) and 1.2 times (95%CI: 1.0-1.3, P = 0.009) more likely to develop cGVHD. PB patients with high biomarkers had increased likelihood to develop cGVHD in both cohorts (22%-32% versus 8%-12%, P = 0.002 and P < 0.001, respectively). Mice showed elevated circulating biomarkers before the signs of cGVHD.CONCLUSIONBiomarker levels at 3 months after HCT identify patients at risk for cGVHD occurrence.FUNDINGNIH grants R01CA168814, R21HL139934, P01CA158505, T32AI007313, and R01CA264921.
To identify diagnostic and prognostic markers of chronic graft-versus-host disease (cGVHD), the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT).
Using ...a quantitative proteomics approach, we compared pooled plasma samples obtained at matched time points after HCT (median, 103 days) from 35 patients with cGVHD and 18 without cGVHD (data are available via ProteomeXchange with identifier PXD002762). Of 105 proteins showing at least a 1.25-fold difference in expression, 22 were selected on the basis of involvement in relevant pathways and enzyme-linked immunosorbent assay availability. Chemokine (C-X-C motif) ligand 9 (CXCL9) and suppression of tumorigenicity 2 (ST2) also were measured on the basis of previously determined associations with GVHD. Concentrations of the four lead biomarkers were measured at or after diagnosis in plasma from two independent verification cohorts (n = 391) to determine their association with cGVHD. Their prognostic ability when measured at approximately day +100 after HCT was evaluated in plasma of a second verification cohort (n = 172).
Of 24 proteins measured in the first verification cohort, nine proteins were associated with cGVHD, and only four (ST2, CXCL9, matrix metalloproteinase 3, and osteopontin) were necessary to compose a four-biomarker panel with an area under the receiver operating characteristic curve (AUC) of 0.89 and significant correlation with cGVHD diagnosis, cGVHD severity, and nonrelapse mortality. In a second verification cohort, this panel distinguished patients with cGVHD (AUC, 0.75), and finally, the panel measured at day +100 could predict cGVHD occurring within the next 3 months with an AUC of 0.67 and 0.79 without and with known clinical risk factors, respectively.
We conclude that the biomarker panel measured at diagnosis or day +100 after HCT may allow patient stratification according to risk of cGVHD.
Cell reprogramming from a quiescent to proliferative state requires coordinate activation of multiple -omic networks. These networks activate histones, increase cellular bioenergetics and the ...synthesis of macromolecules required for cell proliferation. However, mechanisms that coordinate the regulation of these interconnected networks are not fully understood. The oncogene c-Myc (Myc) activates cellular metabolism and global chromatin remodeling. Here we tested for an interconnection between Myc regulation of metabolism and acetylation of histones. Using 13C6glucose and a combination of GC/MS and LC/ESI tandem mass spectrometry, we determined the fractional incorporation of 13C-labeled 2-carbon fragments into the fatty acid palmitate, and acetyl-lysines at the N-terminal tail of histone H4 in myc−/− and myc+/+ Rat1A fibroblasts. Our data demonstrate that Myc increases mitochondrial synthesis of acetyl-CoA, as the de novo synthesis of 13C-labeled palmitate was increased 2-fold in Myc-expressing cells. Additionally, Myc induced a forty percent increase in 13C-labeled acetyl-CoA on H4-K16. This is linked to the capacity of Myc to increase mitochondrial production of acetyl-CoA, as we show that mitochondria provide 50% of the acetyl groups on H4-K16. These data point to a key role for Myc in directing the interconnection of -omic networks, and in particular, epigenetic modification of proteins in response to proliferative signals.
DUX4 is a transcription factor whose misexpression in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). DUX4's transcriptional activity has been extensively characterized, but the ...DUX4-induced proteome remains undescribed. Here, we report concurrent measurement of RNA and protein levels in DUX4-expressing cells via RNA-seq and quantitative mass spectrometry. DUX4 transcriptional targets were robustly translated, confirming the likely clinical relevance of proposed FSHD biomarkers. However, a multitude of mRNAs and proteins exhibited discordant expression changes upon DUX4 expression. Our dataset revealed unexpected proteomic, but not transcriptomic, dysregulation of diverse molecular pathways, including Golgi apparatus fragmentation, as well as extensive post-transcriptional buffering of stress-response genes. Key components of RNA degradation machineries, including UPF1, UPF3B, and XRN1, exhibited suppressed protein, but not mRNA, levels, explaining the build-up of aberrant RNAs that characterizes DUX4-expressing cells. Our results provide a resource for the FSHD community and illustrate the importance of post-transcriptional processes in DUX4-induced pathology.
Genetic Code Expansion (GCE) can use TAG stop codons to guide site-specific incorporation of phosphoserine (pSer) into proteins. To eliminate prematurely truncated peptides, improve yields, and ...enhance the production of multiphosphorylated proteins, Release Factor 1 (RF1)-deficient expression hosts were developed, yet these grew slowly and their use was associated with extensive misincorporation of natural amino acids instead of pSer. Here, we merge a healthy RF1-deficient
cell line with a high-efficiency pSer GCE translation system to produce a versatile pSer GCE platform in which only trace misincorporation of natural amino acids is detected even when five phosphoserines were introduced into one protein. Approximately 400 and 200 mg of singly and doubly phosphorylated GFP per liter of culture were obtained. Importantly, the lack of truncated protein permits expression of oligomeric proteins and the use of N-terminal solubility-enhancing proteins to aid phospho-protein expression and purification. To illustrate the enhanced utility of this system, we produce doubly phosphorylated STING (Stimulator of Interferon Genes), as well as triply phosphorylated BAD (Bcl2-associated agonist of cell death) complexed with 14-3-3, in quantity, purity, and homogeneity sufficient for structural biology applications. We anticipate that the facile access to phosphoproteins enabled by this system, which we call pSer-3.1G, will expand studies of the phospho-proteome.
Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also ...occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae . Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.
Significance Long-lived proteins in extracellular spaces (joints/tissues) or within specialized nondividing cells (eye-lens) are associated with age-related decline. However, aging also occurs in dividing stem cells. Although several hypotheses have been proposed to explain how stem cells age, none have addressed whether long-lived proteins contribute to aging, partially because of technical challenges in identifying such proteins. We developed a method to overcome these limitations in the model system Saccharomyces cerevisiae . We identified two classes of long-lived asymmetrically retained proteins (LARPs). Full-length LARPs remain intact throughout the mother cell lifespan and accumulate in abundance or become posttranslationally modified. Fragmented LARPs are original proteins that are partially degraded, yet retained by the mother cell during aging. We speculate that LARPs contribute to the aging process.
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