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Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Herein we describe the existence of Lys and Arg modifications on histones by a glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.
Copyright © 2018 the Author(s). Published by PNAS.
Studies of regulatory activity and gene expression have revealed an intriguing dichotomy: There is substantial turnover in the regulatory activity of orthologous sequences between species; however, the expression level of orthologous genes is largely conserved. Understanding how distal regulatory elements, for example, enhancers, evolve and function is critical, as alterations in gene expression levels can drive the development of both complex disease and functional divergence between species. In this study, we investigated determinants of the conservation of regulatory enhancer activity for orthologous sequences across mammalian evolution. Using liver enhancers identified from genome-wide histone modification profiles in ten diverse mammalian species, we compared orthologous sequences that exhibited regulatory activity in all species (conserved-activity enhancers) to shared sequences active only in a single species (species-specific-activity enhancers). Conserved-activity enhancers have greater regulatory potential than species-specific-activity enhancers, as quantified by both the density and diversity of transcription factor binding motifs. Consistent with their greater regulatory potential, conserved-activity enhancers have greater regulatory activity in humans than species-specific-activity enhancers: They are active across more cellular contexts, and they regulate more genes than species-specific-activity enhancers. Furthermore, the genes regulated by conserved-activity enhancers are expressed in more tissues and are less tolerant of loss-of-function mutations than those targeted by species-specific-activity enhancers. These consistent results across various stages of gene regulation demonstrate that conserved-activity enhancers are more pleiotropic than their species-specific-activity counterparts. This suggests that pleiotropy is associated with the conservation of regulatory across mammalian evolution.
© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Differentiating osteoclast-rich lesions of bone (giant cell tumor of bone [GCTB], chondroblastoma [CBA], and aneurysmal bone cyst [ABC]) can be challenging, especially in small biopsies or fine-needle aspirations. Mutations affecting codons 34 and 36 of either H3 Histone Family Member 3A (H3F3A) and/or 3B (H3F3B) are characteristically seen in GCTB and CBAs. We devised a simple assay to identify these mutations and evaluated its applicability for routine clinical diagnosis. One hundred twenty-four tissue specimens from 108 patients (43 GCTBs, 38 CBAs and 27 ABCs) were collected from the archives of the Calgary Laboratory Services/University of Calgary and Vanderbilt University Medical Center. Histology was reviewed by an expert orthopedic pathologist. A single base extension assay (SNaPshot) is used to interrogate each nucleotide in codons 34 and 36 of H3F3A and codon 36 of H3F3B. Final diagnoses were generated after re-reviewing cases and incorporating molecular findings. Of 43 GCTBs, 38 (88%) had an H3F3A G34W mutation; 35 of 38 CBAs (92%) had a K36M mutation in either H3F3B (N = 31; 82%) or H3F3A (N = 4; 11%); none of 27 ABCs had a tested mutation. Molecular findings changed the histomorphologic diagnosis in 5 cases (3 GCTB changed to ABC, and 2 ABC changed to GCTB). These findings support the diagnostic utility of mutational analysis for this differential diagnosis in certain challenging cases when clinicoradiologic and histomorphologic features are not definitive, particularly for distinguishing cellular ABC versus GCTB with secondary ABC.
Copyright © 2017 Elsevier Inc. All rights reserved.
Necrosis is induced by ischemic conditions within the core of many solid tumors. Using fluorescent fusion proteins, we provide in vivo evidence of histone trafficking among cancer cells in implanted tumors. In particular, the most abundant H1 isoform (H1.2) was found to be transported from necrotic tumor cells into surrounding viable cells where histones are selectively taken up by energy-dependent endocytosis. We propose that intercellular histone trafficking could function as a target for drug delivery. This concept was validated using an anti-histone antibody that was co-internalized with histones from dead cells into viable ones surrounding the necrotic regions of a tumor, where some of the most chemoresistant cells reside. These findings demonstrate that cellular translocation of conjugated drugs using anti-histone antibodies is a promising strategy for targeted drug delivery to chemoresistant tumors.
Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.
During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic-epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.
ALDH3A1 is a corneal crystallin that protects ocular tissues from ultraviolet radiation through catalytic and non-catalytic functions. In addition, ALDH3A1 plays a functional role in corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation. We have previously shown that Aldh3a1 knockout mice in a C57B6/129sV mixed genetic background develop lens cataracts. In the current study, we evaluated the corneal phenotype of Aldh3a1 knockout mice bred into a C57B/6J congenic background (KO). In vivo confocal microscopy examination of KO and wild-type (WT) corneas revealed KO mice to exhibit corneal haze, manifesting marked light scattering from corneal stroma. This corneal phenotype was further characterized by Imaging Mass Spectrometry (IMS) with spatial resolution that revealed a trilayer structure based on differential lipid localization. In these preliminary studies, no differences were observed in lipid profiles from KO relative to WT mice; however, changes in protein profiles of acyl-CoA binding protein (m/z 9966) and histone H4.4 (m/z 11308) were found to be increased in the corneal epithelial layer of KO mice. This is the first study to use IMS to characterize endogenous proteins and lipids in corneal tissue and to molecularly explore the corneal haze phenotype. Taken together, the current study presents the first genetic animal model of cellular-induced corneal haze due to the loss of a corneal crystallin, and strongly supports the notion that ALDH3A1 is critical to cellular transparency. Finally, IMS represents a valuable new approach to reveal molecular changes underlying corneal disease.
Copyright © 2016. Published by Elsevier B.V.
Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.
Bioactive electrophiles generated from the oxidation of endogenous and exogenous compounds are a contributing factor in numerous disease states. Their toxicity is largely attributed to the covalent modification of cellular nucleophiles, including protein and DNA. With regard to protein modification, the side-chains of Cys, His, Lys, and Arg residues are critical targets. This results in the generation of undesired protein post-translational modifications (PTMs) that can trigger dire cellular consequences. Notably, histones are Lys- and Arg-rich proteins, providing a fertile source for adduction by both exogenous and endogenous electrophiles. The regulation of histone PTMs plays a critical role in the regulation of chromatin structure and thus gene expression. This perspective focuses on the role of electrophilic protein adduction within the context of chromatin and its potential consequences on cellular law and order.
EZH2 is crucial for the progression of prostate cancer (PCa) and castration-resistant prostate cancer (CRPC) through upregulation and activation of progenitor genes, as well as androgen receptor (AR)-target genes. However, the mechanisms by which EZH2 is regulated in PCa and CRPC remain elusive. Here we report that EZH2 is post-transcriptionally regulated by SKP2 in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of Skp2, Ezh2 and histone H3 lysine 27 trimethylation (H3K27me3) in both Pten null mouse embryonic fibroblasts (MEFs) and Pten null mouse prostate tissues. Loss of Skp2 resulted in a striking decrease of Ezh2 levels in Pten/Trp53 double-null MEFs and in prostate tumors of Pten/Trp53 double-null mutant mice. SKP2 knockdown decreased EZH2 levels in human PCa cells through upregulation of TRAF6-mediated and lysine(K) 63-linked ubiquitination of EZH2 for degradation. Ectopic expression of TRAF6 promoted the K63-linked ubiquitination of EZH2 to decrease EZH2 and H3K27me3 levels in PCa cells. In contrast, TRAF6 knockdown resulted in a reduced EZH2 ubiquitination with an increase of EZH2 and H3K27me3 levels in PCa cells. Furthermore, the catalytically dead mutant TRAF6 C70A abolished the TRAF6-mediated polyubiquitination of recombinant human EZH2 in vitro. Most importantly, a concurrent elevation of Skp2 and Ezh2 was found in CRPC tumors of Pten/Trp53 mutant mice, and expression levels of SKP2 and EZH2 were positively correlated in human PCa specimens. Taken together, our findings revealed a novel mechanism on EZH2 ubiquitination and an important signaling network of SKP2-TRAF6-EZH2/H3K27me3, and targeting SKP2-EZH2 pathway may be a promising therapeutic strategy for CRPC treatment.