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The involvement of epigenetic processes in the origin and progression of cancer is now widely appreciated. Consequently, targeting the enzymatic machinery that controls the epigenetic regulation of the genome has emerged as an attractive new strategy for therapeutic intervention. The development of epigenetic drugs requires a detailed knowledge of the processes that govern chromatin regulation. Over the recent years, mass spectrometry (MS) has become an indispensable tool in epigenetics research. In this review, we will give an overview of the applications of MS-based proteomics in studying various aspects of chromatin biology. We will focus on the use of MS in the discovery and mapping of histone modifications and how novel proteomic approaches are being utilized to identify and study chromatin-associated proteins and multi-subunit complexes. Finally, we will discuss the application of proteomic methods in the diagnosis and prognosis of cancer based on epigenetic biomarkers and comment on their future impact on cancer epigenetics. [ABSTRACT FROM AUTHOR]

Summary: Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify “crosstalk” between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding “profile” for proteins regulated by DNA and histone methylation. [Copyright &y& Elsevier]

Apoptotic cell death and survival is controlled by pro- and antiapoptotic proteins. Because these proteins act on each other, cell fate is dictated by the relative activity of pro- versus antiapoptotic proteins. Here we report that BRUCE, a conserved 528 kDa peripheral membrane protein of the trans-Golgi network, protects cells against apoptosis and functions as an inhibitor of apoptosis (IAP). By using wild-type and mutant forms we show that BRUCE inhibits caspase activity and apoptosis depending on its BIR domain. Upon apoptosis induction, BRUCE is antagonized by three mechanisms: first, through binding to Smac; second, by the protease HtrA2; and third, by caspase-mediated cleavage. In addition to its IAP activity BRUCE has the distinctive property of functioning as a chimeric E2/E3 ubiquitin ligase with Smac being a substrate. Our work suggests that, owing to its two activities and its localization, BRUCE may function as a specialized regulator of cell death pathways. [Copyright &y& Elsevier]

One of the main functions of the tumor suppressor p53 is the induction of programmed cell death. Here we investigated in detail the molecular mechanisms that underlay p53 transactivation-dependent apoptosis in the human colon cancer cell line DLD-1. Although p53 upregulated the death receptors Fas, TRAIL-R1 and TRAIL-R2 in this cell line, p53-induced cell death occurred without detectable caspase-8 activation whereas, activation of caspase-9 and caspase-3 was readily observed. In addition to the upregulation of death receptors, p53 induced the pro-apoptotic Bcl-2 family members Bik and Bak and downregulated the anti-apoptotic Bcl-xL protein. Moreover, in RNase protection assay analyses as well as in reporter gene analyses we found a p53-dependent upregulation of the death receptor-inhibitory protein cFLIP. Together, these data argue for a p53-mediated activation of the mitochondrial pathway of apoptosis. In contrast to recently published data obtained in different cellular systems, there was no evidence for an essential role of NF-κB in p53-induced cell death. Moreover, induction of p53 interfered with TNF-induced NF-κB activation independently from apoptosis-induction. Oncogene (2001) 20, 571–580. [ABSTRACT FROM AUTHOR]

Mutations caused by DNA damage are a main driver of cancer. We discovered that recognition of newly synthesised histone H4 directs breast cancer type 1 susceptibility protein (BRCA1) to post-replicative chromatin. The switch from mutagenic to error-free DNA double strand break repair by homologous recombination is therefore controlled by chromatin. [ABSTRACT FROM AUTHOR]

The regulation of chromatin by epigenetic mechanisms plays a central role in gene expression and is essential for development and maintenance of cell identity and function. Aberrant chromatin regulation is observed in many diseases where it leads to defects in epigenetic gene regulation resulting in pathological gene expression programmes. These defects are caused by inherited or acquired mutations in genes encoding enzymes that deposit or remove DNA and histone modifications and that shape chromatin architecture. Chromatin deregulation often results in neurodevelopmental disorders and intellectual disabilities, frequently linked to physical and developmental abnormalities, but can also cause neurodegenerative diseases, immunodeficiency, or muscle wasting syndromes. Epigenetic diseases can either be of monogenic origin or manifest themselves as complex multifactorial diseases such as in congenital heart disease, autism spectrum disorders, or cancer in which mutations in chromatin regulators are contributing factors. The environment directly influences the epigenome and can induce changes that cause or predispose to diseases through risk factors such as stress, malnutrition or exposure to harmful chemicals. The plasticity of chromatin regulation makes targeting the enzymatic machinery an attractive strategy for therapeutic intervention and an increasing number of small molecule inhibitors against a variety of epigenetic regulators are in clinical use or under development. In this review, we will give an overview of the molecular lesions that underlie epigenetic diseases, and we will discuss the impact of the environment and prospects for epigenetic therapies. [ABSTRACT FROM AUTHOR]

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology. [ABSTRACT FROM AUTHOR]

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 a3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 a3 helix play organismal roles in defining chromatin structure. [ABSTRACT FROM AUTHOR]

Summary: Reversible cellular quiescence is critical for developmental processes in metazoan organisms and is characterized by a reduction in cell size and transcriptional activity. We show that the Aurora B kinase and the polycomb protein Ring1B have essential roles in regulating transcriptionally active genes in quiescent lymphocytes. Ring1B and Aurora B bind to a wide range of active promoters in resting B and T cells. Conditional knockout of either protein results in reduced transcription and binding of RNA Pol II to promoter regions and decreased cell viability. Aurora B phosphorylates histone H3S28 at active promoters in resting B cells as well as inhibiting Ring1B-mediated ubiquitination of histone H2A and enhancing binding and activity of the USP16 deubiquitinase at transcribed genes. Our results identify a mechanism for regulating transcription in quiescent cells that has implications for epigenetic regulation of the choice between proliferation and quiescence. [ABSTRACT FROM AUTHOR]

Abstract: The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex. [Copyright &y& Elsevier]

Activation of Janus kinase 2 (JAK2) by chromosomal translocations or point mutations is a frequent event in haematological malignancies. JAK2 is a non-receptor tyrosine kinase that regulates several cellular processes by inducing cytoplasmic signalling cascades. Here we show that human JAK2 is present in the nucleus of haematopoietic cells and directly phosphorylates Tyr 41 (Y41) on histone H3. Heterochromatin protein 1α (HP1α), but not HP1β, specifically binds to this region of H3 through its chromo-shadow domain. Phosphorylation of H3Y41 by JAK2 prevents this binding. Inhibition of JAK2 activity in human leukaemic cells decreases both the expression of the haematopoietic oncogene lmo2 and the phosphorylation of H3Y41 at its promoter, while simultaneously increasing the binding of HP1α at the same site. Τhese results identify a previously unrecognized nuclear role for JAK2 in the phosphorylation of H3Y41 and reveal a direct mechanistic link between two genes, jak2 and lmo2, involved in normal haematopoiesis and leukaemia. [ABSTRACT FROM AUTHOR]

Induction of gene expression in yeast and human cells involves changes in the histone modifications associated with promoters. Here we identify a histone H3 endopeptidase activity in Saccharomyces cerevisiae that may regulate these events. The endopeptidase cleaves H3 after Ala21, generating a histone that lacks the first 21 residues and shows a preference for H3 tails carrying repressive modifications. In vivo, the H3 N terminus is clipped, specifically within the promoters of genes following the induction of transcription. H3 clipping precedes the process of histone eviction seen when genes become fully active. A truncated H3 product is not generated in yeast carrying a mutation of the endopeptidase recognition site (H3 Q19A L20A) and gene induction is defective in these cells. These findings identify clipping of H3 tails as a previously uncharacterized modification of promoter-bound nucleosomes, which may result in the localized clearing of repressive signals during the induction of gene expression. [ABSTRACT FROM AUTHOR]

TNF-related apoptosis-inducing ligand (TRAIL) is a typical member of the tumor necrosis factor (TNF) ligand family that is expressed as a type II membrane protein (memTRAIL) and signals apoptosis via the death domain-containing receptors TRAIL-R1 and -2. Soluble recombinant derivatives of TRAIL (sTRAIL) are considered as novel tumors therapeutics because of their selective apoptosis inducing activity in a variety of human tumors but not in normal cells. Using antagonistic antigen-binding fragment (Fab) preparations of TRAIL-R1- and TRAIL-R2-specific antibodies, we demonstrate in this study that TRAIL-R1 becomes activated by both the soluble and the membrane-bound form of the ligand, whereas TRAIL-R2 becomes only activated by memTRAIL or soluble TRAIL secondarily cross-linked by antibodies. Furthermore, we show that the restricted signal capacity of sTRAIL can be readily converted into a fully signal competent memTRAIL-like molecule, i.e. a TRAIL-R2 stimulating ligand, by genetic fusion to an antibody derivative that allows antigen-dependent ‘immobilization’ of the fusion protein to cell surfaces. We conclude that antibody targeting-dependent activation can be used to design selective therapeutics derived of those ligands of the TNF family that are biologically inactive in their soluble form. Oncogene (2001) 20, 4101–4106. [ABSTRACT FROM AUTHOR]

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition. [Display omitted] • Pol II inhibition induced recruitment of a set of regulatory proteins to chromatin • Many of these changes were also observed upon RNA degradation • RNA binds a set of chromatin modifiers and inhibits interaction with nucleosomes • P-TEFb binds pre-mRNA, and 7SK regulates its interaction with RNA and chromatin Nascent RNA interacts with a wide array of regulatory proteins but its impact on chromatin composition has not been assessed. Skalska and colleagues use proteomics methods to reveal that nascent RNA antagonizes the association of a set of regulatory proteins with chromatin. [ABSTRACT FROM AUTHOR]

DNA replication initiates at genomic locations known as origins of replication, which, in S. cerevisiae , share a common DNA consensus motif. Despite being virtually nucleosome-free, origins of replication are greatly influenced by the surrounding chromatin state. Here, we show that histone H3 lysine 37 mono-methylation (H3K37me1) is catalyzed by Set1p and Set2p and that it regulates replication origin licensing. H3K37me1 is uniformly distributed throughout most of the genome, but it is scarce at replication origins, where it increases according to the timing of their firing. We find that H3K37me1 hinders Mcm2 interaction with chromatin, maintaining low levels of MCM outside of conventional replication origins. Lack of H3K37me1 results in defective DNA replication from canonical origins while promoting replication events at inefficient and non-canonical sites. Collectively, our results indicate that H3K37me1 ensures correct execution of the DNA replication program by protecting the genome from inappropriate origin licensing and spurious DNA replication. [Display omitted] • Set1p and Set2p mono-methylate H3K37 • H3K37me1 hinders MCM replicative helicase interaction with chromatin • Low levels of H3K37me1 at replication origins facilitate origin establishment • H3K37me1 abrogation results in MCM recruitment and licensing of non-replicative ACS Genomes are the template for DNA replication and transcription, which timely coincide. To minimize detrimental collisions between both molecular machineries, only a subset of all replication origins performs as such. Santos-Rosa et al. show that the histone H3K37me1 prevents the establishment of unconventional origins, protecting the genome from spurious replication events. [ABSTRACT FROM AUTHOR]

Summary The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1; however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays, and recombinant chromatin substrates, we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the "backside" of the E2 to stabilize the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation. Graphical Abstract Highlights • The UBL domain of UHRF1 is required for its E3 ubiquitin ligase activity • A hydrophobic patch on the UBL is required to form a stable E2/E3/chromatin complex • The UHRF1 N terminus and UBL hydrophobic patch control targeted H3 ubiquitylation • DNMT1-mediated maintenance DNA methylation requires the UBL hydrophobic patch Foster et al. identify a functional role for the ubiquitin-like domain of UHRF1 in its E3 ubiquitin ligase activity. Biochemical and cell-based assays reveal that a hydrophobic patch on the UBL domain controls targeted histone H3 ubiquitylation that is required for DNMT1 recruitment to newly replicated chromatin and subsequent maintenance of DNA methylation. [ABSTRACT FROM AUTHOR]