Chromatin-associated Proteins Research Articles

DNA damage and oxidative stress in human cells infected by Trypanosoma cruzi.

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. Infected cells with T. cruzi activate several responses that promote unbalance of reactive oxygen species (ROS) that may cause DNA damage that activate cellular responses including DNA repair processes. In this work, HeLa cells and AC16 human cardiomyocyte cell line were infected with T. cruzi to investigate host cell responses at genome level during parasites intracellular life cycle. In fact, alkaline sensitive sites and oxidized DNA bases were detected in the host cell genetic material particularly in early stages of infection. These DNA lesions were accompanied by phosphorylation of the histone H2Ax, inducing γH2Ax, a marker of genotoxic stress. Moreover, Poly [ADP-ribose] polymerase-1 (PARP1) and 8-oxoguanine glycosylase (OGG1) are recruited to host cell nuclei, indicating activation of the DNA repair process. In infected cells, chromatin-associated proteins are carbonylated, as a possible consequence of oxidative stress and the nuclear factor erythroid 2–related factor 2 (NRF2) is induced early after infection, suggesting that the host cell antioxidant defenses are activated. However, at late stages of infection, NRF2 is downregulated. Interestingly, host cells treated with glutathione precursor, N-acetyl cysteine, NRF2 activator (Sulforaphane), and also Benznidonazol (BNZ) reduce parasite burst significantly, and DNA damage. These data indicate that the balance of oxidative stress and DNA damage induction in host cells may play a role during the process of infection itself, and interference in these processes may hamper T. cruzi infection, revealing potential target pathways for the therapy support.

Critical role of high-mobility-group proteins in kidney development/cross-talk Wnt/β-catenin signaling pathway

recovery from critical illness.The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and reduced in the corresponding adult tissues. We used used in study C57BL/6, HMG+/− and HMG−/− mice and found that HMG is expressed in the mouse embryonic kidney at the cortex area. HMG knockout led to enhanced Wnt/β-catenin signaling pathway. Analysis of siRNA-mediated loss-of-function experiments in embryonic kidney culture confirmed the role of HMG as a key regulator of cortex epithelium differentiation.

Critical role of high-mobility-group proteins in kidney development/cross-talk Wnt/β-catenin signaling pathway

The treatment of severe acute kidney injury with dialytic support for renal replacement therapy can be life sustaining and permit recovery from critical illness. The high-mobility-group (HMG) proteins are the most abundant non-histone chromatin-associated proteins. HMG proteins are present at high levels in various undifferentiated tissues during embryonic development and reduced in the corresponding adult tissues. We used used in study C57BL/6, HMG+/− and HMG−/− mice and found that HMG is expressed in the mouse embryonic kidney at the cortex area. HMG knockout led to enhanced Wnt/β-catenin signaling pathway. Analysis of siRNA-mediated loss-of-function experiments in embryonic kidney culture confirmed the role of HMG as a key regulator of cortex epithelium differentiation.

Methylation-dependent SUMOylation of the architectural transcription factor HMGA2

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K66KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function.

'Enhancing' red cell fate through epigenetic mechanisms.

Transcription of erythroid-specific genes is regulated by the three-dimensional (3D) structure and composition of chromatin, which dynamically changes during erythroid differentiation. Chromatin organization and dynamics are regulated by several epigenetic mechanisms involving DNA (de-)methylation, posttranslational modifications (PTMs) of histones, chromatin-associated structural proteins, and higher-order structural changes and interactions. This review addresses examples of recent developments in several areas delineating the interface of chromatin regulation and erythroid-specific lineage transcription. We survey and discuss recent studies that focus on the erythroid chromatin landscape, erythroid enhancer-promotor interactions, super-enhancer functionality, the role of chromatin modifiers and epigenetic crosstalk, as well as the progress in mapping red blood cell (RBC) trait-associated genetic variants within cis-regulatory elements (CREs) identified in genome-wide association study (GWAS) efforts as a step toward determining their impact on erythroid-specific gene expression. As one of the best characterized and accessible cell differentiation systems, erythropoiesis has been at the forefront of studies aiming to conceptualize how chromatin dynamics regulate transcription. New emerging technologies that bring a significantly enhanced spatial and temporal resolution of chromatin structure, and allow investigation of small cell numbers, have advanced our understanding of chromatin dynamics during erythroid differentiation in vivo.

JASMONATE-ZIM DOMAIN proteins engage Polycomb chromatin modifiers to modulate Jasmonate signaling in Arabidopsis

Jasmonate (JA) regulates various aspects of plant growth and development and stress responses, with prominent roles in male reproductive development and defenses against herbivores and necrotrophic pathogens. JASMONATE-ZIM DOMAIN (JAZ) proteins are key regulators in the JA signaling pathway and function to repress the expression of JA-responsive genes. Here, we show that JAZ proteins directly interact with several chromatin-associated Polycomb proteins to mediate repressive chromatin modifications at JA-responsive genes and, thus, their transcriptional repression in Arabidopsis. Genetic analyses revealed that the developmental defects, including anther and pollen abnormalities, resulting from loss or block of JA signaling were partially rescued by loss of Polycomb protein-mediated chromatin silencing (Polycomb repression). We further found that JAZ-mediated transcriptional repression during anther and pollen development requires Polycomb proteins at four key regulatory loci. Analysis of genome-wide occupancy of a Polycomb factor and transcriptome reprogramming in response to JA revealed that Polycomb repression is involved in the repression of various JA-responsive genes. Taken together, our study reveals an important chromatin-based mechanism for JAZ-mediated transcriptional repression and JA signaling in plants.

Chromatin-Associated Protein Complexes Link DNA Base J and Transcription Termination in Leishmania.

ABSTRACTUnlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on posttranscriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-d-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP) tagging and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in the regulation of transcription in other eukaryotes. The downregulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional readthrough at the 3′ end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt the progression of the RNA polymerase. This decoupling of transcription termination from the splicing of individual genes enables the parasites’ unique reliance on polycistronic transcription and posttranscriptional regulation of gene expression.IMPORTANCE Leishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent of those involved in the control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.

Positioning of nucleosomes containing \u03b3-H2AX precedes active DNA demethylation and transcription initiation

In addition to nucleosomes, chromatin contains non-histone chromatin-associated proteins, of which the high-mobility group proteins are the most abundant. Chromatin-mediated regulation of transcription involves DNA methylation and histone modifications. However, the order of events and the precise function of high-mobility group proteins during transcription initiation remain unclear. Here we show that high-mobility group AT-hook 2 protein (HMGA2) induces DNA nicks at the transcription start site, which are required by the histone chaperone FACT complex to incorporate nucleosomes containing the histone variant H2A.X. Further, phosphorylation of H2A.X at S139 (γ-H2AX) is required for repair-mediated DNA demethylation and transcription activation. The relevance of these findings is demonstrated within the context of TGFB1 signaling and idiopathic pulmonary fibrosis, suggesting therapies against this lethal disease. Our data support the concept that chromatin opening during transcriptional initiation involves intermediates with DNA breaks that subsequently require DNA repair mechanisms to ensure genome integrity.

Influence of the disordered domain structure of MeCP2 on its structural stability and dsDNA interaction

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator and a chromatin-associated structural protein. MeCP2 deregulation results in two neurodevelopmental disorders: MeCP2 dysfunction is associated with Rett syndrome, while excess of activity is associated with MeCP2 duplication syndrome. MeCP2 is an intrinsically disordered protein (IDP) constituted by six structural domains with variable, small percentage of well-defined secondary structure. Two domains, methyl-CpG binding domain (MBD) and transcription repressor domain (TRD), are the elements responsible for dsDNA binding ability and recruitment of the gene transcription/silencing machinery, respectively. Previously we studied the influence of the completely disordered, MBD-flanking domains (N-terminal domain, NTD, and intervening domain, ID) on the structural and functional features of the MBD (Claveria-Gimeno, R. et al. Sci Rep. 2017, 7, 41,635). Here we report the biophysical study of the influence of the remaining domains (transcriptional repressor domain, TRD, and C-terminal domains, CTDα and CTDβ) on the structural stability of MBD and the dsDNA binding capabilities of MBD and ID. The influence of distant disordered domains on MBD properties makes it necessary to consider the NTD-MBD-ID variant as the minimal protein construct for studying dsDNA/chromatin binding properties, while the full-length protein should be considered for transcriptional regulation studies.

SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites.

Glucocorticoid receptor (GR) is an essential transcription factor (TF), controlling metabolism, development and immune responses. SUMOylation regulates chromatin occupancy and target gene expression of GR in a locus-selective manner, but the mechanism of regulation has remained elusive. Here, we identify the protein network around chromatin-bound GR by using selective isolation of chromatin-associated proteins and show that the network is affected by receptor SUMOylation, with several nuclear receptor coregulators and chromatin modifiers preferring interaction with SUMOylation-deficient GR and proteins implicated in transcriptional repression preferring interaction with SUMOylation-competent GR. This difference is reflected in our chromatin binding, chromatin accessibility and gene expression data, showing that the SUMOylation-deficient GR is more potent in binding and opening chromatin at glucocorticoid-regulated enhancers and inducing expression of target loci. Blockage of SUMOylation by a SUMO-activating enzyme inhibitor (ML-792) phenocopied to a large extent the consequences of GR SUMOylation deficiency on chromatin binding and target gene expression. Our results thus show that SUMOylation modulates the specificity of GR by regulating its chromatin protein network and accessibility at GR-bound enhancers. We speculate that many other SUMOylated TFs utilize a similar regulatory mechanism.

TrIPP—a method for tracking the inheritance patterns of proteins in living cells—reveals retention of Tup1p, Fpr4p, and Rpd3L in the mother cell

SummaryInheritance of chromatin-bound proteins theoretically plays a role in the epigenetic transmission of cellular phenotypes. Protein segregation during cell division is however poorly understood. We now describe TrIPP (Tracking the Inheritance Patterns of Proteins): a live cell imaging method for tracking maternal proteins during asymmetric cell divisions of budding yeast. Our analysis of the partitioning pattern of a test set of 18 chromatin-associated proteins reveals that abundant and moderately abundant maternal proteins segregate stochastically and symmetrically between the two cells with the exception of Rxt3p, Fpr4p, and Tup1p, which are preferentially retained in the mother. Low abundance proteins also tend to be retained in the mother cell with the exception of Sir2p and the linker histone H1. Our analysis of chromatin protein behavior in single cells reveals potentially general trends such as coupled protein synthesis and decay and a correlation between protein half-lives and cell-cycle duration.

A short ORF-encoded transcriptional regulator

Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Identification of SUMO Targets Associated With the Pluripotent State in Human Stem Cells

To investigate the role of SUMO modification in the maintenance of pluripotent stem cells, we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 resulted in the loss of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with C-terminal TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created three networks of proteins with functions in regulation of gene expression, ribosome biogenesis, and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The rest have roles in transcription and the regulation of chromatin structure. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centered on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins, there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO influences the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

Impact of WIN site inhibitor on the WDR5 interactome

SUMMARYThe chromatin-associated protein WDR5 is a promising pharmacological target in cancer, with most drug discovery efforts directed against an arginine-binding cavity in WDR5 called the WIN site. Despite a clear expectation that WIN site inhibitors will alter the repertoire of WDR5 interaction partners, their impact on the WDR5 interactome remains unknown. Here, we use quantitative proteomics to delineate how the WDR5 interactome is changed by WIN site inhibition. We show that the WIN site inhibitor alters the interaction of WDR5 with dozens of proteins, including those linked to phosphatidylinositol 3-kinase (PI3K) signaling. As proof of concept, we demonstrate that the master kinase PDPK1 is a bona fide high-affinity WIN site binding protein that engages WDR5 to modulate transcription of genes expressed in the G2 phase of the cell cycle. This dataset expands our understanding of WDR5 and serves as a resource for deciphering the action of WIN site inhibitors.

Targeting Chromatin Complexes in Myeloid Malignancies and Beyond: From Basic Mechanisms to Clinical Innovation.

The aberrant function of chromatin regulatory networks (epigenetics) is a hallmark of cancer promoting oncogenic gene expression. A growing body of evidence suggests that the disruption of specific chromatin-associated protein complexes has therapeutic potential in malignant conditions, particularly those that are driven by aberrant chromatin modifiers. Of note, a number of enzymatic inhibitors that block the catalytic function of histone modifying enzymes have been established and entered clinical trials. Unfortunately, many of these molecules do not have potent single-agent activity. One potential explanation for this phenomenon is the fact that those drugs do not profoundly disrupt the integrity of the aberrant network of multiprotein complexes on chromatin. Recent advances in drug development have led to the establishment of novel inhibitors of protein–protein interactions as well as targeted protein degraders that may provide inroads to longstanding effort to physically disrupt oncogenic multiprotein complexes on chromatin. In this review, we summarize some of the current concepts on the role epigenetic modifiers in malignant chromatin states with a specific focus on myeloid malignancies and recent advances in early-phase clinical trials.

Novel ChIP-seq simulating program with superior versatility: isChIP.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is recognized as an extremely powerful tool to study the interaction of numerous transcription factors and other chromatin-associated proteins with DNA. The core problem in the optimization of ChIP-seq protocol and the following computational data analysis is that a 'true' pattern of binding events for a given protein factor is unknown. Computer simulation of the ChIP-seq process based on 'a-priory known binding template' can contribute to a drastically reduce the number of wet lab experiments and finally help achieve radical optimization of the entire processing pipeline. We present a newly developed ChIP-sequencing simulation algorithm implemented in the novel software, in silico ChIP-seq (isChIP). We demonstrate that isChIP closely approximates real ChIP-seq protocols and is able to model data similar to those obtained from experimental sequencing. We validated isChIP using publicly available datasets generated for well-characterized transcription factors Oct4 and Sox2. Although the novel software is compatible with the Illumina protocols by default, it can also successfully perform simulations with a number of alternative sequencing platforms such as Roche454, Ion Torrent and SOLiD as well as model ChIP -Exo. The versatility of isChIP was demonstrated through modelling a wide range of binding events, including those of transcription factors and chromatin modifiers. We also performed a comparative analysis against a few existing ChIP-seq simulators and showed the fundamental superiority of our model. Due to its ability to utilize known binding templates, isChIP can potentially be employed to help investigators choose the most appropriate analytical software through benchmarking of available ChIP-seq programs and optimize the experimental parameters of ChIP-seq protocol. isChIP software is freely available at https://github.com/fnaumenko/isChIP.

G-Quadruplex loops regulate PARP-1 enzymatic activation.

G-Quadruplexes are non-B form DNA structures present at regulatory regions in the genome, such as promoters of proto-oncogenes and telomeres. The prominence in such sites suggests G-quadruplexes serve an important regulatory role in the cell. Indeed, oxidized G-quadruplexes found at regulatory sites are regarded as epigenetic elements and are associated with an interlinking of DNA repair and transcription. PARP-1 binds damaged DNA and non-B form DNA, where it covalently modifies repair enzymes or chromatin-associated proteins respectively with poly(ADP-ribose) (PAR). PAR serves as a signal in regulation of transcription, chromatin remodeling, and DNA repair. PARP-1 is known to bind G-quadruplexes with stimulation of enzymatic activity. We show that PARP-1 binds several G-quadruplex structures with nanomolar affinities, but only a subset promote PARP-1 activity. The G-quadruplex forming sequence found in the proto-oncogene c-KIT promoter stimulates enzymatic activity of PARP-1. The loop-forming characteristics of the c-KIT G-quadruplex sequence regulate PARP-1 catalytic activity, whereas eliminating these loop features reduces PARP-1 activity. Oxidized G-quadruplexes that have been suggested to form unique, looped structures stimulate PARP-1 activity. Our results support a functional interaction between PARP-1 and G-quadruplexes. PARP-1 enzymatic activation by G-quadruplexes is dependent on the loop features and the presence of oxidative damage.

CD36 upregulates DEK transcription and promotes cell migration and invasion via GSK-3β/β-catenin-mediated epithelial-to-mesenchymal transition in gastric cancer.

Evidence indicates that the lipid scavenger receptor CD36 has pro-metastatic functions in several cancers. Although CD36 expression correlates with an unfavorable prognosis in gastric cancer (GC), its specific contribution to disease onset, progression, and/or metastasis remains unclear. Using bioinformatics analyses, we ascertained that CD36 expression was increased in metastatic GC specimens in The Cancer Genome Atlas and Gene Expression Omnibus databases and correlated with poor prognosis. In addition, higher CD36 expression was associated with lymph node metastasis (p < 0.05) and poor prognosis (p = 0.030) in 79 Chinese GC patients. Basal CD36 expression levels correlated positively with migration, invasion, and expression of epithelial-to-mesenchymal transition (EMT) markers in GC cell lines, a relationship confirmed by knockdown and overexpression experiments. Importantly, analysis of gene expression changes in CD36-knockdown GC cells led us to identify the chromatin-associated protein DEK as a c-Myc target that mediates activation of the GSK-3β/β-catenin signaling pathway to trigger EMT. These findings further our understanding of the mechanisms governing metastatic dissemination of GC cells and suggest the therapeutic potential of strategies targeting CD36.

Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response

O-GlcNAc modification plays critical roles in regulating the stress response program and cellular homeostasis. However, systematic and multi-omics studies on the O-GlcNAc regulated mechanism have been limited. Here, comprehensive data are obtained by a chemical reporter-based method to survey O-GlcNAc function in human breast cancer cells stimulated with the genotoxic agent adriamycin. We identify 875 genotoxic stress-induced O-GlcNAc chromatin-associated proteins (OCPs), including 88 O-GlcNAc chromatin-associated transcription factors and cofactors (OCTFs), subsequently map their genomic loci, and construct a comprehensive transcriptional reprogramming network. Notably, genotoxicity-induced O-GlcNAc enhances the genome-wide interactions of OCPs with chromatin. The dynamic binding switch of hundreds of OCPs from enhancers to promoters is identified as a crucial feature in the specific transcriptional activation of genes involved in the adaptation of cancer cells to genotoxic stress. The OCTF nuclear respiratory factor 1 (NRF1) is found to be a key response regulator in O-GlcNAc-modulated cellular homeostasis. These results provide a valuable clue suggesting that OCPs act as stress sensors by regulating the expression of various genes to protect cancer cells from genotoxic stress.