Chromatin-associated Proteins Research Articles

LS1 PRACTICAL APPLICATION AND UNDERLYING BIOLOGY OF TUMOR TREATING FIELDS

Glioblastoma (GBM) is the most common and most aggressive form of brain cancer in adults. For decades, the mainstay of therapeutic intervention was based on surgical resection (when safely feasible), followed by radiotherapy (RT). In 2005, data from the landmark EORTC-NCIC trial changed the standard of care treatment for GBM. This phase III trial demonstrated a survival advantage for concomitant and adjuvant temozolomide (TMZ) chemotherapy when added to the standard course of radiation. In the group of patients assigned to radiation plus TMZ, median survival improved from 12.1 months (radiotherapy alone) to 14.6 months, but all the patient finally relapsed on TMZ. In 2015, Tumor Treating Fields (TTF, Optune) became the first FDA-approved device for the treatment of for newly diagnosed GBM. This approval was based on the EF-14 clinical trial results, in which nearly half of the patients treated with Optune in combination with maintenance temozolomide (TMZ) were alive at 2 years compared with 31% of people on TMZ alone.Optune utilize the natural electrical properties of dividing cancer cells to disrupt mitosis and provide continuous antimitotic action against progression of GBM. TTF-treated tumor cells can exit the process of mitosis aberrantly and release cellular stress signals, such as the endoplasmic reticulum chaperonin calreticulin (CRT) and high mobility group box 1 protein (HMGB1). CRT is important to induce antitumor immune responses because CRT inhibition decreases immunogenicity. HMGB1, an endogenous chromatin-associated protein released from dying tumor cells, also plays a critical role in the activation of HMGB1-mediated toll-like receptor 2 (TLR2) immune signaling. The presence of those signals may facilitate immune activation, and immunogenic induced cell death, and eventually result in tumor destruction.In this presentation, Daniela Bota, MD, PhD, will review the clinical results of Optune in the treatment of GBM, and will discuss the novel biological mechanisms underlying the effects of Optune in controlling tumor growth and promoting the immune responses in GBM.

Formin Links Erythroid Cytoskeleton to Organelle Clearance through Escrt-III Complex during Reticulocyte Maturation

The final stages of mammalian terminal erythropoiesis involve cell cycle exit of orthochromatic erythroblast, enucleation of the condensed nucleus, and organelle clearance of the nascent reticulocytes. Although many critical factors in these processes have been discovered over the past years, several key questions remain unanswered. For example, what are the factors that regulate the exit of the last mitosis of erythroblast for enucleation? How does the nascent reticulocyte separate from the extruded nucleus? What are the signals involved in regulating the clearance of organelles in reticulocyte? Answers to these questions with mechanistic insights are important not only for our understanding of the basic biology of terminal erythropoiesis and pathophysiology of many red cell-related diseases, but also to provide clues for efficient strategies for in vitro or ex vivo generation of red blood cells in transfusion medicine. Our work on formin family proteins, enzymes involved in linear actin filament polymerization, in erythropoiesis may shed light on the clues to these questions. We show in our published work that mDia2, one of the diaphanous-related formins, plays critical roles in enucleation and cytokinesis of erythroblasts. However, the mechanism of how mDia2 regulates these processes is unclear. In this study, we used mDia2 hematopoietic-specific knockout mouse model and revealed that mDia2 controls the motility of the nascent reticulocyte that is required for the detachment of the pyknotic nucleus. Reticulocytes in mDia2 deficient mice are rigid with extended spectrin chains, possibly due to disrupted actin protofilaments. Indeed, a stochastic optical reconstruction (STORM) high resolution microscopy analysis revealed that actin protofilaments were completely disrupted with loss of mDia2 in the reticulocytes. Using immuno-gold stain and electron microscopy, we further found that mDia2 localized at the junctional complex, confirming its critical role in the polymerization of actin protofilaments and maintenance of erythroid cytoskeleton. In addition to the cytoskeleton defects, reticulocytes from mDia2 deficient mice also showed enlarged volume with many organelles failed to be eliminated. Flow cytometry analyses showed that several membrane proteins destined to be downregulated, as well as mitochondria and lysosome markers, remained high in mDia2 deficient reticulocytes. We also performed a tandem mass tagging (TMT) mass spectrometry, which revealed numerous chromatin-associated proteins that failed to be downregulated. Together, these results demonstrated an important role of mDia2 in the reticulocyte maturation. The erythroid phenotypes in mDia2 deficient mice, including failure of cytokinesis and organelle clearance, prompted us to investigate whether there are any defects in ESCRT complexes that are cellular components essential for these processes. Indeed, several ESCRT III complex and associated proteins, including Chmp5, Vta1, and Usp8, were significantly downregulated. mDia2 is known to function though actin polymerization to influence the transcriptional activity of SRF. We found that Chmp5 was a novel target of SRF through ChIP assay. Transplantation of mDia2 knockout c-kit positive progenitors transduced with Chmp5 into the lethally irradiated WT recipient mice dramatically reduced the percentages of bi-nuclear erythroblasts and reverted anemia. Consistent with phenotypes of Chmp5 knockout cells where increased late endosome and lysosome are commonly found, markers for late stage endosome and lysosome were significantly increased in mDia2 deficient erythroblasts and reticulocytes. These data support that mDia2 regulates endosome/MVB and lysosome discharge through Chmp5 during reticulocyte maturation. More importantly, overexpression of Chmp5 largely rescued the defects in lysosome and mitochondria clearance in mDia2 deficient reticulocytes. Our study reveals mDia2 formin as a master regulator of the late stage terminal erythropoiesis in the maintenance of erythroid cytoskeleton and organelle clearance. The novel mDia2-SRF-ESCRT III complex pathway provides the first signaling axis that connects erythroid cytoskeleton to reticulocyte maturation, which may open a new field in signaling networks that modulate enucleation to reticulocyte formation. Disclosures Ji: Longbiopharma: Consultancy.

A Functional Link between Nuclear RNA Decay and Transcriptional Control Mediated by the Polycomb Repressive Complex 2

SummaryPluripotent embryonic stem cells (ESCs) constitute an essential cellular niche sustained by epigenomic and transcriptional regulation. Any role of post-transcriptional processes remains less explored. Here, we identify a link between nuclear RNA levels, regulated by the poly(A) RNA exosome targeting (PAXT) connection, and transcriptional control by the polycomb repressive complex 2 (PRC2). Knockout of the PAXT component ZFC3H1 impairs mouse ESC differentiation. In addition to the upregulation of bona fide PAXT substrates, Zfc3h1−/− cells abnormally express developmental genes usually repressed by PRC2. Such de-repression is paralleled by decreased PRC2 binding to chromatin and low PRC2-directed H3K27 methylation. PRC2 complex stability is compromised in Zfc3h1−/− cells with elevated levels of unspecific RNA bound to PRC2 components. We propose that excess RNA hampers PRC2 function through its sequestration from DNA. Our results highlight the importance of balancing nuclear RNA levels and demonstrate the capacity of bulk RNA to regulate chromatin-associated proteins.

In silico design and molecular basis for the selectivity of Olinone toward the first over the second bromodomain of BRD4.

Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem BrD containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work, we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydropyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations, and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Val49 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity toward BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of small-molecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically improved derivatives.

Evidence That Ion-Based Signaling Initiating at the Cell Surface Can Potentially Influence Chromatin Dynamics and Chromatin-Bound Proteins in the Nucleus.

We have developed tools and performed pilot experiments to test the hypothesis that an intracellular ion-based signaling pathway, provoked by an extracellular stimulus acting at the cell surface, can influence interphase chromosome dynamics and chromatin-bound proteins in the nucleus. The experimental system employs chromosome-specific fluorescent tags and the genome-encoded fluorescent pH sensor SEpHluorinA227D, which has been targeted to various intracellular membranes and soluble compartments in root cells of Arabidopsis thaliana. We are using this system and three-dimensional live cell imaging to visualize whether fluorescent-tagged interphase chromosome sites undergo changes in constrained motion concurrently with reductions in membrane-associated pH elicited by extracellular ATP, which is known to trigger a cascade of events in plant cells including changes in calcium ion concentrations, pH, and membrane potential. To examine possible effects of the proposed ion-based signaling pathway directly at the chromatin level, we generated a pH-sensitive fluorescent DNA-binding protein that allows pH changes to be monitored at specific genomic sites. Results obtained using these tools support the existence of a rapid, ion-based signaling pathway that initiates at the cell surface and reaches the nucleus to induce alterations in interphase chromatin mobility and the surrounding pH of chromatin-bound proteins. Such a pathway could conceivably act under natural circumstances to allow external stimuli to swiftly influence gene expression by affecting interphase chromosome movement and the structures and/or activities of chromatin-associated proteins.

Genome-Wide Profiling of Histone Modifications with ChIP-Seq.

Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) is a widely used method for mapping the genome-wide locations of chromatin-associated proteins. This protocol has been developed and utilized to perform ChIP on histone covalent modifications in various plant species including cereals. DNA and chromatin-associated proteins are crosslinked with formaldehyde. Chromatin is then isolated from nuclei and sheared via sonication. Antibodies targeting the histone modification of interest are incubated with the sheared chromatin and nonspecific interactions are washed away. DNA is purified via phenol-chloroform extraction, end-repaired, ligated to sequencing adapters, and PCR-amplified.

CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis

We introduce CUT&RUNTools as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short-read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. CUT&RUNTools is available at https://bitbucket.org/qzhudfci/cutruntools/.

Association rule mining to identify transcription factor interactions in genomic regions.

Genome regulatory networks have different layers and ways to modulate cellular processes, such as cell differentiation, proliferation, and adaptation to external stimuli. Transcription factors and other chromatin-associated proteins act as combinatorial protein complexes that control gene transcription. Thus, identifying functional interaction networks among these proteins is a fundamental task to understand the genome regulation framework. We developed a novel approach to infer interactions among transcription factors in user-selected genomic regions, by combining the computation of association rules and of a novel Importance Index on ChIP-seq datasets. The hallmark of our method is the definition of the Importance Index, which provides a relevance measure of the interaction among transcription factors found associated in the computed rules. Examples on synthetic data explain the index use and potential. A straightforward pre-processing pipeline enables the easy extraction of input data for our approach from any set of ChIP-seq experiments. Applications on ENCODE ChIP-seq data prove that our approach can reliably detect interactions between transcription factors, including known interactions that validate our approach. A R/Bioconductor package implementing our association rules and Importance Index-based method is available at http://bioconductor.org/packages/release/bioc/html/TFARM.html. Supplementary data are available at Bioinformatics online.

Nuclei Isolation Staining (NIS) Method for Imaging Chromatin-Associated Proteins in Difficult Cell Types.

Spatial distribution of chromatin-associated proteins provides invaluable information for understanding gene regulation. Conventional immunostaining is widely used for labeling chromatin-associated proteins in many cell types. However, for a subset of difficult cell types, such as differentiated human keratinocytes, achieving high-quality immunostaining for nuclear proteins remains challenging. To overcome this technical barrier, we developed the nuclei isolation staining (NIS) method. In brief, NIS involves rapid isolation of nuclei from live cells, followed by fixation and staining of the nuclei directly on coverslips for subsequent high-magnification imaging. By removing the cytoplasmic contents and staining just the nuclei, this NIS method drastically improves antibody labeling efficiency for chromatin-associated proteins. In this article, we describe the development and a step-by-step protocol of NIS, using differentiated human keratinocytes as an example. We also discuss other applications, based on the principle of this NIS method, for understanding cell-type and cell-state specific gene regulation. © 2019 by John Wiley & Sons, Inc.

Generation of CRISPR-cas9 construct for knockout of genes encoding chromatin-associated proteins

BackgroundEukaryotic genomes have a multiscale three-dimensional organization varying from nucleosomes, loops, topologically associating domains, and chromosome territories. Chromatin, DNA wrapped around histone proteins, helps in packaging long DNA within tiny nuclear spaces. We used CRISPR-cas9, which is a system of single-protein and single-guide RNAs for genome engineering and also is simple and target specific.MethodTwo major protein families involved in maintaining and regulating structure and dynamics of chromatin are trithorax group (TrxG) and polycomb group (PcG) proteins. This study was undertaken to generate knockout cell lines of some TrxG and PcG proteins using the CRISPR-based approach in order to study their role in higher order chromatin organization.ResultsFrom TrxG, ISWI and Acf were selected, and from PcG, Pc and Psc were selected. Three pAc-sgRNA-Cas9-puro-vector constructs for ISWI gene, one pAc-sgRNA-cas9-puro-vector construct for Pc, gene and two pAc-sgRNA-cas9-puro-vector constructs for each of the Acf and Psc gene were generated. These constructs were confirmed by PCR and sequencing.ConclusionIn the future, these constructs will be used to study the role of their respective target genes in chromatin organization.

A novel yeast chromatin-enriched fractions purification approach, yChEFs, for the chromatin-associated protein analysis used for chromatin-associated and RNA-dependent chromatin-associated proteome studies from Saccharomyces cerevisiae

We adapted and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. yChEFs allows the easy, reproducible and scalable recovery of proteins associated with chromatin, and bypasses subcellular fractionation requirements that involve using zymolyase to obtain the spheroplast. The use of a small amount of culture cells and small volumes of solutions during the procedure greatly facilitates high-enriched chromatin purification, which is very useful when many samples need to be manipulated. It also reduces costs and efforts. yChEFs can be combined with mass spectrometry for proteomic analyses. We used yChEFs and mass spectrometry to identify the chromatin-associated proteome in Saccharomyces cerevisiae, and detected about 750 chromatin-bound proteins, many of which can be detected by other previous approaches. We also used this methodology to identify the RNA-dependent chromatin-associated proteome and identified about 500 proteins, including: RNA polymerase subunits, transcription regulators, such as helicases Sen1 and Rat1 that participate in transcription termination, components of nucleosome modifying complexes, chromatin remodelers complexes, and some proteins involved in RNA processing, among them, Dcp2, Xrn1 and Dhh1, which act in mRNA processing. Finally, yChEFs identified weak or transient chromatin-associated proteins.

Nuclear Organization in Stress and Aging.

The eukaryotic nucleus controls most cellular processes. It is isolated from the cytoplasm by the nuclear envelope, which plays a prominent role in the structural organization of the cell, including nucleocytoplasmic communication, chromatin positioning, and gene expression. Alterations in nuclear composition and function are eminently pronounced upon stress and during premature and physiological aging. These alterations are often accompanied by epigenetic changes in histone modifications. We review, here, the role of nuclear envelope proteins and histone modifiers in the 3-dimensional organization of the genome and the implications for gene expression. In particular, we focus on the nuclear lamins and the chromatin-associated protein BAF, which are linked to Hutchinson–Gilford and Nestor–Guillermo progeria syndromes, respectively. We also discuss alterations in nuclear organization and the epigenetic landscapes during normal aging and various stress conditions, ranging from yeast to humans.

Multifunctional transcriptional coactivator PC4 is a global co-regulator of p53-dependent stress response and gene regulation.

Human positive coactivator 4 (PC4), a multifunctional chromatin-associated protein, is known to directly interact with p53 and modulate expressions of a few p53-dependent genes. However, the role of PC4 in p53's myriad of other regulatory functions is not known. The p53-PC4 interaction was selectively perturbed by a small peptide which led to abrogation of genotoxic stress-induced up-regulation of many p53-dependent genes and reduction of apoptosis in A549 cells. Over-expression of a PC4 point mutant, incapable of binding p53, recapitulated many of the effects of the peptide. Global gene expression profiling in A549 cells, upon peptide treatment, revealed PC4's involvement in the regulation of many p53-dependent pathways, including the Hippo pathway. Introduction of the peptide in neuronal cells significantly reduced its amyloid-β-induced death. Thus, PC4 emerges as a global co-regulator of p53 and a therapeutic target against pathogeneses where the p53-dependent cell death process plays a crucial role.

Modelling and DNA topology of compact 2-start and 1-start chromatin fibres.

We have investigated the structure of the most compact 30-nm chromatin fibres by modelling those with 2-start or 1-start crossed-linker organisations. Using an iterative procedure we obtained possible structural solutions for fibres of the highest possible compaction permitted by physical constraints, including the helical repeat of linker DNA. We find that this procedure predicts a quantized nucleosome repeat length (NRL) and that only fibres with longer NRLs (≥197 bp) can more likely adopt the 1-start organisation. The transition from 2-start to 1-start fibres is consistent with reported differing binding modes of the linker histone. We also calculate that in 1-start fibres the DNA constrains more torsion (as writhe) than 2-start fibres with the same NRL and that the maximum constraint obtained is in accord with previous experimental results. We posit that the coiling of the fibre is driven by overtwisting of linker DNA which, in the most compact forms - for example, in echinoderm sperm and avian erythrocytes - could adopt a helical repeat of ∼10 bp/turn. We argue that in vivo the total twist of linker DNA could be modulated by interaction with other abundant chromatin-associated proteins and by epigenetic modifications of the C-terminal tail of linker histones.

Can Epigenetics of Endothelial Dysfunction Represent the Key to Precision Medicine in Type 2 Diabetes Mellitus?

In both developing and industrialized Countries, the growing prevalence of Type 2 Diabetes Mellitus (T2DM) and the severity of its related complications make T2DM one of the most challenging metabolic diseases worldwide. The close relationship between genetic and environmental factors suggests that eating habits and unhealthy lifestyles may significantly affect metabolic pathways, resulting in dynamic modifications of chromatin-associated proteins and homeostatic transcriptional responses involved in the progression of T2DM. Epigenetic mechanisms may be implicated in the complex processes linking environmental factors to genetic predisposition to metabolic disturbances, leading to obesity and type 2 diabetes mellitus (T2DM). Endothelial dysfunction represents an earlier marker and an important player in the development of this disease. Dysregulation of the endothelial ability to produce and release vasoactive mediators is recognized as the initial feature of impaired vascular activity under obesity and other insulin resistance conditions and undoubtedly concurs to the accelerated progression of atherosclerotic lesions and overall cardiovascular risk in T2DM patients. This review aims to summarize the most current knowledge regarding the involvement of epigenetic changes associated with endothelial dysfunction in T2DM, in order to identify potential targets that might contribute to pursuing “precision medicine” in the context of diabetic illness.

Pinpointing the Genomic Localizations of Chromatin-Associated Proteins: The Yesterday, Today, and Tomorrow of ChIP-seq.

Chromatin-associated proteins are instrumental for controlling spatiotemporal gene expression. Determining where these proteins bind across the genome is critical for understanding gene regulation. A widely used technique at present is ChIP-seq, which leverages chromatin fragmentation, antibody-mediated enrichment, next-generation sequencing, and data analysis to uncover the genomic sequences and patterns of protein-DNA interactions. In this article, we will provide an overview of how ChIP-seq was developed, the key elements of the experimentation and data analysis pipeline, and the recent variations that push the boundaries of precision and cell number requirements. We will also briefly discuss how future development of ChIP-seq may further advance our understanding of chromatin biology. © 2019 by John Wiley & Sons, Inc.

RNA Targets Ribogenesis Factor WDR43 to Chromatin for Transcription and Pluripotency Control.

Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as achromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of theelongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects inPol II release and pluripotency-associated geneexpression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.

HCF-2 inhibits cell proliferation and activates differentiation-gene expression programs.

HCF-2 is a member of the host-cell-factor protein family, which arose in early vertebrate evolution as a result of gene duplication. Whereas its paralog, HCF-1, is known to act as a versatile chromatin-associated protein required for cell proliferation and differentiation, much less is known about HCF-2. Here, we show that HCF-2 is broadly present in human and mouse cells, and possesses activities distinct from HCF-1. Unlike HCF-1, which is excluded from nucleoli, HCF-2 is nucleolar—an activity conferred by one and a half C-terminal Fibronectin type 3 repeats and inhibited by the HCF-1 nuclear localization signal. Elevated HCF-2 synthesis in HEK-293 cells results in phenotypes reminiscent of HCF-1-depleted cells, including inhibition of cell proliferation and mitotic defects. Furthermore, increased HCF-2 levels in HEK-293 cells lead to inhibition of cell proliferation and metabolism gene-expression programs with parallel activation of differentiation and morphogenesis gene-expression programs. Thus, the HCF ancestor appears to have evolved into a small two-member protein family possessing contrasting nuclear versus nucleolar localization, and cell proliferation and differentiation functions.

HMGB proteins are required for sexual development in Aspergillus nidulans.

Aspergillus nidulans has three high mobility group box (HMGB) proteins, HmbA, HmbB and HmbC that are chromatin-associated architectural proteins involved in DNA-related functions. By creating and studying deletion strains in both veA+ and veA1 background, we have characterized the role of HmbA, HmbB and HmbC in sexual development. Expression of the mating-type MAT1-1 and MAT1-2 coding genes were found to be extremely down-regulated in all three mutants on day 4 of sexual development, which results in deficient ascospore production and/or ascospore viability in the mutants. In addition, we found that HmbA and HmbB play also a role in sensing of and response to environmental signals, while HmbC functionally interacts with VeA, a key regulator of the coordination of asexual and sexual development, as well as of secondary metabolism.

Drugging transcription in heart failure.

Advances in our understanding of the basic biology and biochemistry of chromatin structure and function at genome scales has led to tremendous growth in the fields of epigenomics and transcriptional biology. While it has long been appreciated that transcriptional pathways are dysregulated in failing hearts, only recently has the idea of disrupting altered transcription by targeting chromatin-associated proteins been explored. Here, we provide a brief overview of efforts to drug transcription in the context of heart failure, focusing on the bromo- and extra-terminal domain (BET) family of chromatin co-activator proteins.