Chromatin-associated Complexes Research Articles

Context-defined cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.

The Arabidopsis RRM domain proteins EDM3 and IBM2 coordinate the floral transition and basal immune responses.

The transition from vegetative to reproductive development (floral transition) is a costly process in annual plants requiring increased investments in metabolic resources. The Arabidopsis thaliana (Arabidopsis) PHD finger protein EDM2 and RRM domain proteins EDM3 and IBM2 are known to form chromatin-associated complexes controlling transcript processing. We are reporting that distinct splice isoforms of EDM3 and IBM2 cooperate in the coordination of the floral transition with basal immune responses. These cooperating splice isoforms, termed EDM3L and IBM2L, control the intensity of basal immunity and, via a separate pathway, the timing of the floral transition. During the developmental phase prior to the floral transition expression of EDM3L and IBM2L strongly and gradually increases, while these isoforms simultaneously down-regulate expression of the floral suppressor gene FLC and promote the transition to reproductive growth. At the same time these accumulating EDM3 and IBM2 splice isoforms gradually suppress basal immunity against the virulent Noco2 isolate of the pathogenic oomycete Hyaloperonospora arabidopsidis and down-regulate expression of a set of defense-associated genes and immune receptor genes. We are providing clear evidence for a functional link between the floral transition and basal immunity in the annual plant Arabidopsis. Coordination of these two biological processes, which compete for metabolic resources, is likely critical for plant survival and reproductive success.

Abstract IA020: Therapeutic targeting of MLL complexes in cancer

Abstract Inappropriate expression of genes such as the homeotic (HOX) genes is found in up to 40% of cases of Acute Myelogenous Leukemia (AML). Two well-defined genetic subsets, those with MLL-rearrangements and those with NPM1 mutations, are known to possess high-level expression of HOX and MEIS1 genes and have been shown to be dependent on continued expression of these genes. Recent studies have defined the chromatin-bound protein complexes that maintain this aberrant gene expression. Furthermore, multiple groups including ours have now developed small molecule inhibitors of chromatin-associated complexes. Some of these small molecules, including those targeting DOT1L, EZH2, and Bromodomains are at various stages of clinical development. Another approach to target chromatin associated complexes is via disruption of protein-protein interactions. One such protein-protein interaction that is being targeted is the interaction between the histone methyltransferase MLL1 and Menin, an approach that has also been shown to reverse leukemogenic HOX/MEIS1 gene expression. We have developed novel, potent and orally bioavailable MLL1-Menin inhibitors and have brought one of these small molecules to clinical assessment. Inhibition of the MLL1-Menin interaction in MLL-rearranged and NPM1 mutant AML cells leads to loss of Menin from chromatin, destabilization of the Menin protein and specific changes in chromatin state that include loss of the MLL1 complex at select loci. This results in selective reversal of leukemogenic gene expression, changes in occupancy of multiple other chromatin complexes, rapid cellular differentiation and cell death. Also, we have recently found this interaction to be critical in subtypes of Sarcoma opening up novel potential therapeutic opportunities. These molecules have remarkable preclinical activity in human disease models including patient derived xenograft (PDX) models, show minimal toxicity and are now in phase I/II clinical trials. Inhibition of protein-protein interactions represents a potentially effective approach to destabilize chromatin associated complexes for cancer therapy. Citation Format: Scott A. Armstrong. Therapeutic targeting of MLL complexes in cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA020.

Abstract A015: Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Abstract Interplays between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes encoding nuclear proteins using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 84 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex functions and biochemical interactions, and predict new chromatin crosstalk, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality. Citation Format: Xiao Chen, Yinglu Li, Chao Lu. Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A015.

Topography of histone H3–H4 interaction with the Hat1–Hat2 acetyltransferase complex

Chaperones influence histone conformation and intermolecular interaction in multiprotein complexes, and the structures obtained with full-length histones often provide more accurate and comprehensive views. Here, our structure of the Hat1-Hat2 acetyltransferase complex bound to Asf1-H3-H4 shows that the core domains of H3 and H4 are involved in binding Hat1 and Hat2, and the N-terminal tail of H3 makes extensive interaction with Hat2. These findings expand the knowledge about histone-protein interaction and implicate a function of Hat2/RbAp46/48, which is a versatile histone chaperone found in many chromatin-associated complexes, in the passing of histones between chaperones.

Managing the Steady State Chromatin Landscape by Nucleosome Dynamics.

Gene regulation arises out of dynamic competition between nucleosomes, transcription factors, and other chromatin proteins for the opportunity to bind genomic DNA. The timescales of nucleosome assembly and binding of factors to DNA determine the outcomes of this competition at any given locus. Here, we review how these properties of chromatin proteins and the interplay between the dynamics of different factors are critical for gene regulation. We discuss how molecular structures of large chromatin-associated complexes, kinetic measurements, and high resolution mapping of protein-DNA complexes in vivo set the boundary conditions for chromatin dynamics, leading to models of how the steady state behaviors of regulatory elements arise.

Probing the Interaction Between Chromatin and Chromatin-Associated Complexes with Optical Tweezers.

Single-molecule force spectroscopy is a powerful tool to analyze the architecture and interaction of large macromolecular assemblies that are refractory to high-resolution structural interrogations. Here, we describe an optical tweezers-based platform for extracting the mechanical fingerprints of individual nucleosome arrays bound with chromatin-associated complexes, such as the Polycomb repressive complex 2 (PRC2). This platform comprehensively characterizes the diverse binding modes of PRC2 on chromatin, measures their mechanical strengths, and is broadly applicable to the studies of other epigenetic machineries.

Epigenetic Modifications of Host Genes Induced by Bacterial Infection

Introduction: Epigenetic mechanisms regulate expression of the genome to generate various cell types during development or coordinate cellular responses to external stimulus. While epigenetics is of fundamental importance in eukaryotes, it plays a different role in bacteria. This article uncovers the most important recent data on how bacteria can alter epigenetic marks and can also contribute to and/or result to various diseases. In this review article, Sciencedirect, PubMed, Scopus, Google Scholar databases were used for finding the relevant studies. Research has shown that bacteria can affect the chromatin structure and transcriptional program of host cells by influencing diverse epigenetic factors (i.e., histone modifications, DNA methylation, chromatin-associated complexes, and RNA splicing factors). Therefore, bacterial-induced epigenetic deregulations may affect host cell function either to host defense or to allow pathogen persistence. Hence, pathogenic bacteria can be considered as potential epimutagens able to reshape the epigenome that their effects might leading to cellular dysfunctions, which influenced immunity and might be at the origin of unexplained diseases and this caused presentation or contributed to the development of pathological changes.
 Conclusion: Epigenetic modifications during development and in response to environmental factors contribute to phenotypic variability and susceptibility to a number of diseases, including infectious diseases, cancers, metabolic and autoimmune disorders.

The roles of histone variants in fine-tuning chromatin organization and function.

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.

The Chromatin Regulator ZMYM2 Restricts Human Pluripotent Stem Cell Growth and Is Essential for Teratoma Formation.

SummaryChromatin regulators play fundamental roles in controlling pluripotency and differentiation. We examined the effect of mutations in 703 genes from nearly 70 chromatin-modifying complexes on human embryonic stem cell (ESC) growth. While the vast majority of chromatin-associated complexes are essential for ESC growth, the only complexes that conferred growth advantage upon mutation of their members, were the repressive complexes LSD-CoREST and BHC. Both complexes include the most potent growth-restricting chromatin-related protein, ZMYM2. Interestingly, while ZMYM2 expression is rather low in human blastocysts, its expression peaks in primed ESCs and is again downregulated upon differentiation. ZMYM2-null ESCs overexpress pluripotency genes and show genome-wide promotor-localized histone H3 hyper-acetylation. These mutant cells were also refractory to differentiate in vitro and failed to produce teratomas upon injection into immunodeficient mice. Our results suggest a central role for ZMYM2 in the transcriptional regulation of the undifferentiated state and in the exit-from-pluripotency of human ESCs.

EOMES interacts with RUNX3 and BRG1 to promote innate memory cell formation through epigenetic reprogramming

Memory CD8+ T cells have the ability to provide lifelong immunity against pathogens. Although memory features generally arise after challenge with a foreign antigen, naïve CD8 single positive (SP) thymocytes may acquire phenotypic and functional characteristics of memory cells in response to cytokines such as interleukin-4. This process is associated with the induction of the T-box transcription factor Eomesodermin (EOMES). However, the underlying molecular mechanisms remain ill-defined. Using epigenomic profiling, we show that these innate memory CD8SP cells acquire only a portion of the active enhancer repertoire of conventional memory cells. This reprograming is secondary to EOMES recruitment, mostly to RUNX3-bound enhancers. Furthermore, EOMES is found within chromatin-associated complexes containing BRG1 and promotes the recruitment of this chromatin remodelling factor. Also, the in vivo acquisition of EOMES-dependent program is BRG1-dependent. In conclusion, our results support a strong epigenetic basis for the EOMES-driven establishment of CD8+ T cell innate memory program.

Optimized Cross-Linking Mass Spectrometry for in Situ Interaction Proteomics.

Recent development of mass spectrometer cleavable protein cross-linkers and algorithms for their spectral identification now permits large-scale cross-linking mass spectrometry (XL-MS). Here, we optimized the use of cleavable disuccinimidyl sulfoxide (DSSO) cross-linker for labeling native protein complexes in live human cells. We applied a generalized linear mixture model to calibrate cross-link peptide-spectra matching (CSM) scores to control the sensitivity and specificity of large-scale XL-MS. Using specific CSM score thresholds to control the false discovery rate, we found that higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) can both be effective for large-scale XL-MS protein interaction mapping. We found that the coverage of protein-protein interaction maps is significantly improved through the use of multiple proteases. In addition, the use of focused sample-specific search databases can be used to improve the specificity of cross-linked peptide spectral matching. Application of this approach to human chromatin labeled in live cells recapitulated known and revealed new protein interactions of nucleosomes and other chromatin-associated complexes in situ. This optimized approach for mapping native protein interactions should be useful for a wide range of biological problems.

Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency.

The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 arehighly abundant in 2i/LIF ESCs, and H3K27me3 isdistributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability ortranscriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.

Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases.

PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3

Glioblastoma (GBM) is the most lethal type of human brain cancer, where deletions and mutations in the tumour suppressor gene PTEN (phosphatase and tensin homolog) are frequent events and are associated with therapeutic resistance. Herein, we report a novel chromatin-associated function of PTEN in complex with the histone chaperone DAXX and the histone variant H3.3. We show that PTEN interacts with DAXX and, in turn PTEN directly regulates oncogene expression by modulating DAXX-H3.3 association on the chromatin, independently of PTEN enzymatic activity. Furthermore, DAXX inhibition specifically suppresses tumour growth and improves the survival of orthotopically engrafted mice implanted with human PTEN-deficient glioma samples, associated with global H3.3 genomic distribution changes leading to upregulation of tumour suppressor genes and downregulation of oncogenes. Moreover, DAXX expression anti-correlates with PTEN expression in GBM patient samples. Since loss of chromosome 10 and PTEN are common events in cancer, this synthetic growth defect mediated by DAXX suppression represents a therapeutic opportunity to inhibit tumorigenesis specifically in the context of PTEN deletion.

Mixed-Lineage Leukemia Fusions and Chromatin in Leukemia.

Recent studies have shown the importance of chromatin-modifying complexes in the maintenance of developmental gene expression and human disease. The mixed lineage leukemia gene (MLL1) encodes a chromatin-modifying protein and was discovered as a result of the cloning of translocations involved in human leukemias. MLL1 is a histone lysine 4 (H3K4) methyltransferase that supports transcription of genes that are important for normal development including homeotic (Hox) genes. MLL1 rearrangements result in expression of fusion proteins without H3K4 methylation activity but may gain the ability to recruit other chromatin-associated complexes such as the H3K79 methyltransferase DOT1L and the super elongation complex. Therefore, chromosomal translocations involving MLL1 appear to directly perturb the regulation of multiple chromatin-associated complexes to allow inappropriate expression of developmentally regulated genes and thus drive leukemia development.

Orchestrating the Specific Assembly of Centromeric Nucleosomes.

Centromeres are chromosomal loci that are defined epigenetically in most eukaryotes by incorporation of a centromere-specific nucleosome in which the canonical histone H3 variant is replaced by Centromere Protein A (CENP-A). Therefore, the assembly and propagation of centromeric nucleosomes are critical for maintaining centromere identify and ensuring genomic stability. Centromeres direct chromosome segregation (during mitosis and meiosis) by recruiting the constitutive centromere-associated network of proteins throughout the cell cycle that in turn recruits the kinetochore during mitosis. Assembly of centromere-specific nucleosomes in humans requires the dedicated CENP-A chaperone HJURP, and the Mis18 complex to couple the deposition of new CENP-A to the site of the pre-existing centromere, which is essential for maintaining centromere identity. Human CENP-A deposition occurs specifically in early G1, into pre-existing chromatin, and several additional chromatin-associated complexes regulate CENP-A nucleosome deposition and stability. Here we review the current knowledge on how new CENP-A nucleosomes are assembled selectively at the existing centromere in different species and how this process is controlled to ensure stable epigenetic inheritance of the centromere.

Role of Histone-Modifying Enzymes and Their Complexes in Regulation of Chromatin Biology.

In 1964, Alfrey and colleagues proposed that acetylation and methylation of histones may regulate RNA synthesis and described "the possibility that relatively minor modifications of histone structure, taking place on the intact protein molecule, offer a means of switching-on or off RNA synthesis at different loci along the chromosome" [Allfrey, V., Faulkner, R., and Mirsky, A. (1964) Proc. Natl. Acad. Sci. U.S.A. 51, 786]. Fifty years later, this prescient description provides a simple but conceptually accurate model for the biological role of histone post-translational modifications (PTMs). The basic unit of chromosomes is the nucleosome, with double-stranded DNA wrapped around a histone protein oligomer. The "tails" of histone proteins are post-translationally modified, which alters the physical properties of nucleosomes in a manner that impacts gene accessibility for transcription and replication. Enzymes that catalyze the addition and removal of histone PTMs, histone-modifying enzymes (HMEs), are present in large protein complexes, with DNA-binding proteins, ATP-dependent chromatin remodeling enzymes, and epigenetic reader proteins that bind to post-translationally modified histone residues [Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K., and Schapira, M. (2012) Nat. Rev. Drug Discovery 11, 384-400]. The activity of HME complexes is coordinated with that of other chromatin-associated complexes that, together, regulate gene transcription, DNA repair, and DNA replication. In this context, the enzymes that catalyze addition and removal of histone PTMs are an essential component of the highly regulated mechanism for accessing compacted DNA. To fully understand the function of HMEs, the structure of nucleosomes, their natural substrate, will be described. Each major class of HMEs subsequently will be discussed with regard to its biochemistry, enzymatic mechanism, and biological function in the context of a prototypical HME complex.

The WD40 Domain Protein MSI1 Functions in a Histone Deacetylase Complex to Fine-Tune Abscisic Acid Signaling.

MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis.

Structural Basis for Multi-specificity of MRG Domains

Chromatin-binding proteins play vital roles in the assembly and recruitment of multi-subunit complexes harboring effector proteins to specific genomic loci. MRG15, a chromodomain-containing chromatin-binding protein, recruits diverse chromatin-associated complexes that regulate gene transcription, DNA repair, and RNA splicing. Previous studies with Pf1, another chromatin-binding subunit of the Sin3S/Rpd3S histone deacetylase complex, defined the sequence and structural requirements for interactions with the MRG15 MRG domain, a common target of diverse subunits in the aforementioned complexes. We now show that MRGBP, a member of the Tip60/NuA4 histone acetyltransferase complex, engages the same two surfaces of the MRG domain as Pf1. High-affinity interactions occur via a bipartite structural motif including an FxLP sequence motif. MRGBP shares little sequence and structural similarity with Pf1, yet targets similar pockets on the surface of the MRG domain, mimicking Pf1 in its interactions. Our studies shed light onto how MRG domains have evolved to bind diverse targets.