ABSTRACT
All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.
Subject(s)
Transcription Factors , Ubiquitin-Protein Ligases , Humans , Gene Expression , HEK293 Cells , HeLa Cells , Mutation , Signal Transduction , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/metabolismABSTRACT
Eukaryotic DNA-binding proteins operate in the context of chromatin, where nucleosomes are the elementary building blocks. Nucleosomal DNA is wrapped around a histone core, thereby rendering a large fraction of the DNA surface inaccessible to DNA-binding proteins. Nevertheless, first responders in DNA repair and sequence-specific transcription factors bind DNA target sites obstructed by chromatin. While early studies examined protein binding to histone-free DNA, it is only now beginning to emerge how DNA sequences are interrogated on nucleosomes. These readout strategies range from the release of nucleosomal DNA from histones, to rotational/translation register shifts of the DNA motif, and nucleosome-specific DNA binding modes that differ from those observed on naked DNA. Since DNA motif engagement on nucleosomes strongly depends on position and orientation, we argue that motif location and nucleosome positioning co-determine protein access to DNA in transcription and DNA repair.
Subject(s)
Chromatin/genetics , Genome , Animals , Humans , Models, Biological , Nucleosomes/metabolism , Nucleotide Motifs/genetics , Transcription Factors/metabolismABSTRACT
Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.
Subject(s)
Chromatin , Transcription Factors , Humans , Ligands , Chromatin/genetics , Transcription Factors/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Ubiquitins , Ubiquitin-Protein Ligases/geneticsABSTRACT
The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.
Subject(s)
Phosphopeptides , Telomere-Binding Proteins , BRCA1 Protein/genetics , Carrier Proteins/metabolism , DNA/metabolism , DNA End-Joining Repair , DNA Repair , Phosphopeptides/genetics , Phosphopeptides/metabolism , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , DNA , Histones , ARNTL Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA/genetics , DNA/metabolism , Helix-Loop-Helix Motifs/genetics , Histones/chemistry , Histones/metabolism , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Protein Binding , CLOCK Proteins/chemistry , CLOCK Proteins/metabolism , Proto-Oncogene Proteins c-myc/chemistry , Proto-Oncogene Proteins c-myc/metabolism , Allosteric Regulation , Leucine Zippers , Octamer Transcription Factor-3/metabolism , Protein MultimerizationABSTRACT
Yeast telomeres comprise irregular TG1â3 DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition of telomeres as DNA double-strand breaks. We provide a molecular, biochemical, and functional dissection of the protein backbone at the core of the yeast telosome. The X-ray structures of Rif1 and Rif2 bound to the Rap1 C-terminal domain and that of the Rif1 C terminus are presented. Both Rif1 and Rif2 have separable and independent Rap1-binding epitopes, allowing Rap1 binding over large distances (42-110 Å). We identify tetramerization (Rif1) and polymerization (Rif2) modules that, in conjunction with the long-range binding, give rise to a higher-order architecture that interlinks Rap1 units. This molecular Velcro relies on Rif1 and Rif2 to recruit and stabilize Rap1 on telomeric arrays and is required for telomere homeostasis in vivo.
Subject(s)
Chromosomes, Fungal/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Interaction Maps , Sequence Alignment , Shelterin ComplexABSTRACT
Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.
Subject(s)
Chromatography, High Pressure Liquid , Proteomics/methods , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Ubiquitin-Protein Ligases/metabolism , Cullin Proteins/genetics , Cullin Proteins/metabolism , HEK293 Cells , Humans , Kinetics , Muscle Proteins/genetics , Muscle Proteins/metabolism , NEDD8 Protein/genetics , NEDD8 Protein/metabolism , Protein Interaction Maps , Proteolysis , Signal Transduction , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/geneticsABSTRACT
Thalidomide and its derivatives are powerful cancer therapeutics that are among the best-understood molecular glue degraders (MGDs). These drugs selectively reprogram the E3 ubiquitin ligase cereblon (CRBN) to commit target proteins for degradation by the ubiquitin-proteasome system. MGDs create novel recognition interfaces on the surface of the E3 ligase that engage in induced protein-protein interactions with neosubstrates. Molecular insight into their mechanism of action opens exciting opportunities to engage a plethora of targets through a specific recognition motif, the G-loop. Our analysis shows that current CRBN-based MGDs can in principle recognize over 2,500 proteins in the human proteome that contain a G-loop. We review recent advances in tuning the specificity between CRBN and its MGD-induced neosubstrates and deduce a set of simple rules that govern these interactions. We conclude that rational MGD design efforts will enable selective degradation of many more proteins, expanding this therapeutic modality to more disease areas.
Subject(s)
Thalidomide , Ubiquitin-Protein Ligases , Humans , Thalidomide/pharmacology , Thalidomide/therapeutic use , Proteolysis , Ubiquitin-Protein Ligases/metabolism , Proteasome Endopeptidase Complex/metabolismABSTRACT
Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4DCAF12 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the ß-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.
Subject(s)
Carrier Proteins , Ubiquitin-Protein Ligases , Carrier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Chaperonin Containing TCP-1/metabolismABSTRACT
In mammals, â¼100 deubiquitinases act on â¼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.
Subject(s)
BRCA1 Protein/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , Deubiquitinating Enzymes/genetics , Histone Chaperones/genetics , Neoplasms/genetics , Binding Sites/genetics , Carrier Proteins/genetics , Cell Nucleus/genetics , Cell Nucleus/immunology , Cytoplasm/genetics , Cytoplasm/immunology , DNA Breaks, Double-Stranded , DNA Repair/immunology , Deubiquitinating Enzymes/immunology , HeLa Cells , Humans , Immunity, Cellular/genetics , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Neoplasms/immunology , Nuclear Matrix-Associated Proteins/genetics , Protein Binding/genetics , Ubiquitin/genetics , Ubiquitin-Specific Proteases/genetics , Ubiquitination/geneticsABSTRACT
Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.
Subject(s)
Cyclins , Ubiquitin-Protein Ligases , Cyclins/metabolism , Ubiquitin-Protein Ligases/metabolism , Proteolysis , Structure-Activity RelationshipABSTRACT
The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.
Subject(s)
Ubiquitin-Protein Ligases/chemistry , Animals , Crystallography, X-Ray , Cullin Proteins/chemistry , DNA Damage , DNA-Binding Proteins/chemistry , Enzyme Activation , Humans , Models, Molecular , Ubiquitin-Protein Ligases/metabolism , Xeroderma Pigmentosum Group A Protein/chemistryABSTRACT
The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons 2-7. cGAS is also present inside the cell nucleus, which is replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.
Subject(s)
Cryoelectron Microscopy , Nucleosomes/metabolism , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/chemistry , HeLa Cells , Histones/metabolism , Humans , Models, Molecular , Mutation , Nucleosomes/chemistry , Nucleosomes/ultrastructure , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/ultrastructureABSTRACT
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Subject(s)
Cyclins/deficiency , Cyclins/metabolism , Proteolysis/drug effects , Purines/chemistry , Purines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , Cyclins/chemistry , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Proteasome Endopeptidase Complex/metabolism , Protein Binding/drug effects , Purines/toxicity , Pyridines/toxicity , Small Molecule Libraries/analysis , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitination/drug effectsABSTRACT
The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the Ë29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
Subject(s)
Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cryoelectron Microscopy , Cullin Proteins/metabolism , Humans , Models, Molecular , Mutation , Protein Domains , Protein Multimerization , Protein Serine-Threonine Kinases/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , vpr Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.
ABSTRACT
Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.
Subject(s)
DNA Damage , DNA-Binding Proteins/metabolism , DNA/metabolism , DNA/ultrastructure , Nucleosomes/metabolism , Nucleosomes/ultrastructure , Pyrimidine Dimers/analysis , Cryoelectron Microscopy , DNA/chemistry , DNA/radiation effects , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/ultrastructure , Histones/chemistry , Histones/metabolism , Histones/ultrastructure , Humans , Models, Molecular , Nucleosomes/genetics , Nucleosomes/radiation effects , Pyrimidine Dimers/chemistry , Pyrimidine Dimers/genetics , Thermodynamics , Ultraviolet Rays/adverse effectsABSTRACT
The Fanconi anemia (FA) core complex is the ~0.9-mDa ubiquitin ligase most frequently mutated in patients with FA. New cryo-electron microscopy (cryo-EM) data from Shakeel et al. reveals a surprisingly complex ubiquitin ligase architecture, providing unprecedented insight into this critical hub at the interface of DNA crosslink detection and repair.
Subject(s)
Fanconi Anemia , Cryoelectron Microscopy , Fanconi Anemia Complementation Group D2 Protein/genetics , Fanconi Anemia Complementation Group D2 Protein/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Humans , Ligases , UbiquitinationABSTRACT
De novo mutations in ADNP, which encodes activity-dependent neuroprotective protein (ADNP), have recently been found to underlie Helsmoortel-Van der Aa syndrome, a complex neurological developmental disorder that also affects several other organ functions 1 . ADNP is a putative transcription factor that is essential for embryonic development 2 . However, its precise roles in transcriptional regulation and development are not understood. Here we show that ADNP interacts with the chromatin remodeller CHD4 and the chromatin architectural protein HP1 to form a stable complex, which we refer to as ChAHP. Besides mediating complex assembly, ADNP recognizes DNA motifs that specify binding of ChAHP to euchromatin. Genetic ablation of ChAHP components in mouse embryonic stem cells results in spontaneous differentiation concomitant with premature activation of lineage-specific genes and in a failure to differentiate towards the neuronal lineage. Molecularly, ChAHP-mediated repression is fundamentally different from canonical HP1-mediated silencing: HP1 proteins, in conjunction with histone H3 lysine 9 trimethylation (H3K9me3), are thought to assemble broad heterochromatin domains that are refractory to transcription. ChAHP-mediated repression, however, acts in a locally restricted manner by establishing inaccessible chromatin around its DNA-binding sites and does not depend on H3K9me3-modified nucleosomes. Together, our results reveal that ADNP, via the recruitment of HP1 and CHD4, regulates the expression of genes that are crucial for maintaining distinct cellular states and assures accurate cell fate decisions upon external cues. Such a general role of ChAHP in governing cell fate plasticity may explain why ADNP mutations affect several organs and body functions and contribute to cancer progression1,3,4. Notably, we found that the integrity of the ChAHP complex is disrupted by nonsense mutations identified in patients with Helsmoortel-Van der Aa syndrome, and this could be rescued by aminoglycosides that suppress translation termination 5 . Therefore, patients might benefit from therapeutic agents that are being developed to promote ribosomal read-through of premature stop codons6,7.