Subject(s)
Histones , Histones/metabolism , Histones/chemistry , Protein Binding , Models, Molecular , Protein Domains , Humans , Transcriptional Elongation Factors/metabolism , Transcriptional Elongation Factors/chemistry , Transcriptional Elongation Factors/genetics , Molecular Chaperones/metabolism , Molecular Chaperones/chemistry , Minichromosome Maintenance Complex Component 2/metabolism , Minichromosome Maintenance Complex Component 2/chemistry , Minichromosome Maintenance Complex Component 2/geneticsABSTRACT
Salinity is one of the most severe abiotic stresses that adversely affect plant growth and agricultural productivity. The plant Na+/H+ antiporter Salt Overly Sensitive 1 (SOS1) located in the plasma membrane extrudes excess Na+ out of cells in response to salt stress and confers salt tolerance. However, the molecular mechanism underlying SOS1 activation remains largely elusive. Here we elucidate two cryo-electron microscopy structures of rice (Oryza sativa) SOS1, a full-length protein in an auto-inhibited state and a truncated version in an active state. The SOS1 forms a dimeric architecture, with an NhaA-folded transmembrane domain portion in the membrane and an elongated cytosolic portion of multiple regulatory domains in the cytoplasm. The structural comparison shows that SOS1 adopts an elevator transport mechanism accompanied by a conformational transition of the highly conserved Pro148 in the unwound transmembrane helix 5 (TM5), switching from an occluded conformation in the auto-inhibited state to a conducting conformation in the active state. These findings allow us to propose an inhibition-release mechanism for SOS1 activation and elucidate how SOS1 controls Na+ homeostasis in response to salt stress.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Oryza , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Oryza/metabolism , Antiporters/metabolism , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Cryoelectron Microscopy , Sodium/metabolism , Gene Expression Regulation, PlantABSTRACT
Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes. However, the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified. In this study, we found that the previously identified PWWP-EPCR-ARID-TRB (PEAT) complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2 (HAM1/2) to form a larger version of PEAT complex in Arabidopsis thaliana. UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo. HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase complex, and are responsible for histone H4K5 acetylation. Within the PEAT complex, the PWWP components (PWWP1, PWWP2, and PWWP3) directly interact with UBP5 and are necessary for UBP5-mediated H2A deubiquitination, while the EPCR components (EPCR1 and EPCR2) directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5 acetylation. Collectively, our study not only identifies dual roles of the PEAT complex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.
Subject(s)
Arabidopsis , Histones , Histones/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Endothelial Protein C Receptor , Acetylation , Histone Acetyltransferases/genetics , Histone Acetyltransferases/metabolism , Deubiquitinating Enzymes , SoilABSTRACT
Histone variant H3.3 differs from the canonical histone H3.1 by only five amino acids, yet its chaperone death domain-associated protein (DAXX) can specifically recognize H3.3 over H3.1, despite having a large DAXX-interacting surface on the H3.3-H4 heterodimer common to that on the H3.1-H4 complex. This observation gives rise to the question of, from the binding energy point view, how high binding specificity may be achieved with small differences of the overall binding energy for protein-protein interactions in general. Here we investigate the mechanism of coupling of binding specificity and affinity in protein-protein interactions using the DAXX-H3.3-H4 complex as a model. Using a multi-scale method, we found that the hydrophobic interactions between DAXX and the H3.3-specific region contributed to their initial binding process. And the structural flexibility of the interacting partners contributed to the binding affinity after their encounter. By quantifying the free energy landscape, we revealed that the interaction between the specific residues of H3.3 and DAXX decreased the encounter barrier height while the folding of H3.3-H4 and DAXX increased the depth of the free energy basin of the final binding state. The encounter barrier height, which is not coupled to the thermodynamic stability of the final binding state, had a marked effect on the initial binding rate of flexible histones and chaperones. Based on the energy landscape theory, we found that the intrinsic binding energy funnel of this uncoupled recognition process was affected by the structural flexibility and the flexibility modulated the degree of coupling between binding specificity and affinity. Our work offers a biophysical explanation of the specific recognition between the histones and their chaperones, and also extends the use of energy landscape theory for understanding molecular recognitions in general.
Subject(s)
Histones , Nuclear Proteins , Histones/chemistry , Nuclear Proteins/chemistry , Histone Chaperones/chemistry , Histone Chaperones/metabolism , Molecular Chaperones , Protein BindingABSTRACT
This study aimed to explore the role and mechanism of DYRK1a regulating ferroptosis of cardiomyocytes during myocardial ischemia-reperfusion injury (MIRI). H9c2 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R) were used as MIRI cell models and transfected with sh-DYRK1a or/and erastin. Cell viability, apoptosis, and DYRK1a mRNA/protein expression were measured accordingly. The levels of reactive oxygen species (ROS), iron, malondialdehyde (MDA), and glutathione (GSH) were determined. The expression of ferroptosis-related proteins (GPX4, SLC7A11, ACSL4, and TFR1) was detected using western blotting. The MIRI rat model was established to explore the possible role of DYRK1a suppression in cell injury and ferroptosis. OGD/R cells showed elevated mRNA and protein expression for DYRK1a. OGD/R cells transfected with sh-DYRK1a showed elevated cell viability, GSH content, increased GPX4 and SLC7A11 expression, suppressed iron content, MDA, ROS, ACSL4, and TFR1 expression, and reduced apoptosis rate, whereas co-transfection of sh-DYRK1a with erastin reversed the attenuation of sh-DYRK1a on MIRI. The suppressive effect of sh-DYRK1a on MI/R injury was confirmed in an MIRI rat model. DYRK1a mediates ferroptosis of cardiomyocytes to deteriorate MIRI progression.
Subject(s)
Ferroptosis , Myocardial Reperfusion Injury , Reperfusion Injury , Animals , Rats , Ferroptosis/genetics , Glucose , Glutathione , Iron , Myocardial Reperfusion Injury/genetics , Myocytes, Cardiac , Oxygen , Reactive Oxygen Species , RNA, Messenger/geneticsABSTRACT
Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.
Subject(s)
Chromatin Assembly Factor-1 , Histones , Nucleosomes , Humans , Chromatin , Chromatin Assembly Factor-1/chemistry , Cryoelectron Microscopy , Histones/chemistry , Crystallography, X-Ray , Protein DomainsABSTRACT
Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.
Subject(s)
Deubiquitinating Enzymes , Histones , Polycomb Repressive Complex 1 , Polycomb-Group Proteins , Humans , Cryoelectron Microscopy , Histones/chemistry , Histones/metabolism , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Polycomb Repressive Complex 1/chemistry , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 1/ultrastructure , Polycomb-Group Proteins/chemistry , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/ultrastructure , Ubiquitin/metabolism , Ubiquitin Thiolesterase/chemistry , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/ultrastructure , Ubiquitination , Repressor Proteins/chemistry , Repressor Proteins/metabolism , Repressor Proteins/ultrastructure , Catalytic Domain , Deubiquitinating Enzymes/classification , Deubiquitinating Enzymes/metabolism , Deubiquitinating Enzymes/ultrastructure , Substrate Specificity , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/ultrastructureABSTRACT
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.
Subject(s)
Histone Acetyltransferases , Histones , Acetylation , Cell Cycle Proteins/metabolism , Chromatin , Histone Acetyltransferases/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Molecular Chaperones/geneticsABSTRACT
Bivalent genes are ready for activation upon the arrival of developmental cues. Here, we report that BEND3 is a CpG island (CGI)-binding protein that is enriched at regulatory elements. The cocrystal structure of BEND3 in complex with its target DNA reveals the structural basis for its DNA methylation-sensitive binding property. Mouse embryos ablated of Bend3 died at the pregastrulation stage. Bend3 null embryonic stem cells (ESCs) exhibited severe defects in differentiation, during which hundreds of CGI-containing bivalent genes were prematurely activated. BEND3 is required for the stable association of polycomb repressive complex 2 (PRC2) at bivalent genes that are highly occupied by BEND3, which suggests a reining function of BEND3 in maintaining high levels of H3K27me3 at these bivalent genes in ESCs to prevent their premature activation in the forthcoming developmental stage.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/metabolism , Repressor Proteins/metabolism , Transcriptional Activation , Animals , Chromatin/metabolism , CpG Islands , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Methylation , Embryonic Stem Cells/cytology , Enhancer Elements, Genetic , Gene Deletion , Gene Expression Regulation, Developmental , Histones/metabolism , Methylation , Mice , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic , Protein Binding , Protein Domains , RNA-Seq , Repressor Proteins/chemistry , Repressor Proteins/genetics , Up-RegulationABSTRACT
Chromosomal duplication requires de novo assembly of nucleosomes from newly synthesized histones, and the process involves a dynamic network of interactions between histones and histone chaperones. sNASP and ASF1 are two major histone H3-H4 chaperones found in distinct and common complexes, yet how sNASP binds H3-H4 in the presence and absence of ASF1 remains unclear. Here we show that, in the presence of ASF1, sNASP principally recognizes a partially unfolded Nα region of histone H3, and in the absence of ASF1, an additional sNASP binding site becomes available in the core domain of the H3-H4 complex. Our study also implicates a critical role of the C-terminal tail of H4 in the transfer of H3-H4 between sNASP and ASF1 and the coiled-coil domain of sNASP in nucleosome assembly. These findings provide mechanistic insights into coordinated histone binding and transfer by histone chaperones.
Subject(s)
Histone Chaperones , Histones , Binding Sites , Cell Cycle Proteins/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Nucleosomes , Protein BindingABSTRACT
CENP-A (centromeric protein A), a histone H3 variant, specifies centromere identity and is essential to centromere maintenance. Little is known about how protein levels of CENP-A are controlled in mammalian cells. Here, we report that the phosphorylation of CENP-A Ser68 primes the ubiquitin-proteasome-mediated proteolysis of CENP-A during mitotic phase in human cultured cells. We identify two major polyubiquitination sites that are responsible for this phosphorylation-dependent degradation. Substituting the two residues, Lys49 and Lys124, with arginines abrogates proper CENP-A degradation and results in CENP-A mislocalization to non-centromeric regions. Furthermore, we find that DCAF11 (DDB1 and CUL4 associated factor 11/WDR23) is the E3 ligase that specifically mediates the observed polyubiquitination. Deletion of DCAF11 hampers CENP-A degradation and causes its mislocalization. We conclude that the Ser68 phosphorylation plays an important role in regulating cellular CENP-A homeostasis via DCAF11-mediated degradation to prevent ectopic localization of CENP-A during the cell cycle.
Subject(s)
Cell Cycle , Centromere Protein A/metabolism , Cullin Proteins/metabolism , DNA-Binding Proteins/metabolism , Serine/metabolism , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitination , Animals , Centromere , Centromere Protein A/chemistry , Centromere Protein A/genetics , Chromatin/genetics , Chromatin/metabolism , Cullin Proteins/genetics , DNA-Binding Proteins/genetics , Female , Histones/genetics , Histones/metabolism , Male , Mice , Mice, Inbred C57BL , Nucleosomes , Phosphorylation , Proteolysis , Serine/chemistry , Serine/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligase Complexes/geneticsABSTRACT
Metastasis is the primary cause of cancer-related mortality in colorectal cancer (CRC) patients. How to improve therapeutic options for patients with metastatic CRC is the core question for CRC treatment. However, the complexity and diversity of stromal context of the tumor microenvironment (TME) in liver metastases of CRC have not been fully understood, and the influence of stromal cells on response to chemotherapy is unclear. Here we performed an in-depth analysis of the transcriptional landscape of primary CRC, matched liver metastases and blood at single-cell resolution, and a systematic examination of transcriptional changes and phenotypic alterations of the TME in response to preoperative chemotherapy (PC). Based on 111,292 single-cell transcriptomes, our study reveals that TME of treatment-naïve tumors is characterized by the higher abundance of less-activated B cells and higher heterogeneity of tumor-associated macrophages (TAMs). By contrast, in tumors treated with PC, we found activation of B cells, lower diversity of TAMs with immature and less activated phenotype, lower abundance of both dysfunctional T cells and ECM-remodeling cancer-associated fibroblasts, and an accumulation of myofibroblasts. Our study provides a foundation for future investigation of the cellular mechanisms underlying liver metastasis of CRC and its response to PC, and opens up new possibilities for the development of therapeutic strategies for CRC.
ABSTRACT
The SLX1-SLX4 structure-specific endonuclease complex is involved in processing diverse DNA damage intermediates, including resolution of Holliday junctions, collapse of stalled replication forks and removal of DNA flaps. The nuclease subunit SLX1 is inactive on its own, but become activated upon binding to SLX4 via its conserved C-terminal domain (CCD). Yet, how the SLX1-SLX4 complex recognizes specific DNA structure and chooses cleavage sites remains unknown. Here we show, through a combination of structural, biochemical and computational analyses, that the SAP domain of SLX4 is critical for efficient and accurate processing of 5'-flap DNA. It binds the minor groove of DNA about one turn away from the flap junction, and the 5'-flap is implicated in binding the core domain of SLX1. This binding mode accounts for specific recognition of 5'-flap DNA and specification of cleavage site by the SLX1-SLX4 complex.
Subject(s)
Endodeoxyribonucleases/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Catalytic Domain , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , Endodeoxyribonucleases/metabolism , Models, Molecular , Protein Binding , Protein Domains , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Activation of cyclic GMP-AMP synthase (cGAS) through sensing cytosolic double stranded DNA (dsDNA) plays a pivotal role in innate immunity against exogenous infection as well as cellular regulation under stress. Aberrant activation of cGAS induced by self-DNA is related to autoimmune diseases. cGAS accumulates at chromosomes during mitosis or spontaneously in the nucleus. Binding of cGAS to the nucleosome competitively attenuates the dsDNA-mediated cGAS activation, but the molecular mechanism of the attenuation is still poorly understood. Here, we report two cryo-electron microscopy structures of cGAS-nucleosome complexes. The structures reveal that cGAS interacts with the nucleosome as a monomer, forming 1:1 and 2:2 complexes, respectively. cGAS contacts the nucleosomal acidic patch formed by the H2A-H2B heterodimer through the dsDNA-binding site B in both complexes, and could interact with the DNA from the other symmetrically placed nucleosome via the dsDNA-binding site C in the 2:2 complex. The bound nucleosome inhibits the activation of cGAS through blocking the interaction of cGAS with ligand dsDNA and disrupting cGAS dimerization. R236A or R255A mutation of cGAS impairs the binding between cGAS and the nucleosome, and largely relieves the nucleosome-mediated inhibition of cGAS activity. Our study provides structural insights into the inhibition of cGAS activity by the nucleosome, and advances the understanding of the mechanism by which hosts avoid the autoimmune attack caused by cGAS.
Subject(s)
Nucleosomes/ultrastructure , Nucleotidyltransferases/chemistry , Nucleotidyltransferases/ultrastructure , Binding Sites , Cryoelectron Microscopy , DNA/metabolism , Histones/metabolism , Humans , Models, Molecular , Nucleosomes/metabolism , Nucleotidyltransferases/antagonists & inhibitors , Nucleotidyltransferases/metabolism , Protein MultimerizationABSTRACT
How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication-linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.
Subject(s)
DNA Polymerase I , Histones , Animals , DNA/genetics , DNA Polymerase I/genetics , DNA Polymerase I/metabolism , DNA Replication , Histones/genetics , Histones/metabolism , Mice , Nucleosomes/metabolism , Saccharomyces cerevisiae/geneticsABSTRACT
As approximately 70% of human breast tumors are estrogen receptor α (ERα)-positive, estrogen and ERα play essential roles in breast cancer development. By interrupting the ERα signaling pathway, endocrine therapy has been proven to be an effective therapeutic strategy. In this study, we identified a mechanism by which Transcription Start Site (TSS)-associated histone H3K27 acetylation signals the Super Elongation Complex (SEC) to regulate transcriptional elongation of the ESR1 (ERα) gene. SEC interacts with H3K27ac on ESR1 TSS through its scaffold protein AFF4. Depletion of AFF4 by siRNA or CRISPR/Cas9 dramatically reduces expression of ESR1 and its target genes, consequently inhibiting breast cancer cell growth. More importantly, a AFF4 mutant which lacks H3K27ac interaction failed to rescue ESR1 gene expression, suggesting H3K27 acetylation at TSS region is a key mark bridging the transition from transcriptional initiation to elongation, and perturbing SEC function can be an alternative strategy for targeting ERα signaling pathway at chromatin level.
Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor alpha/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Transcription Elongation, Genetic , Acetylation , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation , Estrogen Receptor alpha/genetics , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Histones/genetics , Humans , MCF-7 Cells , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , Signal Transduction , Transcription Factor AP-2/genetics , Transcription Factor AP-2/metabolism , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolismABSTRACT
A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.
Subject(s)
MicroRNAs/chemistry , MicroRNAs/metabolism , Ribonuclease III/chemistry , Ribonuclease III/metabolism , Animals , Cryoelectron Microscopy , Humans , Models, Molecular , Protein Conformation , Protein Domains , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonuclease III/genetics , Spodoptera/cytologyABSTRACT
Chromosomal translocations of MLL1 (Mixed Lineage Leukemia 1) yield oncogenic chimeric proteins containing the N-terminal portion of MLL1 fused with distinct partners. The MLL1-AF10 fusion causes leukemia through recruiting the H3K79 histone methyltransferase DOT1L via AF10's octapeptide and leucine zipper (OM-LZ) motifs. Yet, the precise interaction sites in DOT1L, detailed interaction modes between AF10 and DOT1L, and the functional configuration of MLL1-AF10 in leukeomogenesis remain unknown. Through a combined approach of structural and functional analyses, we found that the LZ domain of AF10 interacts with the coiled-coil domains of DOT1L through a conserved binding mode and discovered that the C-terminal end of the LZ domain and the OM domain of AF10 mediate the formation of a DOT1L-AF10 octamer via tetramerization of the binary complex. We reveal that the oligomerization ability of the DOT1L-AF10 complex is essential for MLL1-AF10's leukemogenic function. These findings provide insights into the molecular basis of pathogenesis by MLL1 rearrangements.
Subject(s)
Gene Expression Regulation, Leukemic , Histone-Lysine N-Methyltransferase/metabolism , Leukemia/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Transcription Factors/metabolism , Cell Nucleus/metabolism , Cell Transformation, Neoplastic , Escherichia coli/metabolism , Humans , Leucine Zippers , Leukemia/pathology , Mutation , Oncogene Proteins, Fusion/metabolism , Protein Binding , Protein Domains , Protein Interaction Mapping , Protein Multimerization , Protein Structure, SecondaryABSTRACT
Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger-type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.