RESUMO
The mixed-lineage leukemia (MLL)-AF10 fusion oncoprotein recruits DOT1L to the homeobox A (HOXA) gene cluster through its octapeptide motif leucine zipper (OM-LZ), thereby inducing and maintaining the MLL-AF10-associated leukemogenesis. However, the recognition mechanism between DOT1L and MLL-AF10 is unclear. Here, we present the crystal structures of both apo AF10OM-LZ and its complex with the coiled-coil domain of DOT1L. Disruption of the DOT1L-AF10 interface abrogates MLL-AF10-associated leukemic transformation. We further show that zinc stabilizes the DOT1L-AF10 complex and may be involved in the regulation of the HOXA gene expression. Our studies may also pave the way for the rational design of therapeutic drugs against MLL-rearranged leukemia.
Assuntos
Transformação Celular Neoplásica/patologia , Metiltransferases , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide , Fatores de Transcrição , Cristalização , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase , Proteínas de Homeodomínio/genética , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Zinco/químicaRESUMO
The MLL1 gene is a frequent target for recurrent chromosomal translocations, resulting in transformation of hematopoietic precursors into leukemia stem cells. Here, we report on structure-function studies that elucidate molecular events in MLL1 binding of histone H3K4me3/2 marks and recruitment of the cyclophilin CyP33. CyP33 contains a PPIase and a RRM domain and regulates MLL1 function through HDAC recruitment. We find that the PPIase domain of CyP33 regulates the conformation of MLL1 through proline isomerization within the PHD3-Bromo linker, thereby disrupting the PHD3-Bromo interface and facilitating binding of the MLL1-PHD3 domain to the CyP33-RRM domain. H3K4me3/2 and CyP33-RRM target different surfaces of MLL1-PHD3 and can bind simultaneously to form a ternary complex. Furthermore, the MLL1-CyP33 interaction is required for repression of HOXA9 and HOXC8 genes in vivo. Our results highlight the role of PHD3-Bromo cassette as a regulatory platform, orchestrating MLL1 binding of H3K4me3/2 marks and cyclophilin-mediated repression through HDAC recruitment.
Assuntos
Ciclofilinas/metabolismo , Histona Desacetilases/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Sequência de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Prolina/química , Domínios e Motivos de Interação entre ProteínasRESUMO
Methyltransferases of the mixed-lineage leukaemia (MLL) family-which include MLL1, MLL2, MLL3, MLL4, SET1A and SET1B-implement methylation of histone H3 on lysine 4 (H3K4), and have critical and distinct roles in the regulation of transcription in haematopoiesis, adipogenesis and development1-6. The C-terminal catalytic SET (Su(var.)3-9, enhancer of zeste and trithorax) domains of MLL proteins are associated with a common set of regulatory factors (WDR5, RBBP5, ASH2L and DPY30) to achieve specific activities7-9. Current knowledge of the regulation of MLL activity is limited to the catalysis of histone H3 peptides, and how H3K4 methyl marks are deposited on nucleosomes is poorly understood. H3K4 methylation is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120ub1), a prevalent histone H2B mark that disrupts chromatin compaction and favours open chromatin structures, but the underlying mechanism remains unknown10-12. Here we report cryo-electron microscopy structures of human MLL1 and MLL3 catalytic modules associated with nucleosome core particles that contain H2BK120ub1 or unmodified H2BK120. These structures demonstrate that the MLL1 and MLL3 complexes both make extensive contacts with the histone-fold and DNA regions of the nucleosome; this allows ease of access to the histone H3 tail, which is essential for the efficient methylation of H3K4. The H2B-conjugated ubiquitin binds directly to RBBP5, orienting the association between MLL1 or MLL3 and the nucleosome. The MLL1 and MLL3 complexes display different structural organizations at the interface between the WDR5, RBBP5 and MLL1 (or the corresponding MLL3) subunits, which accounts for the opposite roles of WDR5 in regulating the activity of the two enzymes. These findings transform our understanding of the structural basis for the regulation of MLL activity at the nucleosome level, and highlight the pivotal role of nucleosome regulation in histone-tail modification.
Assuntos
Proteínas de Ligação a DNA/química , Histona-Lisina N-Metiltransferase/metabolismo , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Nucleossomos/química , Nucleossomos/metabolismo , Microscopia Crioeletrônica , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Histona-Lisina N-Metiltransferase/química , Histonas/química , Histonas/metabolismo , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Estrutura Quaternária de ProteínaRESUMO
The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Células Germinativas/citologia , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Especiação Genética , Células Germinativas/metabolismo , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas , Domínios ProteicosRESUMO
Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.
Assuntos
Histona-Lisina N-Metiltransferase , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Nucleossomos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Histonas/química , Histonas/genética , Microscopia Crioeletrônica/métodosRESUMO
The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.
Assuntos
Histona-Lisina N-Metiltransferase , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Ubiquitinação , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Nucleossomos/enzimologia , Ligação ProteicaRESUMO
The MLL/KMT2 family enzymes are frequently mutated in human cancers and congenital diseases. They deposit the majority of histone 3 lysine 4 (H3K4) mono-, di-, or tri-methylation in mammals and are tightly associated with gene activation. Structural and biochemical studies in recent years provide in-depth understanding of how the MLL1 and homologous yeast SET1 complexes interact with the nucleosome core particle (NCP) and how their activities for H3K4 methylation are regulated by the conserved core components. Here, we will discuss the recent single molecule cryo-EM studies on the MLL1 and ySET1 complexes bound on the NCP. These studies highlight the dynamic regulation of the MLL/SET1 family lysine methyltransferases with unique features as compared with other histone lysine methyltransferases. These studies provide insights for loci-specific regulation of H3K4 methylation states in cells. The mechanistic studies on the MLL1 complex have already led to the development of the MLL1 inhibitors that show efficacy in acute leukemia and metastatic breast cancers. Future studies on the MLL/SET1 family enzymes will continue to bring to light potential therapeutic opportunities.
Assuntos
Histonas , Proteína de Leucina Linfoide-Mieloide , Animais , Humanos , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Lisina/metabolismo , Núcleo Celular/metabolismo , Histona-Lisina N-Metiltransferase/genética , Mamíferos/metabolismoRESUMO
Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.
Assuntos
Antígenos Virais/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Sarcoma de Kaposi/genética , Latência Viral/genética , Antígenos Virais/química , Antígenos Virais/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/fisiologia , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica , Sarcoma de Kaposi/virologiaRESUMO
Human lysine methyltransferase 2D (hKMT2D) is an epigenetic writer catalyzing the methylation of histone 3 lysine 4. hKMT2D by itself has little catalytic activity and reaches full activation as part of the WRAD2 complex, additionally comprising binding partners WDR5, RbBP5, Ash2L, and DPY30. Here, a detailed mechanistic study of the hKMT2D SET domain and its WRAD2 interactions is described. We characterized the WRAD2 subcomplexes containing full-length components and the hKMT2D SET domain. By performing steady-state analysis as a function of WRAD2 concentration, we identified the inner stoichiometry and determined the binding affinities for complex formation. Ash2L and RbBP5 were identified as the binding partners critical for the full catalytic activity of the SET domain. Contrary to a previous report, product and dead-end inhibitor studies identified hKMT2D as a rapid equilibrium random Bi-Bi mechanism with EAP and EBQ dead-end complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) analysis showed that hKMT2D uses a distributive mechanism and gives further insights into how the WRAD2 components affect mono-, di-, and trimethylation. We also conclude that the Win motif of hKMT2D is not essential in complex formation, unlike other hKMT2 proteins.
Assuntos
Histona-Lisina N-Metiltransferase , Lisina , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Lisina/metabolismo , Metilação , Proteína de Leucina Linfoide-Mieloide/químicaRESUMO
Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/ultraestrutura , Histonas/metabolismo , Humanos , Metilação , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/ultraestrutura , Ligação Proteica , Conformação ProteicaRESUMO
The mixed lineage leukaemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the molecular mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochemical and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Ativação Enzimática , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
Assuntos
Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Leucemia Aguda Bifenotípica/enzimologia , Proteína de Leucina Linfoide-Mieloide/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Oligopeptídeos/química , Oligopeptídeos/fisiologia , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
The nine-amino-acid activation domain (9aaTAD) is defined by a short amino acid pattern including two hydrophobic regions (positions p3-4 and p6-7). The KIX domain of mediator transcription CBP interacts with the 9aaTAD domains of transcription factors MLL, E2A, NF-kB, and p53. In this study, we analyzed the 9aaTADs-KIX interactions by nuclear magnetic resonance. The positions of three KIX helixes α1-α2-α3 are influenced by sterically-associated hydrophobic I611, L628, and I660 residues that are exposed to solvent. The positions of two rigid KIX helixes α1 and α2 generate conditions for structural folding in the flexible KIX-L12-G2 regions localized between them. The three KIX I611, L628, and I660 residues interact with two 9aaTAD hydrophobic residues in positions p3 and p4 and together build a hydrophobic core of five residues (5R). Numerous residues in 9aaTAD position p3 and p4 could provide this interaction. Following binding of the 9aaTAD to KIX, the hydrophobic I611, L628, and I660 residues are no longer exposed to solvent and their position changes inside the hydrophobic core together with position of KIX α1-α2-α3 helixes. The new positions of the KIX helixes α1 and α2 allow the KIX-L12-G2 enhanced formation. The second hydrophobic region of the 9aaTAD (positions p6 and p7) provides strong binding with the KIX-L12-G2 region. Similarly, multiple residues in 9aaTAD position p6 and p7 could provide this interaction. In conclusion, both 9aaTAD regions p3, p4 and p6, p7 provide co-operative and highly universal binding to mediator KIX. The hydrophobic core 5R formation allows new positions of the rigid KIX α-helixes and enables the enhanced formation of the KIX-L12-G2 region. This contributes to free energy and is the key for the KIX-9aaTAD binding. Therefore, the 9aaTAD-KIX interactions do not operate under the rigid key-and-lock mechanism what explains the 9aaTAD natural variability.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína de Ligação a CREB/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , NF-kappa B/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Motivos de Aminoácidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Sítios de Ligação , Proteína de Ligação a CREB/química , Histona-Lisina N-Metiltransferase/química , Humanos , Proteína de Leucina Linfoide-Mieloide/química , NF-kappa B/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/químicaRESUMO
Crosstalk between H2B ubiquitylation (H2Bub) and H3 K4 methylation plays important roles in coordinating functions of diverse cofactors during transcription activation. The underlying mechanism for this trans-tail signaling pathway is poorly defined in higher eukaryotes. Here, we show the following: (1) ASH2L in the MLL complex is essential for H2Bub-dependent H3 K4 methylation. Deleting or mutating K99 of the N-terminal winged helix (WH) motif in ASH2L abrogates H2Bub-dependent regulation. (2) Crosstalk can occur in trans and does not require ubiquitin to be on nucleosomes or histones to exert regulatory effects. (3) trans-regulation by ubiquitin promotes MLL activity for all three methylation states. (4) MLL3, an MLL homolog, does not respond to H2Bub, highlighting regulatory specificity for MLL family histone methyltransferases. Altogether, our results potentially expand the classic histone crosstalk to nonhistone proteins, which broadens the scope of chromatin regulation by ubiquitylation signaling.
Assuntos
Proteínas de Ligação a DNA/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Proteína de Leucina Linfoide-Mieloide/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Ubiquitinação , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Ligação a DNA/genética , Estabilidade Enzimática , Expressão Gênica , Células HeLa , Histona Metiltransferases , Histonas/fisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metilação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteína Meis1 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Nucleossomos , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Fatores de Transcrição/genética , Ubiquitina C/química , Enzimas de Conjugação de Ubiquitina/química , Xenopus , Proteínas de Xenopus/químicaRESUMO
Histone H3K4 methylation is an epigenetic mark associated with actively transcribed genes. This modification is catalyzed by the mixed lineage leukaemia (MLL) family of histone methyltransferases including MLL1, MLL2, MLL3, MLL4, SET1A and SET1B. The catalytic activity of this family is dependent on interactions with additional conserved proteins, but the structural basis for subunit assembly and the mechanism of regulation is not well understood. We used a hybrid methods approach to study the assembly and biochemical function of the minimally active MLL1 complex (MLL1, WDR5 and RbBP5). A combination of small angle X-ray scattering, cross-linking mass spectrometry, nuclear magnetic resonance spectroscopy and computational modeling were used to generate a dynamic ensemble model in which subunits are assembled via multiple weak interaction sites. We identified a new interaction site between the MLL1 SET domain and the WD40 ß-propeller domain of RbBP5, and demonstrate the susceptibility of the catalytic function of the complex to disruption of individual interaction sites.
Assuntos
Proteínas de Ligação a DNA/química , Histona-Lisina N-Metiltransferase/química , Histonas/química , Proteína de Leucina Linfoide-Mieloide/química , Catálise , Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisina/genética , Metilação , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína de Leucina Linfoide-Mieloide/genética , Domínios PR-SET/genética , Conformação Proteica , Mapas de Interação de Proteínas/genética , Repetições WD40/genéticaRESUMO
The Mixed Lineage Leukemia protein 1 (MLL1) plays an essential role in the maintenance of the histone H3 lysine 4 (H3K4) methylation status for gene expression during differentiation and development. The methyltransferase activity of MLL1 is regulated by three conserved core subunits, WDR5, RBBP5 and ASH2L. Here, we determined the structure of human RBBP5 and demonstrated its role in the assembly and regulation of the MLL1 complex. We identified an internal interaction between the WD40 propeller and the C-terminal distal region in RBBP5, which assisted the maintenance of the compact conformation of the MLL1 complex. We also discovered a vertebrate-specific motif in the C-terminal distal region of RBBP5 that contributed to nucleosome recognition and methylation of nucleosomes by the MLL1 complex. Our results provide new insights into functional conservation and evolutionary plasticity of the scaffold protein RBBP5 in the regulation of KMT2-family methyltransferase complexes.
Assuntos
Proteínas de Ligação a DNA/ultraestrutura , Histona-Lisina N-Metiltransferase/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Proteína de Leucina Linfoide-Mieloide/ultraestrutura , Fatores de Transcrição/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Conformação Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Nucleares/genética , Ligação Proteica/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Fatores de Transcrição/químicaRESUMO
The mammalian CLOCK:BMAL1 transcription factor complex and its coactivators CREB-binding protein (CBP)/p300 and mixed-lineage leukemia 1 (MLL1) critically regulate circadian transcription and chromatin modification. Circadian oscillations are regulated by interactions of BMAL1's C-terminal transactivation domain (TAD) with the KIX domain of CBP/p300 (activating) and with the clock protein CRY1 (repressing) as well as by the BMAL1 G-region preceding the TAD. Circadian acetylation of Lys537 within the G-region enhances repressive BMAL1-TAD-CRY1 interactions. Here, we characterized the interaction of the CBP-KIX domain with BMAL1 proteins, including the BMAL1-TAD, parts of the G-region, and Lys537 Tethering the small compound 1-10 in the MLL-binding pocket of the CBP-KIX domain weakened BMAL1 binding, and MLL1-bound KIX did not form a ternary complex with BMAL1, indicating that the MLL-binding pocket is important for KIX-BMAL1 interactions. Small-angle X-ray scattering (SAXS) models of BMAL1 and BMAL1:KIX complexes revealed that the N-terminal BMAL1 G-region including Lys537 forms elongated extensions emerging from the bulkier BMAL1-TAD:KIX core complex. Fitting high-resolution KIX domain structures into the SAXS-derived envelopes suggested that the G-region emerges near the MLL-binding pocket, further supporting a role of this pocket in BMAL1 binding. Additionally, mutations in the second CREB-pKID/c-Myb-binding pocket of the KIX domain moderately impacted BMAL1 binding. The BMAL1(K537Q) mutation mimicking Lys537 acetylation, however, did not affect the KIX-binding affinity, in contrast to its enhancing effect on CRY1 binding. Our results significantly advance the mechanistic understanding of the protein interaction networks controlling CLOCK:BMAL1- and CBP-dependent gene regulation in the mammalian circadian clock.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteína de Ligação a CREB/metabolismo , Relógios Circadianos , Fatores de Transcrição ARNTL/química , Fatores de Transcrição ARNTL/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína de Ligação a CREB/química , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
Mixed lineage leukemia protein (MLL1 protein) recognizes the CpG site via its CXXC domain and is frequently associated with leukemia. The specific recognition is abolished by C1188D mutation, which also prevents MLL-related leukemia. In this paper, multiple molecular dynamic (MD) simulations were performed to investigate the mechanism of recognition and influences of C1188D mutation. Started from fully dissociated DNA and MLL1-CXXC domain, remarkably, the center of mass (COM) of MLL1-CXXC domain quickly concentrates on the vicinity of the CpG site in all 53 short MD simulations. Extended simulations of the wild type showed that the native complex formed in 500 ns among 4 of 53 simulations. In contrast, the C1188D mutant COM distributed broadly around the DNA and the native complex was not observed in any of the extended simulations. Simulations on the apo MLL1-CXXC domain further suggest that the wild type protein remained predominantly in an open form that closely resembles its structure in the native complex whereas C1188D mutant formed predominantly compact structures in which the N- terminal bends to D1188. This conformational switch hinders the formation of encounter complex, thus abolishes the recognition. Our study also provides clues to the study mechanism of recognition, by the CXXC domain from proteins like DNA methyltransferase and ten-eleven translocation enzymes.
Assuntos
Ácido Aspártico/química , Cisteína/química , DNA/química , Histona-Lisina N-Metiltransferase/química , Mutação , Proteína de Leucina Linfoide-Mieloide/química , Substituição de Aminoácidos , Ácido Aspártico/metabolismo , Sítios de Ligação , Ilhas de CpG , Cisteína/metabolismo , DNA/genética , DNA/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Simulação de Dinâmica Molecular , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , TermodinâmicaRESUMO
The mixed lineage leukemia (MLL) family of proteins became known initially for the leukemia link of its founding member. Over the decades, the MLL family has been recognized as an important class of histone H3 lysine 4 (H3K4) methyltransferases that control key aspects of normal cell physiology and development. Here, we provide a brief history of the discovery and study of this family of proteins. We address two main questions: why are there so many H3K4 methyltransferases in mammals; and is H3K4 methylation their key function?
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Animais , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/classificação , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/classificação , Isoformas de Proteínas/metabolismoRESUMO
The highly-conserved Polycomb group (PcG) and trithorax group (trxG) proteins play major roles in regulating gene expression and maintaining developmental states in many organisms. However, neither the recruitment of Polycomb repressive complexes (PRC) nor the mechanisms of PcG and trxG-mediated gene silencing and activation are well understood. Recent progress in Arabidopsis research challenges the dominant model of PRC2-dependent recruitment of PRC1 to target genes. Moreover, evidence indicates that diverse forms of PRC1, with shared components, are a common theme in plants and mammals. Although trxG is known to antagonize PcG, emerging data reveal that trxG can also repress gene expression, acting cooperatively with PcG. We discuss these recent findings and highlight the employment of diverse epigenetic mechanisms during development in plants and animals.