RESUMO
Histone deacylases are erasers of Nε-acyl-lysine post-translational modifications and have been targeted for decades for the treatment of cancer, neurodegeneration and other disorders. Due to their relatively promiscuous activity on peptide substrates in vitro, it has been challenging to determine the individual targets and substrate identification mechanisms of each isozyme, and they have been considered redundant regulators. In recent years, biochemical and biophysical studies have incorporated the use of reconstituted nucleosomes, which has revealed a diverse and complex arsenal of recognition mechanisms by which histone deacylases may differentiate themselves in vivo. In this review, we first present the peptide-based tools that have helped characterize histone deacylases in vitro to date, and we discuss the new insights that nucleosome tools are providing into their recognition of histone substrates within chromatin. Then, we summarize the powerful semi-synthetic approaches that are moving forward the study of chromatin-associated factors, both in vitro by detailed single-molecule mechanistic studies, and in cells by live chromatin modification. We finally offer our perspective on how these new techniques would advance the study of histone deacylases. We envision that such studies will help elucidate the role of individual isozymes in disease and provide a platform for the development of the next generation of therapeutics.
Assuntos
Cromatina , Histonas , Histonas/metabolismo , Nucleossomos , Processamento de Proteína Pós-Traducional , Peptídeos , AcetilaçãoRESUMO
Since Strahl and Allis proposed the "language of covalent histone modifications", a host of experimental studies have shed light on the different facets of chromatin regulation by epigenetic mechanisms. Initially proposed as a concept for controlling gene transcription, the regulation of deposition and removal of histone post-translational modifications (PTMs), such as acetylation, methylation, and phosphorylation, have been implicated in many chromatin regulation pathways. However, large PTMs such as ubiquitylation challenge research on many levels due to their chemical complexity. In recent years, chemical tools have been developed to generate chromatin in defined ubiquitylation states in vitro. Chemical biology approaches are now used to link specific histone ubiquitylation marks with downstream chromatin regulation events on the molecular level. Here, we want to highlight how chemical biology approaches have empowered the mechanistic study of chromatin ubiquitylation in the context of gene regulation and DNA repair with attention to future challenges.
Assuntos
Cromatina , Histonas , Ubiquitinação , Cromatina/química , Cromatina/metabolismo , Histonas/química , Histonas/metabolismo , Transcrição GênicaRESUMO
Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Nucleossomos , DNA/metabolismo , Autoantígenos/metabolismoRESUMO
Rapid and efficient cyclization methods that form structurally novel peptidic macrocycles are of high importance for medicinal chemistry. Herein, we report the first gold(I)-catalyzed macrocyclization of peptide-EBXs (ethynylbenziodoxolones) via C2-Trp C-H activation. This reaction was carried out in the presence of protecting group free peptide sequences and is enabled by a simple commercial gold catalyst (AuCl·Me2S). The method displayed a rapid reaction rate (within 10 min), wide functional group tolerance (27 unprotected peptides were cyclized), and up to 86% isolated yield. The obtained highly conjugated cyclic peptide linker, formed through C-H alkynylation, can be directly applied to live-cell imaging as a fluorescent probe without further attachment of fluorophores.
Assuntos
Peptídeos Cíclicos , Peptídeos , Sequência de Aminoácidos , Ciclização , Catálise , Corantes FluorescentesRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , DNA , Histonas , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/genética , DNA/metabolismo , Sequências Hélice-Alça-Hélice/genética , Histonas/química , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Proteínas CLOCK/química , Proteínas CLOCK/metabolismo , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/metabolismo , Regulação Alostérica , Zíper de Leucina , Fator 3 de Transcrição de Octâmero/metabolismo , Multimerização ProteicaRESUMO
Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.
Assuntos
Microtúbulos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Processamento de Proteína Pós-Traducional , Ligação ProteicaRESUMO
Physical interactions between proteins are essential for most biological processes governing life1. However, the molecular determinants of such interactions have been challenging to understand, even as genomic, proteomic and structural data increase. This knowledge gap has been a major obstacle for the comprehensive understanding of cellular protein-protein interaction networks and for the de novo design of protein binders that are crucial for synthetic biology and translational applications2-9. Here we use a geometric deep-learning framework operating on protein surfaces that generates fingerprints to describe geometric and chemical features that are critical to drive protein-protein interactions10. We hypothesized that these fingerprints capture the key aspects of molecular recognition that represent a new paradigm in the computational design of novel protein interactions. As a proof of principle, we computationally designed several de novo protein binders to engage four protein targets: SARS-CoV-2 spike, PD-1, PD-L1 and CTLA-4. Several designs were experimentally optimized, whereas others were generated purely in silico, reaching nanomolar affinity with structural and mutational characterization showing highly accurate predictions. Overall, our surface-centric approach captures the physical and chemical determinants of molecular recognition, enabling an approach for the de novo design of protein interactions and, more broadly, of artificial proteins with function.
Assuntos
Simulação por Computador , Aprendizado Profundo , Ligação Proteica , Proteínas , Humanos , Proteínas/química , Proteínas/metabolismo , Proteômica , Mapas de Interação de Proteínas , Sítios de Ligação , Biologia SintéticaRESUMO
Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.
Assuntos
Proteínas Cromossômicas não Histona , Heterocromatina , Animais , Camundongos , Histonas/metabolismo , Cromatina , Homólogo 5 da Proteína Cromobox , Peptídeos/metabolismoRESUMO
The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.
Assuntos
Cromatina , Proteínas de Drosophila , Animais , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Histonas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Nucleossomos , Drosophila/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismoRESUMO
Highly charged intrinsically disordered proteins are essential regulators of chromatin structure and transcriptional activity. Here we identify a surprising mechanism of molecular competition that relies on the pronounced dynamical disorder present in these polyelectrolytes and their complexes. The highly positively charged human linker histone H1.0 (H1) binds to nucleosomes with ultrahigh affinity, implying residence times incompatible with efficient biological regulation. However, we show that the disordered regions of H1 retain their large-amplitude dynamics when bound to the nucleosome, which enables the highly negatively charged and disordered histone chaperone prothymosin α to efficiently invade the H1-nucleosome complex and displace H1 via a competitive substitution mechanism, vastly accelerating H1 dissociation. By integrating experiments and simulations, we establish a molecular model that rationalizes the remarkable kinetics of this process structurally and dynamically. Given the abundance of polyelectrolyte sequences in the nuclear proteome, this mechanism is likely to be widespread in cellular regulation.
Assuntos
Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Nucleossomos/metabolismo , Polieletrólitos/metabolismo , HumanosRESUMO
Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.
Assuntos
NucleossomosRESUMO
The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.
Assuntos
Proteínas de Ciclo Celular , Heterocromatina , Código das Histonas/genética , Histona-Lisina N-Metiltransferase , Histonas , Proteínas de Schizosaccharomyces pombe , Domínio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilação de DNA/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinação/genéticaRESUMO
Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein's endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein-DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.
Assuntos
Acetilglucosamina/metabolismo , Dano ao DNA , Proteína HMGB1/metabolismo , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Sistema Livre de Células , Reparo do DNA , Proteína HMGB1/genética , Humanos , MutaçãoRESUMO
Epigenetics research focuses on the study of heritable gene regulatory mechanisms that do not involve changes of the DNA sequence. Such mechanisms include post-translational modifications of histone proteins that organize the genome in the nucleus into a nucleoprotein complex called chromatin, and which are of key importance in development and disease. Chemical biology tools as developed by my group, in particular synthetic peptide and protein chemistry, have been critical to elucidate epigenetic signaling mechanisms. As outlined below, they allow the reconstitution of chromatin carrying defined modifications and thus the elucidation of detailed molecular mechanisms.
Assuntos
Epigênese Genética , Processamento de Proteína Pós-Traducional , Cromatina , Histonas/genética , Histonas/metabolismo , PeptídeosRESUMO
The centromere is an essential chromatin domain required for kinetochore recruitment and chromosome segregation in eukaryotes. To perform this role, centro-chromatin adopts a unique structure that provides access to kinetochore proteins and maintains stability under tension during mitosis. This is achieved by the presence of nucleosomes containing the H3 variant CENP-A, which also acts as the epigenetic mark defining the centromere. In this review, we discuss the role of CENP-A on the structure and dynamics of centromeric chromatin. We further discuss the impact of the CENP-A binding proteins CENP-C, CENP-N, and CENP-B on modulating centro-chromatin structure. Based on these findings we provide an overview of the higher order structure of the centromere.
Assuntos
Proteína Centromérica A/química , Proteína B de Centrômero/química , Centrômero/ultraestrutura , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Humanos , Mitose , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
The inner nuclear membrane is functionalized by diverse transmembrane proteins that associate with nuclear lamins and/or chromatin. When cells enter mitosis, membrane-chromatin contacts must be broken to allow for proper chromosome segregation; yet how this occurs remains ill-understood. Unexpectedly, we observed that an imbalance in the levels of the lamina-associated polypeptide 1 (LAP1), an activator of ER-resident Torsin AAA+-ATPases, causes a failure in membrane removal from mitotic chromatin, accompanied by chromosome segregation errors and changes in post-mitotic nuclear morphology. These defects are dependent on a hitherto unknown chromatin-binding region of LAP1 that we have delineated. LAP1-induced NE abnormalities are efficiently suppressed by expression of wild-type but not ATPase-deficient Torsins. Furthermore, a dominant-negative Torsin induces chromosome segregation defects in a LAP1-dependent manner. These results indicate that association of LAP1 with chromatin in the nucleus can be modulated by Torsins in the perinuclear space, shedding new light on the LAP1-Torsin interplay.
Assuntos
Cromatina/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Choque Térmico HSC70/metabolismo , Mitose/fisiologia , Chaperonas Moleculares/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Células HCT116 , Proteínas de Choque Térmico HSC70/genética , Células HeLa , Células Hep G2 , Humanos , Chaperonas Moleculares/genética , Membrana Nuclear/metabolismoRESUMO
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.
Assuntos
Microscopia Crioeletrônica , Nucleossomos/metabolismo , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Células HeLa , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutação , Nucleossomos/química , Nucleossomos/ultraestrutura , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/ultraestruturaRESUMO
The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/imunologia , Imunidade Inata , Nucleotidiltransferases/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Membrana Nuclear/metabolismoRESUMO
The essential human enzyme lysine specific demethylase 1 (LSD1) silences genes by demethylating mono- and dimethylated lysine 4 in histone H3 (H3K4me1/2). Studies of the minimal requirements for LSD1 activity are complicated by the heterogeneity of histone modification states in cells. We overcame this challenge by generating homogeneous mononucleosome substrates containing semisynthetic H3K4me2. Biophysical and biochemical assays with full-length LSD1 revealed its ability to bind and demethylate nucleosomes. Consistent with a requirement for nucleosome binding prior to demethylation, a competing nucleosome-binding peptide from the high-mobility group protein effectively inhibited LSD1 activity. Thus, our studies provide the first glimpse of nucleosome demethylation by LSD1 in the absence of other scaffolding proteins.
Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , Histona Desmetilases/química , Histona Desmetilases/isolamento & purificação , Histonas/química , Humanos , Metilação , Modelos Moleculares , Nucleossomos/química , Ligação ProteicaRESUMO
Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.