RESUMEN
In nature, individual histones in the same nucleosome can carry identical (symmetric) or different (asymmetric) post-translational modification (PTM) patterns, increasing the combinatorial complexity. Embryonic stem cells exhibit "bivalent" nucleosomes, some of which are marked by an asymmetric arrangement of H3K36me3 (an activating PTM) and H3K27me3 (a repressive PTM). Here we describe a modular synthetic method to access such asymmetrically modified nucleosomes and show that H3K36me3 inhibits the activity of the methyltransferase PRC2 locally while still prolonging its chromatin binding time.
Asunto(s)
Histonas/química , Nucleosomas/química , Disulfuros/síntesis química , Disulfuros/química , Histonas/síntesis química , Histonas/metabolismo , Humanos , Lisina/química , Metilación , Nucleosomas/metabolismo , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Unión ProteicaRESUMEN
The cellular response to DNA damage results in a signaling cascade that primes chromatin for repair. Combinatorial post-translational modifications (PTMs) play an important role in this process by altering the physical properties of chromatin and recruiting downstream factors. One key signal integrator is the histone variant H2A.X, which is phosphorylated at a C-terminal serine (S139ph), and ubiquitylated within its N-terminal tail at lysines 13 and 15 (K13/15ub). How these PTMs directly impact chromatin structure and thereby facilitate DNA repair is not well understood. Detailed studies require synthetic access to such N- and C-terminally modified proteins. This is complicated by the requirement for protecting groups allowing multi-fragment assembly. Here, we report a semi-synthetic route to generate simultaneously N- and C-terminally modified proteins using genetically encoded orthogonal masking groups. Applied to H2A.X, expression of a central protein fragment, containing a protected N-terminal cysteine and a C-terminal thioester masked as a split intein, enables sequential C- and N-terminal protein modification and results in the convergent production of H2A.X carrying K15ub and S139ph. Using single-molecule FRET between defined nucleosomes in synthetic chromatin fibers, we then show that K15 ubiquitylation (but not S139ph) impairs nucleosome stacking in tetranucleosome units, opening chromatin during DNA repair.
RESUMEN
The regulation of fundamental processes such as gene expression or cell differentiation involves chromatin states, demarcated by combinatorial histone post-translational modification (PTM) patterns. The subnuclear organization and dynamics of chromatin states is not well understood, as tools for their detection and modulation in live cells are lacking. Here, we report the development of genetically encoded chromatin-sensing multivalent probes, cMAPs, selective for bivalent chromatin, a PTM pattern associated with pluripotency in embryonic stem cells (ESCs). cMAPs were engineered from a set of PTM-binding (reader) proteins and optimized using synthetic nucleosomes carrying defined PTMs. Applied in live ESCs, cMAPs formed discrete subnuclear foci, revealing the organization of bivalent chromatin into local clusters. Moreover, cMAPs enabled direct monitoring of the loss of bivalency upon treatment with small-molecule epigenetic modulators. cMAPs thus provide a versatile platform to monitor chromatin state dynamics in live cells.
Asunto(s)
Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Proteínas Luminiscentes/metabolismo , Ingeniería de Proteínas , Cromatina/genética , Humanos , Proteínas Luminiscentes/genética , Estructura Molecular , Procesamiento Proteico-PostraduccionalRESUMEN
Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.
Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Código de Histonas/fisiología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Homólogo de la Proteína Chromobox 5 , Epigénesis Genética , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Cinética , Ratones , Células 3T3 NIH , Fosforilación , Unión Proteica , Imagen Individual de MoléculaRESUMEN
In stem cells, H4 proteins carrying different modifications coexist within single nucleosomes. For functional studies, we report the synthesis of such asymmetric nucleosomes. Asymmetry is achieved by transiently crosslinking H4 by a traceless, protease-removable tag introduced via an isopeptide linkage. These nucleosomes are used to study Set8 activity, a key methyltransferase.
RESUMEN
Nucleosomes carry extensive post-translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysineâ 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27-specific lysine methyltransferase (KMT) polycomb repressive complexâ 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3-mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.
Asunto(s)
Nucleosomas/química , Electroforesis en Gel de Poliacrilamida , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Nucleosomas/metabolismoRESUMEN
Biomineralization processes leading to complex solid structures of inorganic material in biological systems are constantly gaining attention in biotechnology and biomedical research. An outstanding example for biomineral morphogenesis is the formation of highly elaborate, nano-patterned silica shells by diatoms. Among the organic macromolecules that have been closely linked to the tightly controlled precipitation of silica in diatoms, silaffins play an extraordinary role. These peptides typically occur as complex posttranslationally modified variants and are directly involved in the silica deposition process in diatoms. However, even in vitro silaffin-based peptides alone, with and without posttranslational modifications, can efficiently mediate biomimetic silica precipitation leading to silica material with different properties as well as with encapsulated cargo molecules of a large size range. In this review, the biomineralization process of silica in diatoms is summarized with a specific focus on silaffins and their in vitro silica precipitation properties. Applications in the area of bio- and nanotechnology as well as in diagnostics and therapy are discussed.
Asunto(s)
Materiales Biomiméticos/química , Péptidos/química , Dióxido de Silicio/química , Animales , Materiales Biomiméticos/farmacología , Biomimética/métodos , Biotecnología/métodos , Diatomeas/química , Nanotecnología/métodos , Péptidos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
Immobilisation of proteins on solid supports such as silica is commonly applied to improve performance of enzymes under detrimental conditions and to allow enzyme recycling. Silica biomineralisation processes occurring in nature have recently inspired approaches towards mild, biomimetic silica formation. In diatoms, complex posttranslationally modified silaffin peptides are directly involved in formation and patterning of silica cell walls. Here, chemically modified silaffin peptides are used to establish a novel strategy for silica immobilisation of target proteins. Silaffin variants carrying different modifications are covalently linked to eGFP and thioredoxin using expressed protein ligation. Covalent eGFP- and thioredoxin-silaffin conjugates are able to efficiently precipitate silica and control silica properties by choice of different silaffin modifications leading to functional encapsulation of these proteins in silica particles. Covalent protein-silaffin conjugates lead to a distinctly more efficient and homogenous encapsulation of proteins in silica, superior to random protein entrapment resulting from simple co-precipitation. Silica-immobilised proteins are confirmed to be fully active and stabilised against denaturation.
Asunto(s)
Proteínas Inmovilizadas/química , Péptidos/química , Desnaturalización Proteica/efectos de los fármacos , Dióxido de Silicio/química , Tiorredoxinas/química , Fenómenos Bioquímicos , Biomimética/métodos , Proteínas Inmovilizadas/metabolismo , Péptidos/metabolismo , Dióxido de Silicio/metabolismo , Tiorredoxinas/metabolismoRESUMEN
The R5 peptide is derived from silaffin peptides naturally occurring in the diatom Cylindrotheca fusiformis and exhibits outstanding activity in silica precipitation. Because of its ability to cause silicification under mild conditions, several biotechnological applications based on R5-mediated biomimetic silica formation have already been reported. Yet a more detailed understanding of the R5 peptide and its intrinsic silica precipitation activity will help the rational design of R5 peptide variants as efficient agents for defined silica precipitation. The herein presented analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation. Furthermore, a tetra amino acid motif (RRIL) has to be present within the R5 sequence, but in contrast to previous reports, we demonstrate that localization of the RRIL motif shows minor impact on silica precipitation activity but rather on morphology of the resulting silica material. The amino acid sequence of silaffin peptides is a well-balanced arrangement in terms of charges, functional groups and distances. The impact of this pattern of charges and functionalities was highlighted by the disturbed morphology of silica spheres resulting from R5 variants with scrambled sequences. A detailed understanding of the highly evolved silaffin sequence(s) will contribute to unravel the intriguing process of silica biomineralization in diatoms.
Asunto(s)
Fragmentos de Péptidos/química , Péptidos/química , Precursores de Proteínas/química , Ácido Silícico/química , Dióxido de Silicio/química , Microscopía Electrónica de Rastreo , Modelos Moleculares , Dióxido de Silicio/análisisRESUMEN
Biomimetic silica formation has attracted increasing interest over the last decade for numerous biotechnological applications due to the favorable mild reaction conditions. Inspired from silica biogenesis in diatoms, peptide variants derived from native silaffins have been used for silica formation in vitro. Here a generally applicable route for covalently linking a cargo molecule to the R5 silaffin peptide via a disulfide linkage is established. The peptide CG12AB, a peptide ligand of the epidermal growth factor receptor, was chosen as model. The ability of such silaffin-cargo conjugates to encapsulate the cargo molecule during silaffin-mediated silica precipitation is demonstrated. Cargo release from silica material under different conditions was analyzed. The results obtained here provide a rational basis for developing engineered R5 silaffin peptides into efficient tools for silica precipitation as well as for entrapment and release of cargo molecules under physiological conditions.