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Histones: at the crossroads of peptide and protein chemistry.

Müller MM, Muir TW - Chem. Rev. (2014)

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Princeton University, Frick Laboratory, Princeton, New Jersey 08544, United States.

ABSTRACT

Peptide and protein chemistry have become an integral part of chromatinresearch. Methods ranging from solid-phase synthesis to recombinanttechnology are available to construct site-specifically modified histonepeptides and chromatin templates with distinct patterns. In particular,a plethora of histones carrying PTMs and their analogues have beengenerated in a chemically defined fashion (Figure 60). These reagents can directly feed into cutting edge biochemicaland biophysical pipelines, including transcription assays341 or structural studies based on X-ray crystallography,282,488,489 electron microscopy,283,490 or NMR spectroscopy.491,492 Given these advances,what are the remaining challenges and opportunities for protein chemiststo further contribute to unraveling the mechanism of histone-basedsignaling?

Severalpeptide- and mononucleosome based approaches have been developedto biochemically address the enormous combinatorial possibilitiesof histone PTM combinations. Nevertheless, methods to synthesize hundredsof proteins in parallel are still lacking. Innovative purificationschemes or reliable fragment condensation protocols that allow bypassingof individual workup steps are needed to attain the level of throughputthat peptide synthesis can achieve. Furthermore, can the resultinghistone libraries be incorporated into templates that more closelyreflect the heteropolymeric nature of chromatin fibers? Such arrayswill enable dissection of spatial components that underlie the controlof chromatin-templated processes, both on a biophysical and on a biochemicallevel. Specific aspects that remain largely unanswered include questionsconcerning how, which, and if defined PTM patterns alter structuressynergistically, and whether these structural perturbations are propagatedalong the chromatin fiber beyond the actual installation site. Transcriptionis a vectorial process, and chromatin architecture contributes todefining the coordinates of the origin and direction of polymeraseaction. At which level do histone PTM gradients facilitate guidanceof the transcription machinery, and in what ways do these modificationpatterns also contribute to local memory of transcriptional states?We believe that some of these issues can be addressed with next-generationchromatin biochemistry on the foundation of designer histones.

The vast majorityof contributions that protein chemistry has so far made in the chromatinbiochemistry area have revolved around histone modifications. Yet,many other chromatin-associated proteins are hubs for PTMs, in particularRNA polymerase and coactivators such as p53. In addition, many enzymescharacterized as histone methyl- and acetyltransferases also act onnonhistone targets. Thus, elucidating the mechanism of these processesrequires that the chemical toolkit, originating in basic research,and since refined for histone synthesis, be extended to the manufactureof other cellular factors that are considerably larger than histones.Semisyntheses of p53 (ref (493)), a bacterial RNA polymerase,494 as well as the p300 HAT domain495 havealready been achieved, laying the groundwork for systems-wide analysisof how PTM-based nuclear signaling affects transcription.

Modifiedhistones have contributed immensely to biochemical andbiophysical analyses in vitro. What are the prospects of implementingthese reagents in vivo to elucidate the mechanism of their action?Currently, access to specifically modified histones in vivo is mainlylimited to genetic strategies. Typical examples include site-directedmutagenesis of a target histone residue, such as Lys-to-Gln or Lys-to-Argsubstitutions to mimic acetyllysine side-chains and preclude methylationor acetylation at that position, respectively.290,496 Alternatively, overexpression of a histone-modifying enzyme canresult in global accumulation of a desired PTM. Upon targeting enzymesto specific genomic sites (using the Gal4 system, or perhaps CRISPR-CAS9),perturbations can be localized to genetic reporters, enabling moredefined functional assignments of histone modifying activities. Inconjunction with reversible dimerization modules, these targetingstrategies can shed light on the kinetics of the formation and interpretationof histone PTMs.497 Artificial expansionof the genetic code further diversifies the scope of genetic approachesto study the effect of histone modifications, as exemplified by therecent success in tracking structural consequences of mitotic histonephosphorylation.380 A multitude of bioorthogonalreactions,498 as well as the ability toperform protein trans-splicing in vivo,499,500 might also aid in generating designer chromatin in living cells.

Fueled by diverse success stories since the late 1960s, the journeyfor chemical biologists into the chromatin field continues. Many excitingmilestones still lie ahead, with key challenges involving the vastnessof the combinatorial landscape of histone modifications and the complexityof their interpretation in a cellular context. Whether these routeslead to high-throughput biochemistry or meander into cells, the journeypromises to be extremely fruitful. These efforts will likely contributeto the rich tradition at the intersection of peptide and protein chemistrywith histone biology, and target a systems-level understanding ofhow cellular signaling converges on chromatin and is relayed intofunctional outputs.

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K56ac (black) increasesbreathing of nucleosomal DNA.
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fig33: K56ac (black) increasesbreathing of nucleosomal DNA.

Mentions: In the context of histones, the ability to genetically encode residuescontaining PTMs has been tremendously useful. In particular, acetyllysinecan be integrated into ribosomal protein synthesis using an engineeredaaRS originally dedicated to pyrrolysine in Methanosarcinabarkeri.350 Neumann etal. harnessed the amber suppression strategy to define the biophysicaland biochemical effects of H3K56 acetylation.351 By replacing the codon that specifies lysine 56 with anamber stop codon, and supplementing the growth medium with acetyllysine,the authors were able to produce H3 homogeneously modified with K56acin E. coli carrying the orthogonaltRNACUA and the evolved aaRS. This protein was subsequentlyincorporated into nucleosomes and nucleosome arrays using standardtechniques. Surprisingly, K56ac did not alter chromatin compaction,and only had minor effects on chromatin remodeling by bromodomain-containingmotor proteins. In contrast, single-molecule FRET measurements revealedthat DNA “breathing” was enhanced in K56ac-containingmononucleosomes as compared to unmodified versions, consistent withthe position of K56 close to the DNA entry/exit site (Figure 33).351 K56ac marks alsofacilitated binding of the pluripotency factor Oct4 to nucleosomesin vitro,352 yet inhibited interactionswith the components of the yeast silencing apparatus Sir2–4.353


Histones: at the crossroads of peptide and protein chemistry.

Müller MM, Muir TW - Chem. Rev. (2014)

K56ac (black) increasesbreathing of nucleosomal DNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4378460&req=5

fig33: K56ac (black) increasesbreathing of nucleosomal DNA.
Mentions: In the context of histones, the ability to genetically encode residuescontaining PTMs has been tremendously useful. In particular, acetyllysinecan be integrated into ribosomal protein synthesis using an engineeredaaRS originally dedicated to pyrrolysine in Methanosarcinabarkeri.350 Neumann etal. harnessed the amber suppression strategy to define the biophysicaland biochemical effects of H3K56 acetylation.351 By replacing the codon that specifies lysine 56 with anamber stop codon, and supplementing the growth medium with acetyllysine,the authors were able to produce H3 homogeneously modified with K56acin E. coli carrying the orthogonaltRNACUA and the evolved aaRS. This protein was subsequentlyincorporated into nucleosomes and nucleosome arrays using standardtechniques. Surprisingly, K56ac did not alter chromatin compaction,and only had minor effects on chromatin remodeling by bromodomain-containingmotor proteins. In contrast, single-molecule FRET measurements revealedthat DNA “breathing” was enhanced in K56ac-containingmononucleosomes as compared to unmodified versions, consistent withthe position of K56 close to the DNA entry/exit site (Figure 33).351 K56ac marks alsofacilitated binding of the pluripotency factor Oct4 to nucleosomesin vitro,352 yet inhibited interactionswith the components of the yeast silencing apparatus Sir2–4.353

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Princeton University, Frick Laboratory, Princeton, New Jersey 08544, United States.

ABSTRACT

Peptide and protein chemistry have become an integral part of chromatinresearch. Methods ranging from solid-phase synthesis to recombinanttechnology are available to construct site-specifically modified histonepeptides and chromatin templates with distinct patterns. In particular,a plethora of histones carrying PTMs and their analogues have beengenerated in a chemically defined fashion (Figure 60). These reagents can directly feed into cutting edge biochemicaland biophysical pipelines, including transcription assays341 or structural studies based on X-ray crystallography,282,488,489 electron microscopy,283,490 or NMR spectroscopy.491,492 Given these advances,what are the remaining challenges and opportunities for protein chemiststo further contribute to unraveling the mechanism of histone-basedsignaling?

Severalpeptide- and mononucleosome based approaches have been developedto biochemically address the enormous combinatorial possibilitiesof histone PTM combinations. Nevertheless, methods to synthesize hundredsof proteins in parallel are still lacking. Innovative purificationschemes or reliable fragment condensation protocols that allow bypassingof individual workup steps are needed to attain the level of throughputthat peptide synthesis can achieve. Furthermore, can the resultinghistone libraries be incorporated into templates that more closelyreflect the heteropolymeric nature of chromatin fibers? Such arrayswill enable dissection of spatial components that underlie the controlof chromatin-templated processes, both on a biophysical and on a biochemicallevel. Specific aspects that remain largely unanswered include questionsconcerning how, which, and if defined PTM patterns alter structuressynergistically, and whether these structural perturbations are propagatedalong the chromatin fiber beyond the actual installation site. Transcriptionis a vectorial process, and chromatin architecture contributes todefining the coordinates of the origin and direction of polymeraseaction. At which level do histone PTM gradients facilitate guidanceof the transcription machinery, and in what ways do these modificationpatterns also contribute to local memory of transcriptional states?We believe that some of these issues can be addressed with next-generationchromatin biochemistry on the foundation of designer histones.

The vast majorityof contributions that protein chemistry has so far made in the chromatinbiochemistry area have revolved around histone modifications. Yet,many other chromatin-associated proteins are hubs for PTMs, in particularRNA polymerase and coactivators such as p53. In addition, many enzymescharacterized as histone methyl- and acetyltransferases also act onnonhistone targets. Thus, elucidating the mechanism of these processesrequires that the chemical toolkit, originating in basic research,and since refined for histone synthesis, be extended to the manufactureof other cellular factors that are considerably larger than histones.Semisyntheses of p53 (ref (493)), a bacterial RNA polymerase,494 as well as the p300 HAT domain495 havealready been achieved, laying the groundwork for systems-wide analysisof how PTM-based nuclear signaling affects transcription.

Modifiedhistones have contributed immensely to biochemical andbiophysical analyses in vitro. What are the prospects of implementingthese reagents in vivo to elucidate the mechanism of their action?Currently, access to specifically modified histones in vivo is mainlylimited to genetic strategies. Typical examples include site-directedmutagenesis of a target histone residue, such as Lys-to-Gln or Lys-to-Argsubstitutions to mimic acetyllysine side-chains and preclude methylationor acetylation at that position, respectively.290,496 Alternatively, overexpression of a histone-modifying enzyme canresult in global accumulation of a desired PTM. Upon targeting enzymesto specific genomic sites (using the Gal4 system, or perhaps CRISPR-CAS9),perturbations can be localized to genetic reporters, enabling moredefined functional assignments of histone modifying activities. Inconjunction with reversible dimerization modules, these targetingstrategies can shed light on the kinetics of the formation and interpretationof histone PTMs.497 Artificial expansionof the genetic code further diversifies the scope of genetic approachesto study the effect of histone modifications, as exemplified by therecent success in tracking structural consequences of mitotic histonephosphorylation.380 A multitude of bioorthogonalreactions,498 as well as the ability toperform protein trans-splicing in vivo,499,500 might also aid in generating designer chromatin in living cells.

Fueled by diverse success stories since the late 1960s, the journeyfor chemical biologists into the chromatin field continues. Many excitingmilestones still lie ahead, with key challenges involving the vastnessof the combinatorial landscape of histone modifications and the complexityof their interpretation in a cellular context. Whether these routeslead to high-throughput biochemistry or meander into cells, the journeypromises to be extremely fruitful. These efforts will likely contributeto the rich tradition at the intersection of peptide and protein chemistrywith histone biology, and target a systems-level understanding ofhow cellular signaling converges on chromatin and is relayed intofunctional outputs.

Show MeSH