Unnatural Amino Acid Crosslinking for Increased Spatiotemporal Resolution of Chromatin Dynamics.

The utilization of an expanded genetic code and in vivo unnatural amino acid crosslinking has grown significantly in the past decade, proving to be a reliable system for the examination of protein-protein interactions. Perhaps the most utilized amino acid crosslinker, p-benzoyl-(l)-phenylalanine (pBPA), has delivered a vast compendium of structural and mechanistic data, placing it firmly in the upper echelons of protein analytical techniques. pBPA contains a benzophenone group that is activated with low energy radiation (~365 nm), initiating a diradical state that can lead to hydrogen abstraction and radical recombination in the form of a covalent bond to a neighboring protein. Importantly, the expanded genetic code system provides for site-specific encoding of the crosslinker, yielding spatial control for protein surface mapping capabilities. Paired with UV-activation, this process offers a practical means for spatiotemporal understanding of protein-protein dynamics in the living cell. The chromatin field has benefitted particularly well from this technique, providing detailed mapping and mechanistic insight for numerous chromatin-related pathways. We provide here a brief history of unnatural amino acid crosslinking in chromatin studies and outlooks into future applications of the system for increased spatiotemporal resolution in chromatin related research.

Spanning the gap: unraveling RSC dynamics in vivo.

Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.

Interaction of RSC Chromatin Remodeling Complex with Nucleosomes Is Modulated by H3 K14 Acetylation and H2B SUMOylation In Vivo.

Chromatin remodeling complexes are multi-subunit nucleosome translocases that reorganize chromatin in the context of DNA replication, repair, and transcription. To understand how these complexes find their target sites on chromatin, we use genetically encoded photo-cross-linker amino acids to map the footprint of Sth1, the catalytic subunit of the RSC complex, on nucleosomes in living yeast. We find that H3 K14 acetylation induces the interaction of the Sth1 bromodomain with the H3 tail and mediates the interaction of RSC with neighboring nucleosomes rather than recruiting it to chromatin. RSC preferentially resides on H2B SUMOylated nucleosomes in vivo and shows a moderately enhanced affinity due to this modification in vitro. Furthermore, RSC is not ejected from chromatin in mitosis, but changes its mode of nucleosome binding. Our in vivo analyses show that RSC recruitment to specific chromatin targets involves multiple histone modifications likely in combination with histone variants and transcription factors.

Axial contraction and short-range compaction of chromatin synergistically promote mitotic chromosome condensation.

The segregation of eukaryotic chromosomes during mitosis requires their extensive folding into units of manageable size for the mitotic spindle. Here, we report on how phosphorylation at serine 10 of histone H3 (H3 S10) contributes to this process. Using a fluorescence-based assay to study local compaction of the chromatin fiber in living yeast cells, we show that chromosome condensation entails two temporally and mechanistically distinct processes. Initially, nucleosome-nucleosome interaction triggered by H3 S10 phosphorylation and deacetylation of histone H4 promote short-range compaction of chromatin during early anaphase. Independently, condensin mediates the axial contraction of chromosome arms, a process peaking later in anaphase. Whereas defects in chromatin compaction have no observable effect on axial contraction and condensin inactivation does not affect short-range chromatin compaction, inactivation of both pathways causes synergistic defects in chromosome segregation and cell viability. Furthermore, both pathways rely at least partially on the deacetylase Hst2, suggesting that this protein helps coordinating chromatin compaction and axial contraction to properly shape mitotic chromosomes.

Genetically encoding lysine modifications on histone H4.

Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.

A cascade of histone modifications induces chromatin condensation in mitosis.

Metaphase chromosomes are visible hallmarks of mitosis, yet our understanding of their structure and of the forces shaping them is rudimentary. Phosphorylation of histone H3 serine 10 (H3 S10) by Aurora B kinase is a signature event of mitosis, but its function in chromatin condensation is unclear. Using genetically encoded ultraviolet light-inducible cross-linkers, we monitored protein-protein interactions with spatiotemporal resolution in living yeast to identify the molecular details of the pathway downstream of H3 S10 phosphorylation. This modification leads to the recruitment of the histone deacetylase Hst2p that subsequently removes an acetyl group from histone H4 lysine 16, freeing the H4 tail to interact with the surface of neighboring nucleosomes and promoting fiber condensation. This cascade of events provides a condensin-independent driving force of chromatin hypercondensation during mitosis.

Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids.

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.

Site-specific incorporation of fluorotyrosines into proteins in Escherichia coli by photochemical disguise.

Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the total synthesis of proteins. As a general solution to this problem, we genetically encoded the unnatural amino acids o-nitrobenzyl-2-fluorotyrosine, -3-fluorotyrosine, and -2,6-difluorotyrosine in Escherichia coli. These amino acids are disguised from recognition by the endogenous protein biosynthetic machinery, effectively preventing global incorporation of fluorotyrosine into proteins.

Photochemical control of FlAsH labeling of proteins.

Spatiotemporal control of protein fluorescence is a powerful tool in tracking protein movements within cells. Here we report an approach to using genetically encoded photo-caged amino acids to control labeling protein tetracysteine tags with biarsenical fluorescein dyes (FlAsH).

Peptide mass fingerprinting using isotopically encoded photo-crosslinking amino acids.

When isotopically labelled photo-crosslinking amino acids are site-specifically incorporated into proteins, in combination with the corresponding non-labeled analogue, cross-linked tryptic peptides are easily identified in mass spectra via characteristic "doublet" patterns.