Parental histone transfer caught at the replication fork.

In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.

New advisory board members speak on challenges and opportunities in chemical biology.

Cell Chemical Biology has recently added several advisory board members from China in an effort to better represent our authorship and readership. In this Voices piece, a few of the new members introduce themselves, give their perspective on challenges and opportunities in chemical biology, and discuss how they plan to contribute to the field through their new position.

Synergism between CMG helicase and leading strand DNA polymerase at replication fork.

The replisome that replicates the eukaryotic genome consists of at least three engines: the Cdc45-MCM-GINS (CMG) helicase that separates duplex DNA at the replication fork and two DNA polymerases, one on each strand, that replicate the unwound DNA. Here, we determined a series of cryo-electron microscopy structures of a yeast replisome comprising CMG, leading-strand polymerase Polε and three accessory factors on a forked DNA. In these structures, Polε engages or disengages with the motor domains of the CMG by occupying two alternative positions, which closely correlate with the rotational movement of the single-stranded DNA around the MCM pore. During this process, the polymerase remains stably coupled to the helicase using Psf1 as a hinge. This synergism is modulated by a concerted rearrangement of ATPase sites to drive DNA translocation. The Polε-MCM coupling is not only required for CMG formation to initiate DNA replication but also facilitates the leading-strand DNA synthesis mediated by Polε. Our study elucidates a mechanism intrinsic to the replisome that coordinates the activities of CMG and Polε to negotiate any roadblocks, DNA damage, and epigenetic marks encountered during translocation along replication forks.

Recent advances in the development of peptide-based inhibitors targeting epigenetic readers of histone lysine acetylation and methylation marks.

Inhibitors for epigenetic readers of histone modifications are useful chemical probes to interrogate the functional roles played by their cognate targets in epigenetic regulation and can even serve as drugs for the treatment of diseases associated with the dysregulated targets. However, many epigenetic readers are intractable to small molecules, as the recognition of modified histone peptides commonly involves flat and extended protein surfaces. In contrast, the relatively large sizes and structural complexity of peptides help them achieve tight and specific binding to the target proteins. Increasing efforts have been made to target epigenetic readers using peptide-based inhibitors that can complement small molecules. In this review, we discuss the recent advances in the development of peptide-based inhibitors of lysine acetylation and methylation readers.

Integrated regulation of tubulin tyrosination and microtubule stability by human α-tubulin isotypes.

Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based in vitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.

Development of multifunctional synthetic nucleosomes to interrogate chromatin-mediated protein interactions.

Various proteins bind to chromatin to regulate DNA and its associated processes such as replication, transcription, and damage repair. The identification and characterization of these chromatin-associating proteins remain a challenge, as their interactions with chromatin often occur within the context of the local nucleosome or chromatin structure, which makes conventional peptide-based strategies unsuitable. Here, we developed a simple and robust protein labeling chemistry to prepare synthetic multifunctional nucleosomes that carry a photoreactive group, a biorthogonal handle, and a disulfide moiety to examine chromatin-protein interactions in a nucleosomal context. Using the prepared protein- and nucleosome-based photoaffinity probes, we examined a number of protein-protein and protein-nucleosome interactions. In particular, we (i) mapped the binding sites for the HMGN2-nucleosome interaction, (ii) provided the evidence for transition between the active and poised states of DOT1L in recognizing H3K79 within the nucleosome, and (iii) identified OARD1 and LAP2α as nucleosome acidic patch-associating proteins. This study provides powerful and versatile chemical tools for interrogating chromatin-associating proteins.

Autoinhibitory mechanism controls binding of centrosomin motif 1 to γ-tubulin ring complex.

The γ-tubulin ring complex (γTuRC) is the principal nucleator of cellular microtubules, and the microtubule-nucleating activity of the complex is stimulated by binding to the γTuRC-mediated nucleation activator (γTuNA) motif. The γTuNA is part of the centrosomin motif 1 (CM1), which is widely found in γTuRC stimulators, including CDK5RAP2. Here, we show that a conserved segment within CM1 binds to the γTuNA and blocks its association with γTuRCs; therefore, we refer to this segment as the γTuNA inhibitor (γTuNA-In). Mutational disruption of the interaction between the γTuNA and the γTuNA-In results in a loss of autoinhibition, which consequently augments microtubule nucleation on centrosomes and the Golgi complex, the two major microtubule-organizing centers. This also causes centrosome repositioning, leads to defects in Golgi assembly and organization, and affects cell polarization. Remarkably, phosphorylation of the γTuNA-In, probably by Nek2, counteracts the autoinhibition by disrupting the γTuNA‒γTuNA-In interaction. Together, our data reveal an on-site mechanism for controlling γTuNA function.

Menin "reads" H3K79me2 mark in a nucleosomal context.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

Photo-ANA enables profiling of host-bacteria protein interactions during infection.

Bacterial pathogens rapidly change and adapt their proteome to cope with the environment in host cells and secrete effector proteins to hijack host targets and ensure their survival and proliferation during infection. Excessive host proteins make it difficult to profile pathogens' proteome dynamics by conventional proteomics. It is even more challenging to map pathogen-host protein-protein interactions in real time, given the low abundance of bacterial effectors and weak and transient interactions in which they may be involved. Here we report a method for selectively labeling bacterial proteomes using a bifunctional amino acid, photo-ANA, equipped with a bio-orthogonal handle and a photoreactive warhead, which enables simultaneous analysis of bacterial proteome reprogramming and pathogen-host protein interactions of Salmonella enterica serovar Typhimurium (S. Typhimurium) during infection. Using photo-ANA, we identified FLOT1/2 as host interactors of S. Typhimurium effector PipB2 in late-stage infection and globally profiled the extensive interactions between host proteins and pathogens during infection.

Chemical Synthesis of Proteins with Base-Labile Posttranslational Modifications Enabled by a Boc-SPPS Based General Strategy Towards Peptide C-Terminal Salicylaldehyde Esters.

Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H2 O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.

YEATS Domains as Novel Epigenetic Readers: Structures, Functions, and Inhibitor Development.

Interpretation of the histone posttranslational modifications (PTMs) by effector proteins, or readers, is an important epigenetic mechanism to regulate gene function. YEATS domains have been recently identified as novel readers of histone lysine acetylation and a variety of nonacetyl acylation marks. Accumulating evidence has revealed the association of dysregulated interactions between YEATS domains and histone PTMs with human diseases, suggesting the therapeutic potential of YEATS domain inhibition. Here, we discuss the molecular mechanisms adopted by YEATS domains in recognizing their preferred histone marks and the biological significance of such recognitions in normal cell physiology and pathogenesis of human diseases. Recent progress in the development of YEATS domain inhibitors is also discussed.

Photo-Cross-Linking To Delineate Epigenetic Interactome.

Covalent modifications of DNA and histones are key cellular epigenetic marks to regulate gene functions. Most of these epigenetic marks are added or removed by corresponding enzymes known as writers and erasers, whose catalytic activities normally rely on the presence of cellular metabolites as cofactors. Epigenetic marks can either directly alter the chromatin structure and dynamics through changing the intra-/internucleosomal histone-histone and histone-DNA interactions or recruit readers that further bring in other proteins with chromatin-modifying/remodeling activities to reshape the local and regional chromatin organization. In these two ways, epigenetic modifications modulate diverse DNA-templated processes, such as gene transcription, DNA replication, and DNA damage repair. Therefore, elucidation of the regulatory mechanisms and biological significance of epigenetic marks requires the identification and characterization of the protein-protein, protein-nucleic acid, and protein-small molecule interactions that control the underlying epigenetic processes. Here, we review the recent advances in using photo-cross-linking strategies to interrogate the epigenetic interactome, focusing on the protein-protein interactions mediated by epigenetic marks in histone tails. We also discuss future directions of developing photo-cross-linking-based tools and methods toward the investigation of the binding events in nucleosomal/chromatinic contexts, and toward the in situ capture of the epigenetic interactome in live cells or even organisms.

Preparation of Site-Specific Succinylated Histone Mimics to Investigate Its Impact on Nucleosome Dynamics.

Posttranslational modifications (PTMs) of histones have been demonstrated to be the key regulating mechanism of nucleosome dynamics and chromatin structure. Lysine succinylation is a recently discovered PTM that plays critical roles in metabolism, epigenetic signaling, and is correlated with several diseases. One significant challenge in studying the effects of this modification on nucleosome dynamics is to obtain site-specifically modified histones. Here, we report the rapid site-specific incorporation of a succinylation mimic into histones, which facilitates the characterization of its impact on nucleosome dynamics with a Förster resonance energy transfer (FRET) approach.

Roles of Negatively Charged Histone Lysine Acylations in Regulating Nucleosome Structure and Dynamics.

The nucleosome, the basic repeating unit of chromatin, is a dynamic structure that consists of DNA and histones. Insights derived from biochemical and biophysical approaches have revealed that histones posttranslational modifications (PTMs) are key regulators of nucleosome structure and dynamics. Mounting evidence suggests that the newly identified negatively charged histone lysine acylations play significant roles in altering nucleosome and chromatin dynamics, subsequently affecting downstream DNA-templated processes including gene transcription and DNA damage repair. Here, we present an overview of the dynamic changes of nucleosome and chromatin structures in response to negatively charged histone lysine acylations, including lysine malonylation, lysine succinylation, and lysine glutarylation.

Integrative Chemical Biology Approaches to Deciphering the Histone Code: A Problem-Driven Journey.

The hereditary blueprint of a eukaryotic cell is encoded in its genomic DNA that is tightly compacted into chromatin together with histone proteins. The basic repeating units of chromatin fibers are nucleosomes, in which approximately 1.7 turns of DNA wrap around a proteinaceous octamer consisting of two copies of histones H2A, H2B, H3, and H4. Histones are extensively decorated by a variety of posttranslational modifications (PTMs, e.g., methylation, acetylation, ubiquitylation, phosphorylation, etc.), serving as one of the cellular mechanisms that regulates DNA-templated processes, including but not limited to gene transcription, DNA replication, and DNA damage repair. Most of the histone PTMs exist in dynamic fluctuations, and their on and off states are exquisitely regulated by enzymes known as "writers" and "erasers", respectively. When installed at certain sites, histone PTMs can change the local physicochemical environment and thereby directly influence the nucleosome and chromatin structures. Alternatively, histone PTMs can recruit effectors (or "readers") to signal the downstream events. A "histone code" hypothesis has been proposed in which the combinatory actions of different histone PTMs orchestrate the epigenetic landscape of cells, modulating the activity of the underlying DNA and maintaining the genome stability between generations. Accumulating evidence also suggests that malfunctions of histone PTMs are associated with the pathogenesis of human diseases, such as cancer. It is therefore important to fully decipher the histone code, namely, to dissect the regulatory mechanisms and biological functions of histone PTMs.Owing to the advances in state-of-the-art mass spectrometry, dozens of novel histone modifications have been archived during the past decade. However, most of these newly identified histone PTMs remain poorly explored. To unravel the roles played by these PTMs in histone code, key questions that have driven our study are (i) how to detect the novel histone PTMs; (ii) how to identify the enzymes that catalyze the addition (writers) and removal (erasers) of the histone PTMs along with the regulating mechanisms; (iii) what is the biological significance of the histone PTMs and how do they function, by affecting the nucleosome and chromatin dynamics or by recruiting readers; and (iv) how to develop chemical probes to interrogate the histone PTMs or even serve as potential leads for the drug discovery campaigns to treat diseases caused by abnormalities in the regulation of histone PTMs.This Account focuses on our efforts in developing and applying chemical tools and methods to answer the above questions. Specifically, we review the detection of negatively charged histone acylations by developing and applying chemical reporters; preparing homogeneous nucleosomes carrying negatively charged acylations by protein chemistry approaches and the in vitro biophysical analyses of the effects of the acylations on nucleosome structures; investigating the negatively charged acylations' influence on chromatin dynamics in vivo using yeast genetic approaches; identifying and characterizing protein-protein interactions (PPIs) mediated by histone PTMs in different biological contexts (i.e., to identify the readers and erasers) by establishing a chemical proteomics platform that is enabled by photo-cross-linking chemistry and quantitative proteomics strategies; and manipulating PTM-mediated PPIs by the structure-guided design of inhibitors. We also discuss possible future directions in our journey to fully decipher the histone code.

Lysine succinylation on non-histone chromosomal protein HMG-17 (HMGN2) regulates nucleosomal DNA accessibility by disrupting the HMGN2-nucleosome association.

Lysine succinylation (Ksucc) is a novel posttranslational modification that frequently occurs on chromatin proteins including histones and non-histone proteins. Histone Ksucc affects nucleosome dynamics by increasing the DNA unwrapping rate and accessibility. However, very little is known about the regulation and functions of Ksucc located on non-histone chromosomal proteins. Here, we site-specifically installed a succinyl lysine analogue (Kcsucc) onto the non-histone chromosomal protein HMG-17 (HMGN2) to mimic the natural succinylated protein. We found that the incorporation of Kcsucc into HMGN2 at the K30 site (HMGN2Kc30succ), which is located within the nucleosome-binding domain (NBD), leads to significantly decreased HMGN2 binding to the mononucleosome. HMGN2Kc30succ also increased the nucleosomal DNA accessibility by promoting nucleosomal DNA unwrapping in the entry/exit region. This study reveals a novel mechanism of non-histone protein succinylation on altering chromatin recruitment, which can further affect nucleosome and chromatin dynamics.

Concise solid-phase synthesis enables derivatisation of YEATS domain cyclopeptide inhibitors for improved cellular uptake.

YEATS domains, which are newly identified epigenetic readers of histone lysine acetylation and crotonylation, have emerged as promising anti-cancer drug targets. We recently developed AF9 YEATS domain-selective cyclopeptide inhibitors. However, the cumbersome and time-consuming synthesis of the cyclopeptides limited further structural derivatisation and applications. Here, we reported a concise method for the solid-phase synthesis of the cyclopeptides, which substantially reduced the amount of time required for the preparation of the cyclopeptides and led to a higher overall yield. Moreover, this new synthetic route also allowed further derivatisation of the cyclopeptides with various functional modules, including fluorescent dye and cell-penetrating peptide. We demonstrated that the conjugation of the cyclopeptide with cell-penetrating peptide TAT led to a significantly increased cellular uptake.

AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana.

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation-sequencing (ChIP-Seq), we mapped the genome-wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild-type and hda6 mutant strains. Results show pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an 'eraser' enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

Protocol for the preparation of site-specific succinylated histone mimics to investigate the impact on nucleosome dynamics.

Lysine succinylation is a recently discovered posttranslational modification that plays critical roles in metabolism, epigenetic signaling, and human diseases. To investigate the effects of site-specific histone lysine succinylation on nucleosome dynamics requires the generation of homogeneously modified histones, which is a significant challenge. Here, we report a protocol for the rapid site-specific installation of a succinyl lysine analog onto histone. We then use a Förster resonance energy transfer approach to characterize the impact on nucleosome dynamics. For complete details on the use and execution of this protocol, please refer to Jing et al. (2018).