Chemical biology develops molecular tools for studying biological processes, setting the basis for new diagnostics and therapeutics, and relies heavily on the ability to modify selectively biomolecules. In our work, we introduce hypervalent iodine bonds into peptides and proteins, via functionalization of cysteine, by using unique cyclic reagents developed in our group. The hypervalent bond can then be selectively modified in the presence of both natural and synthetic functional groups, opening new opportunities for applications in chemical biology.

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.

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.

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.

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.Nucleosomes, the basic unit of chromatin, organize 147 bp of DNA wrapped around two of each core histone H3, H4, H2A and H2B.1 The histone proteins carry combinations of post-translational modifications (PTMs, or marks), which are implicated in regulating chromatin function. 2 In particular, methylation of lysine residues on H3 and H4 has clearly defined roles in gene activation and repression, with implication for cell differentiation, development and disease. 3Detailed MS studies found that key methyl-marks in embryonic stem cells (ESCs) exist in asymmetric nucleosomes, i.e. nucleosomes carrying two differently modified copies of H3 or H4. 4 These PTMs include H4 monomethylated at lysine 20 (H4K20me1), as well as H3K4me3, H3K36me3 and H3K27me3. 5 H4K20 methylation is a critical modification involved in heterochromatin silencing, and also in DNA replication and the DNA damage response. 6 The methyltransferase Set8 (also known as PR-Set7 or KMT5A) is responsible for H4K20 monomethylation, whereas two further methyltransferases, Suv4-20h1 and Suv4-20h2, catalyze di-and trimethylation of this residue. 6 Together, H4K20 methylation is involved in chromatin structure regulation, 7 and serves as a binding site for chromatin regulators, including 53BP1 8 and L3MBTL1. The establishment, maintenance and function of nucleosomes asymmetrically modified on H4 is not well understood. This is mainly due to the fact that such nucleosomes are not easily available for detailed in vitro mechanistic studies. Here, we thus developed a synthetic strategy enabling the traceless synthesis of nucleosomes containing differentially modified H4 proteins, with a focus on H4K20me1. While expressed protein ligation (EPL) methods readily enable the installation of combinations of marks on nucleosomes, 10 the reconstitution of asymmetric chromatin is not straightforward and is largely based on the attachment of affinity tags, followed by multi-step purification schemes. 4,11 We recently developed a chemical method to address this problem and control supramolecular nucleosome assembly. 12 Our synthetic approach was based on the inclusion of a link-and-cut (lnc)-tag that enabled transient crosslinking (by a disulfide bond) of H3 species during nucleosome reconstitution. This allowed us to synthesize asymmetrically modified nucleosomes, carrying H3K4me3 and H3K27me3 on different H3 copies. 12 After the formation of nucleosomes, the crosslink was reversed and the lnc-tag was removed using tobacco etch virus (TEV) protease (Scheme 1A-C).Based on these studies, we thus wondered if our synthetic method could be adapted to synthesize crosslinked versions of differentially mo...