Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.

Multivalent Peptide Ligands To Probe the Chromocenter Microenvironment in Living Cells.

Chromatin is spatially organized into functional states that are defined by both the presence of specific histone post-translational modifications (PTMs) and a defined set of chromatin-associated "reader" proteins. Different models for the underlying mechanism of such compartmentalization have been proposed, including liquid-liquid phase separation (LLPS) of chromatin-associated proteins to drive spatial organization. Heterochromatin, characterized by lysine 9 methylation on histone H3 (H3K9me3) and the presence of heterochromatin protein 1 (HP1) as a multivalent reader, represents a prime example of a spatially defined chromatin state. Heterochromatin foci exhibit features of protein condensates driven by LLPS; however, the exact nature of the physicochemical environment within heterochromatin in different cell types is not completely understood. Here we present tools to interrogate the environment of chromatin subcompartments in the form of modular, cell-permeable, multivalent, and fluorescent peptide probes. These probes can be tuned to target specific chromatin states by providing binding sites to reader proteins and can thereby integrate into the PTM-reader interaction network. Here we generate probes specific to HP1, directing them to heterochromatin at chromocenters in mouse fibroblasts. Moreover, we use a polarity-sensing photoactivatable probe that photoconverts to a fluorescent state in phase-separated protein droplets and thereby reports on the local microenvironment. Equipped with this dye, our probes indeed turn fluorescent in murine chromocenters. Image analysis and single-molecule tracking experiments reveal that the compartments are less dense and more dynamic than HP1 condensates obtained in vitro. Our results thus demonstrate that the local organization of heterochromatin in chromocenters is internally more complex than an HP1 condensate.

SUV39 SET domains mediate crosstalk of heterochromatic histone marks.

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.

Semisynthesis and Reconstitution of Nucleosomes Carrying Asymmetric Histone Modifications.

Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.

A Modular Ligation Strategy for Asymmetric Bivalent Nucleosomes Trimethylated at K36 and K27.

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.

Engineered Multivalent Sensors to Detect Coexisting Histone Modifications in Living Stem Cells.

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.

Controlling the supramolecular assembly of nucleosomes asymmetrically modified on H4.

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.