Set1/COMPASS repels heterochromatin invasion at euchromatic sites by disrupting Suv39/Clr4 activity and nucleosome stability.

Protection of euchromatin from invasion by gene-repressive heterochromatin is critical for cellular health and viability. In addition to constitutive loci such as pericentromeres and subtelomeres, heterochromatin can be found interspersed in gene-rich euchromatin, where it regulates gene expression pertinent to cell fate. While heterochromatin and euchromatin are globally poised for mutual antagonism, the mechanisms underlying precise spatial encoding of heterochromatin containment within euchromatic sites remain opaque. We investigated ectopic heterochromatin invasion by manipulating the fission yeast mating type locus boundary using a single-cell spreading reporter system. We found that heterochromatin repulsion is locally encoded by Set1/COMPASS on certain actively transcribed genes and that this protective role is most prominent at heterochromatin islands, small domains interspersed in euchromatin that regulate cell fate specifiers. Sensitivity to invasion by heterochromatin, surprisingly, is not dependent on Set1 altering overall gene expression levels. Rather, the gene-protective effect is strictly dependent on Set1's catalytic activity. H3K4 methylation, the Set1 product, antagonizes spreading in two ways: directly inhibiting catalysis by Suv39/Clr4 and locally disrupting nucleosome stability. Taken together, these results describe a mechanism for spatial encoding of euchromatic signals that repel heterochromatin invasion.

Heterodimerization of H3K9 histone methyltransferases G9a and GLP activates methyl reading and writing capabilities.

Unique among metazoan repressive histone methyltransferases, G9a and GLP, which chiefly target histone 3 lysine 9 (H3K9), require dimerization for productive H3K9 mono (me1)- and dimethylation (me2) in vivo. Intriguingly, even though each enzyme can independently methylate H3K9, the predominant active form in vivo is a heterodimer of G9a and GLP. How dimerization influences the central H3K9 methyl binding ("reading") and deposition ("writing") activity of G9a and GLP and why heterodimerization is essential in vivo remains opaque. Here, we examine the H3K9me "reading" and "writing" activities of defined, recombinantly produced homo- and heterodimers of G9a and GLP. We find that both reading and writing are significantly enhanced in the heterodimer. Compared with the homodimers, the heterodimer has higher recognition of H3K9me2, and a striking ∼10-fold increased turnover rate for nucleosomal substrates under multiple turnover conditions, which is not evident on histone tail peptide substrates. Cross-linking Mass Spectrometry suggests that differences between the homodimers and the unique activity of the heterodimer may be encoded in altered ground state conformations, as each dimer displays different domain contacts. Our results indicate that heterodimerization may be required to relieve autoinhibition of H3K9me reading and chromatin methylation evident in G9a and GLP homodimers. Relieving this inhibition may be particularly important in early differentiation when large tracts of H3K9me2 are typically deposited by G9a-GLP, which may require a more active form of the enzyme.

Middle-down mass spectrometry enables characterization of branched ubiquitin chains.

Protein ubiquitylation, one of the most prevalent post-translational modifications in eukaryotes, is involved in regulating nearly every cellular signaling pathway. The vast functional range of ubiquitylation has largely been attributed to the formation of a diverse array of polymeric ubiquitin (polyUb) chains. Methods that enable the characterization of these diverse chains are necessary to fully understand how differences in structure relate to function. Here, we describe a method for the detection of enzymatically derived branched polyUb conjugates in which a single Ub subunit is modified by two Ub molecules at distinct lysine residues. Using a middle-down mass spectrometry approach in which restricted trypsin-mediated digestion is coupled with mass spectrometric analysis, we characterize the polyUb chains produced by bacterial effector E3 ligases NleL (non-Lee-encoded effector ligase from enterohemorrhagic Escherichia coli O157:H7) and IpaH9.8 (from Shigella flexneri). Because Ub is largely intact after minimal trypsinolysis, multiple modifications on a single Ub moiety can be detected. Analysis of NleL- and IpaH9.8-derived polyUb chains reveals branch points are present in approximately 10% of the overall chain population. When unanchored, well-defined polyUb chains are added to reaction mixtures containing NleL, longer chains are more likely to be modified internally, forming branch points rather than extending from the end of the chain. These results suggest that middle-down mass spectrometry can be used to assess the extent to which branched polyUb chains are formed by various enzymatic systems and potentially evaluate the presence of these atypical conjugates in cell and tissue extracts.

Forging isopeptide bonds using thiol-ene chemistry: site-specific coupling of ubiquitin molecules for studying the activity of isopeptidases.

Chemical methods for modifying proteins can enable studies aimed at uncovering biochemical function. Herein, we describe the use of thiol-ene coupling (TEC) chemistry to report on the function of branched (also referred to as forked) ubiquitin trimers. We show how site-specific isopeptide (Nε-Gly-L-homothiaLys) bonds are forged between two molecules of Ub, demonstrating the power of TEC in protein conjugation. Moreover, we demonstrate that the Nε-Gly-L-homothiaLys isopeptide bond is processed to a similar extent by deubiquitinases (DUBs) as that of a native Nε-Gly-L-Lys isopeptide bond, thereby establishing the utility of TEC in the generation of Ub-Ub linkages. TEC is then applied to the synthesis of branched Ub trimers. Interrogation of these branched derivatives with DUBs reveals that the relative orientation of the two Ub units has a dramatic impact on how they are hydrolyzed. In particular, cleavage of K48C-linkages is suppressed when the central Ub unit is also conjugated through K6C, whereas cleavage proceeds normally when the central unit is conjugated through either K11C or K63C. The results of this work presage a role for branched polymeric Ub chains in regulating linkage-selective interactions.