Cellular proteins continuously undergo non-enzymatic covalent modifications (NECMs) that accumulate under normal physiological conditions and are stimulated by changes in the cellular microenvironment. Glycation, the hallmark of diabetes, is a prevalent NECM associated with an array of pathologies. Histone proteins are particularly susceptible to NECMs due to their long half-lives and nucleophilic disordered tails that undergo extensive regulatory modifications; however, histone NECMs remain poorly understood. Here we perform a detailed analysis of histone glycation in vitro and in vivo and find it has global ramifications on histone enzymatic PTMs, the assembly and stability of nucleosomes, and chromatin architecture. Importantly, we identify a physiologic regulation mechanism, the enzyme DJ-1, which functions as a potent histone deglycase. Finally, we detect intense histone glycation and DJ-1 overexpression in breast cancer tumors. Collectively, our results suggest an additional mechanism for cellular metabolic damage through epigenetic perturbation, with implications in pathogenesis.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction 1 , although their biological functions are poorly understood. Histone H1 ( HIST1H1B-E ) mutations are highly recurrent in B-cell lymphomas, but their cancer relevance and mechanism are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in profound architectural remodeling of the genome characterized by large-scale, yet focal shifts of chromatin from a compacted, to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily due to gain of histone H3 lysine 36 dimethylation, and/or loss of repressive H3 lysine 27 trimethylation. These changes unlock expression of stem cell genes that are normally silenced during early development. Loss of H1c and H1e alleles in mice conferred enhanced fitness and self-renewal properties to germinal center B-cells, ultimately leading to aggressive lymphoma with enhanced repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We furthermore establish H1 as a bona fide tumor suppressor, whose mutation drives malignant transformation primarily through three-dimensional genome reorganization, followed by epigenetic reprogramming and derepression of developmentally silenced genes.

Elucidating the molecular details of how chromatin-associated factors deposit, remove and recognize histone posttranslational modification (‘PTM’) signatures remains a daunting task in the epigenetics field. Here, we introduce a versatile platform that greatly accelerates biochemical investigations into chromatin recognition and signaling. This technology is based on the streamlined semi-synthesis of DNA-barcoded nucleosome libraries with distinct combinations of PTMs. Chromatin immunoprecipitation of these libraries treated with purified chromatin effectors or the combined chromatin recognizing and modifying activities of the nuclear proteome is followed by multiplexed DNA-barcode sequencing. This ultrasensitive workflow allowed us to collect thousands of biochemical data points revealing the binding preferences of various nuclear factors for PTM patterns and how pre-existing PTMs, alone or synergistically, affect further PTM deposition via crosstalk mechanisms. We anticipate that the high-throughput and -sensitivity of the technology will help accelerate the decryption of the diverse molecular controls that operate at the level of chromatin.

Post-translational modification of the histone proteins in chromatin plays a central role in epigenetic control of DNA-templated processes in eukaryotic cells. Developing methods that enable the structure of histones to be manipulated is therefore essential to understand the biochemical mechanisms underlying genomic regulation. Here we present a synthetic biology method to engineer histones bearing site-specific modifications on cellular chromatin using protein trans-splicing. We genetically fused the N-terminal fragment of ultrafast split-intein to the C-terminus of histone H2B, which upon reaction with a complementary synthetic C-intein, generated labeled histone. Using this approach, we incorporated various non-native chemical modifications to chromatin in vivo with temporal control. Furthermore, the time and concentration dependence of protein trans-splicing performed in nucleo enabled us to examine differences in the accessibility of the euchromatin and heterochromatin regions of the epigenome. Finally, we used protein trans-splicing to semi-synthesize a native histone modification, H2BK120 ubiquitination, in isolated nuclei, and show that this can trigger downstream epigenetic cross-talk of H3K79 methylation.

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.In eukaryotic cells, most proteins are degraded by the 26 S proteasome, which hydrolyzes in an ATP-dependant manner, both ubiquitin-conjugated and certain non-ubiquitinated proteins. In addition to its role in the turnover of damaged or misfolded proteins, the proteasome controls the cell cycle and other processes through the degradation of critical regulatory components and transcription factors (1-3). Upon association of ubiquitinated targets with the proteasome, ubiquitin molecules are proteolytically removed for reuse, whereas the unfolded substrates are fed into the 20 S catalytic core, where they are digested into small peptides (4, 5).Protein ubiquitination is a multistep process orchestrated by the concerted action of three enzymes. The chain reaction begins with a ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin. Next, a thioester bond is formed between the activated C terminus of ubiquitin and a cysteine residue of the E1. A ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a transthioesterification reaction. Finally, a RING ubiquitin-protein ligase (E3) recruits the substrate and guides the transfer of the ubiquitin from the E2 active site cysteine to the substrate. An ⑀-amine of a lysine residue on the substrate (or of additional ubiquitin) attacks the thioester bond between the ubiquitin and the E2 enzyme, forming an isopeptide bond with the C-terminal glycine of the ubiquitin (6 -8). Alternatively, when a HECT E3 catalyzes the transfer of the ubiquitin from the E2 to the target, an intermediate complex, of the activated ubiquitin and the active site cysteine of the HECT domain E3, is formed (9).

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Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.H istone posttranslational modifications (PTMs) modulate chromatin structure and function either by directly altering the intrinsic physical properties of the chromatin fiber or by nucleating the recruitment and activity of a host of transacting nuclear factors (1-3). The chemical diversity, differential dynamics, and sheer number (currently over 100) (4, 5) of these PTMs, along with their combinatorial occurrence at the level of the nucleosome, create a complex and nonstatic molecular architecture in which all chromatin-related processes function. A central challenge in the field of epigenetics is to disentangle how distinct chromatin states control biochemical outputs, which requires the elucidation of the critical determinants governing histone PTM readout.A particularly fascinating histone PTM is the ubiquitylation of H2B at lysine 120 (H2B-Ub). H2B-Ub is enriched near the 5′ end of highly expressed genes and has been implicated in transcriptional elongation, as well as chromatin structure definition (6-9). Moreover, H2B-Ub directly regulates the H3K4 and H3K79 methyltransferases Set1 and Dot1, respectively (10-13). The mechanistic principles underlying these various phenomena remain poorly understood. At 8.5 kDa, ubiquitin is nearly as large as the histone to which it is linked (13.8 kDa in the case of H2B), increasing the nucleosome surface by as much as 4,800 A 2 (14). Thus, compared with smaller PTMs such as acetylation and methylation, ubiquitin is "information rich" in that it alters the steric and electrostatic properties around its attachment site, as well as presenting a large surface area for the recruitment of binding factors. Structural and biochemical studies of ubiquitin-ligand complexes, including the ubiquitylation of histone H2A at lysine 15, have revealed a canonical binding hotspot on ubiquitin involving a hydrophobic patch centered on Leu8/I...

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono-and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains. Protein ubiquitination is a multistep process accomplished by the concerted action of three enzymes. The reaction begins with the ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue in its active site. Subsequently, a ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a trans-thioesterification reaction. Finally, a ubiquitin-protein ligase (E3) recruits a target protein and guides the transfer of the activated ubiquitin from the E2 to the substrate (1-3). Ubiquitin transfer from the E2 enzyme to the substrate is catalyzed directly by really interesting new gene (RING) 3 finger-containing E3s or indirectly when a homologous to E6-AP C terminus (HECT) domain E3 is mediating the transfer (4). Several forms of ubiquitination have been identified (5). Monoubiquitination or multiple monoubiquitinations are referred to as the conjugation of single or multiple ubiquitin moieties to distinct lysine residues on the substrate. These forms of ubiquitination were implicated in various cellular pathways, which include endocytosis and sorting of proteins to different cellular compartments (6, 7), as well as in several cases of proteasomal activity, such as the processing of the p105 precursor of the transcription regulator NF-B (8). However, polyubiquitination is the most common post-translational modification of proteins destined for degradation (9).In polyubiquitination assembly, ubiquitin conjugation was originally thought to be repeated in a cyclic manner whereby in each step a new moiety of ubiquitin is linked to one of the lysine residues of the previously conjugated ubiquitin. However, in view of recent findings, several alternative mechanisms have been proposed (10). Li et al. (11) demonstrated in a recon...