Site-specific modified histone half-synthesis and histone probes of chromatin-modifying enzymes (2023)


Eukaryotic chromosomes are made up of a large nucleoprotein complex called chromatin. Chromatin contains millions of repeating nucleosome subunits, each consisting of approximately 150 base pairs of double-stranded (ds) DNA wrapped around several highly conserved core structural proteins. These proteins, called core histones, form a flattened cylindrical coil that serves to compress dsDNA for storage in the microscopic cell nucleus. The core nucleosome contains two copies of each of the core histones H2A, H2B, H3 and H4, with histone H1 attached to the ends of the DNA entering and exiting the nucleosome [1], [2]. While most of each histone strand is structured and involved in protein-protein or protein-DNA contacts, the first twenty N-terminal histone residues, called histones, arecruzThey are not structured. Reversible post-translational modifications (PTM) of histone tails, also called histonesto noticethey play a key role in the regulation of higher-order chromatin structure and gene expression [3]. A large number of different histonesto noticeare presented as the basis of ahiston-codein eukaryotes, in which different sets of histone PTMs act as signals for specific DNA patterning processes. This histone code is implemented by a large familyauteurproteins that set up chemically distinct PTMs. family ofdocentproteins interpret different histone MTPs andgomproteins remove any PTM when the histone code needs to be erased and rewritten. Many, but not all, histone MTPs are located on lysine side-chain amines at the N- and C-termini of histones; including methylation [4], acetylation [5], ubiquitylation [6] and sumoylation [7]. In fact, the small ubiquitin-like modifier (SUMO) is the largest histone PTM known to date. The consequences of some of themto noticeon chromatin structure and gene transcription were assessed with homogeneously modified semi-synthetic histones generated by different synthetic pathways [8], [9], [10]. A common theme in these semi-synthetic approaches was the splitting of the target histone into two fragments. A smaller N- or C-terminal peptide fragment containing the PTM was prepared by solid phase peptide synthesis (SPPS), and a second larger histone fragment was heterologously expressed and purified from bacteria. The two fragments merged, orconnected, using the Native Chemical Ligation (NCL) technique [11]. for largeto noticelike ubiquitin or SUMO, a recombinant protein modifier is installed on a specific lysine side chain with an additional ligation reaction [8].

We used semi-synthetic histones to determine the effectsto noticeabout chromatin structure, gene transcription and biochemical cross-talk with othersto notice(Figure 1) [10], [12], [13], [14], [15], [16], [17], [18], [19]. Sedimentation rate analysis with reconstituted arrays of nucleosomes containing modified synthetic histones showed that SUMO conjugated to histone H4 on lysine 12, H4K12su [16] and ubiquitin bound to histone H2B on lysine 120 [16], H2BK120ub [20], inhibit chromatin compaction . Cell-free transcription assays with chromatinized plasmids containing semisynthetic histones also revealed that H4K12su directly suppresses gene transcription [19]. In addition, demethylation assays with H4K12-containing mononucleosomes showed inhibition of H3K4 methylation and stimulation of lysine-specific demethylase 1 (LSD1) activity at H3 lysine 4, H3K4me2 [19]. Many hypothesis-driven experiments, based on observations in different cell types and tissues, have used semi-synthetic histones to elucidate the biophysical and biochemical basis of the histone code. However, new approaches based on chemical biology are also needed to complement genetic experiments that can identify key enzymes that write and erase the dynamic histone code.

Although mutations in the active site of a known chromatin-modifying enzyme have been used to enhance binding to histones [21], it remains difficult to identify all, or even most, of the histone-writer/eraser interactions for specific modifications. To address this gap in our understanding of histone PTMs, particularly for histone ubiquitylation, we now report the synthesis of covalent histone-based probes that can be used to capture chromatin-binding enzymes. The covalent warheads we present have already been successful in investigating the biochemistry of ubiquitin [13] and in the characterization of binding-specific deubiquitylase enzymes [21], [22], [23], [24]. Therefore, we present a general semi-synthetic method to obtain homogeneously ubiquitous histones with an oxidizable cysteine ​​residue or an electrophilic dehydroalanine residue at the H2B-ubiquitin junction in H2BK120ub that can be used to generate active site thiols in chromatin-associated enzymes. to catch. which operate on H2BK120ub.

fragments section

Demethylation of the H3K4me2 character

Lysine ε-amines on histones can be modified by mono-, di-, or trimethylation, and these modification states occur at different loci on chromatin [25]. Each methylation state is interpreted by specific sets of reading enzymes [26], [27]. Dimethylation at lysine 4 on histone H3 (H3K4me2) is enriched at the transcription start site (TSS) of many actively transcribed genes [25], [28], [29], [30]. Set1-associated protein complex (COMPASS) is responsible for H3K4 methylation in yeast,


Half-synthesis of H2BK120ub(G76A), H2BK120ub(G76C) and H2BK120ub(G76Dha) can be achieved in three to four linear steps with two identical peptide and protein fragments. This provides rapid access to any desired protein form for biochemical and structural studies. For example, H2BK120ub(G76A) was first used in mechanistic studies of the biochemical cross-talk between H3K79 ubiquitylation and methylation by Dot1L and can be easily obtained in one step from H2BK120ub(G76C) as


Ophiston-codefor gene regulation was first proposed at the beginning of the 21st centuryto callcentury. Over the past two decades, we have collectively gained a strong understanding of the critical role that epigenetic marks, including approximately 30 chemically distinct histone PTMs and DNA methylation, play in orchestrating numerous DNA-directed processes in cells. eukaryotes. Although most of the histone marks discovered to date have been examined by site-directed mutagenesis, Western blots with site and

Data Availability

No external data were used for the research described in the article. All information mentioned in the manuscript is available for inspection upon reasoned request.


Work in our laboratory is supported by National Institutes of Health R01GM110430, R01HD097408, and National Science Foundation CHE-2107525.

(Video) Regulation of Bivalent Domains by Histone Modifications

no reference provided


Statement on CRediT's contribution

Madeline F. Currie:Writing: original draft, Writing: revision and editing.Sumeet K. Singh:Conceptualization, research, writing - review and publication.Meihuan-ji:Research, writing - reviewing and editing.Champak Chatterjee:Conceptualization, financing, mentoring, writing: revision and publication.

Declaration of Interest in the Contest

The authors declare that they have no conflicting financial interests or known personal relationships that could affect the work reported in this document.

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