Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins

David, Yael; Vila-Perelló, Miquel; Verma, Shivam; Muir, Tom W.

doi:10.1038/nchem.2224

Cited by 140 publications

(144 citation statements)

References 46 publications

Supporting

Mentioning

140

Contrasting

Unclassified

Order By: Relevance

“…We confirmed the importance of these residues for endogenous hDot1L activity in isolated nuclei through monitoring H3K79 methylation levels subsequent to the site-specific incorporation of a ubiquitin mutant, lacking these residues, into chromatin (26). Extending our findings to other well-established H2B-Ub processes, we find that this ubiquitin hotspot is also necessary for the stimulation of ySet1-mediated H3K4 methylation.…”

Section: Significancesupporting

confidence: 69%

“…This approach uses naturally split inteins [referred to herein as the N-terminal intein (Int N ) and C-terminal intein (Int C )] to assemble the desired H2B-Ub construct through an in nucleo protein ligation procedure (SI Appendix, Fig. S11A) (26). This reaction results in attachment of ubiquitin to H2BK120 through an isopeptide linkage that is identical to that used in our in vitro studies and, importantly, without mutating any of the other histones.…”

Section: The Leu71/leu73 Patch Is Required For Hdot1l Stimulation Onmentioning

confidence: 99%

See 1 more Smart Citation

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Holt

David

Pollock

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: Significancesupporting

confidence: 69%

Section: The Leu71/leu73 Patch Is Required For Hdot1l Stimulation Onmentioning

confidence: 99%

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Holt

David

Pollock

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…We demonstrated that the Cfa GEP intein improves the scope of two such applications: protein cyclization and the in nucleo semisynthesis of chemically tailored chromatin (22,25). The improved cyclization enabled by Cfa GEP may be further extended to a SICLOPPS library and selection system to identify cyclic peptides that bind or inhibit a target enzyme (23).…”

Section: Discussionmentioning

confidence: 99%

“…Recently, split inteins have been used to chemically modify cellular chromatin (25). This in nucleo protein semisynthesis strategy provides a means to validate in vitro observations of histone biochemistry in the context of a native cellular chromatin (26).…”

Section: Resultsmentioning

confidence: 99%

A promiscuous split intein with expanded protein engineering applications

Stevens

Sekar

Shah

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

114

119

View full text Add to dashboard Cite

The protein trans-splicing (PTS) activity of naturally split inteins has found widespread use in chemical biology and biotechnology. However, currently used naturally split inteins suffer from an "extein dependence," whereby residues surrounding the splice junction strongly affect splicing efficiency, limiting the general applicability of many PTS-based methods. To address this, we describe a mechanism-guided protein engineering approach that imbues ultrafast DnaE split inteins with minimal extein dependence. The resulting "promiscuous" inteins are shown to be superior reagents for protein cyclization and protein semisynthesis, with the latter illustrated through the modification of native cellular chromatin. The promiscuous inteins reported here thus improve the applicability of existing PTS methods and should enable future efforts to engineer promiscuity into other naturally split inteins. A n intein is an intervening protein domain that undergoes a unique posttranslational autoprocessing event, termed protein splicing. In this spontaneous process, the intein excises itself from the host protein and, in the process, ligates together the flanking N-and C-terminal residues (exteins) to form a native peptide bond (SI Appendix, Fig. S1A) (1, 2). Although inteins are most frequently found as a contiguous domain, some exist in a naturally split form. In this case, the two fragments are expressed as separate polypeptides and must associate before splicing takes place, so-called protein trans-splicing (SI Appendix, Fig. S1B). Unlike many well-characterized contiguous inteins that splice slowly, several naturally split inteins demonstrate rapid splicing kinetics (3-6). Indeed, the discovery of these ultrafast split inteins has enabled the development of numerous tools for both synthetic and biological applications (1).A major caveat to splicing-based methods is that all characterized inteins exhibit a sequence preference at extein residues adjacent to the splice site. In addition to a mandatory catalytic Cys, Ser, or Thr residue at position +1 (i.e., the first residue within the C-extein), there is a bias for residues resembling the proximal N-and C-extein sequence found in the native insertion site. Deviation from this preferred sequence context leads to a marked reduction in splicing activity, limiting the applicability of protein trans-splicing (PTS)-based methods (3, 7-11). Accordingly, there is a need for split inteins whose activities are minimally affected by local sequence environment. Although efforts have previously been made to engineer promiscuous inteins (12, 13), these have not focused on naturally split inteins, which have superior fragment association and splicing kinetics (4-6).Here, we report engineered versions of naturally split inteins that possess greatly improved extein tolerance. Guided by our understanding of active site interactions critical for efficient protein splicing, we carried out targeted saturation mutagenesis of an ultrafast split intein and then used a cell-based selection system to...

show abstract

“…Deoxynucleotide analogues are used to chemically modify DNA, which thereby becomes site-specifically photoreactive. Moreover, the availability of reconstituted nucleosomes and their semi-synthetic variants bearing specific chemical modifications has enabled the generation of large nucleosome libraries for analytical experiments on binding properties (Montel et al, 2007;Simon, 2010;Maltby et al, 2012;Pichler et al, 2012;Yun et al, 2012;Lee et al, 2013;Rogge et al, 2013;Al-Ani et al, 2014b;David et al, 2015). The EMBO Journal Mechanisms of nucleosome recognition Valentina Speranzini et al the nucleosome is formed by an octamer of tightly associated histone proteins (H2A-H2B) 2 (H3-H4) 2 , and~150 bp of DNA wrapped around the octamer to define a left-handed superhelical fragment (Luger et al, 1997), where the core of the histone proteins is well defined, while the tails mostly lack a defined structure.…”

Section: Chemical Probes and Nucleosome Librariesmentioning

confidence: 99%

Touch, act and go: landing and operating on nucleosomes

et al. 2016

View full text Add to dashboard Cite

Chromatin-associated enzymes are responsible for the installation, removal and reading of precise post-translation modifications on DNA and histone proteins. They are specifically recruited to the target gene by associated factors, and as a result of their activity, they contribute in modulating cell identity and differentiation. Structural and biophysical approaches are broadening our knowledge on these processes, demonstrating that DNA, histone tails and histone surfaces can each function as distinct yet functionally interconnected anchoring points promoting nucleosome binding and modification. The mechanisms underlying nucleosome recognition have been described for many histone modifiers and related readers. Here, we review the recent literature on the structural organization of these nucleosome-associated proteins, the binding properties that drive nucleosome modification and the methodological advances in their analysis. The overarching conclusion is that besides acting on the same substrate (the nucleosome), each system functions through characteristic modes of action, which bring about specific biological functions in gene expression regulation.

show abstract

Chemical tagging and customizing of cellular chromatin states using ultrafast trans-splicing inteins

Cited by 140 publications

References 46 publications

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

A promiscuous split intein with expanded protein engineering applications

Touch, act and go: landing and operating on nucleosomes

Contact Info

Product

Resources

About