A Robust Method for the Purification and Characterization of Recombinant Human Histone H1 Variants

Osunsade, Adewola; Prescott, Nicholas Adrian; Hebert, Jakob M; Ray, Devin M.; Jmeian, Yazen; Lorenz, Ivo C.; David, Yael

doi:10.1021/acs.biochem.8b01060

Cited by 20 publications

(23 citation statements)

References 39 publications

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“…Moreover, intrinsic characteristics have even hindered in vitro analyses of modified H1 following recombinant expression. In particular, the long, highly unstructured and lysine-rich CTD is prone to degradation and yields insoluble and truncated proteins 17 . Yet, there is growing evidence that linker histone H1 regulates cellular functions by direct protein-protein interactions [18][19][20][21] .…”

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confidence: 99%

Site-specific ubiquitylation acts as a regulator of linker histone H1

et al. 2021

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Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. This is well studied for PTMs of core histones but not for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the chemical synthesis of site-specifically mono-ubiquitylated H1.2 and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that site-specific ubiquitylation of H1 at position K64 modulates interactions with deubiquitylating enzymes and the deacetylase SIRT1. Moreover, it affects H1-dependent chromatosome assembly and phase separation resulting in a more open chromatosome conformation generally associated with a transcriptionally active chromatin state. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

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confidence: 99%

Site-specific ubiquitylation acts as a regulator of linker histone H1

et al. 2021

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“…Additionally, use of the SPI strategy may not be limited to just improving yields of peptides that undergo degradation. Osunsade et al used a similar strategy to produce a human histone H1 protein, which had previously been difficult due to the intrinsically disordered and basic C‐terminal domain that led to insolubility or truncation 34 . Taken together, their results and ours suggest a “sandwiching” strategy may be generally effective to prevent degradation of proteins or peptides of varying sizes during heterologous expression.…”

Section: Resultsmentioning

confidence: 57%

SPI “sandwich”: Combined SUMO‐Peptide‐Intein expression system and isolation procedure for improved stability and yield of peptides

et al. 2022

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Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin-like modifier protein (SUMO), maltose-binding protein (MBP), glutathione S-transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a "sandwiched" fusion protein of the form: His 6 -SUMO-peptideintein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17-fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.

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“…His-Sumo-H1.4 A4C -GyrA-His was expressed and purified as described previously 33 with minor adjustments. Briefly, the construct was expressed in Rosetta DE3 cells overnight at 16 o C. Cells were lysed and lysate incubated with Ni-NTA beads (Bio-Rad).…”

Section: Linker Histone H1mentioning

confidence: 99%

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

Nguyen

Chang

Watters

et al. 2022

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Formation of biomolecular condensates constitutes an emerging mechanism for transcriptional regulation. Recent studies suggest that the co-condensation between transcription factors (TFs) and DNA can generate mechanical forces driving genome rearrangements. However, the reported forces generated by such protein-DNA co-condensation are typically below one piconewton (pN), questioning its physiological significance. Moreover, the force-generating capacity of these condensates in the chromatin context remains unknown. Using single-molecule biophysical techniques, we show that Sox2, a nucleosome-binding pioneer TF, forms co-condensates with DNA, thereby exerting considerable mechanical tension on DNA strands both in cis and trans. Sox2 can generate forces up to 7 pN—similar in magnitude to other cellular forces. Sox2:DNA condensates are highly stable, withstanding disruptive forces high enough to melt DNA. We find that the disordered domains of Sox2 are required for maximum force generation but not condensate formation per se. Finally, we show that nucleosomes dramatically attenuate the mechanical stress exerted by Sox2 via sequestering it from coalescing on bare DNA. Our findings reveal that TF-mediated DNA condensation can exert significant mechanical stress which can nonetheless be alleviated by the chromatin organization, suggesting a new function of eukaryotic chromatin in protecting the genome from potentially deleterious nuclear forces.

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A Robust Method for the Purification and Characterization of Recombinant Human Histone H1 Variants

Cited by 20 publications

References 39 publications

Site-specific ubiquitylation acts as a regulator of linker histone H1

Site-specific ubiquitylation acts as a regulator of linker histone H1

SPI “sandwich”: Combined SUMO‐Peptide‐Intein expression system and isolation procedure for improved stability and yield of peptides

Chromatin sequesters pioneer transcription factor Sox2 from exerting force on DNA

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