Highly disordered histone H1−DNA model complexes and their condensates

Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies.

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Histone H1 and Chromatin Higher-Order Structure

Critical Reviews in Eukaryotic Gene Expression ◽

10.1615/critreveukargeneexpr.v7.i3.20 ◽

1997 ◽

Vol 7 (3) ◽

pp. 215-230 ◽

Cited By ~ 82

Author(s):

V. Ramakrishnan

Keyword(s):

Histone H1 ◽

Higher Order ◽

Order Structure

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Faculty Opinions recommendation of ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1090600.543889 ◽

2007 ◽

Author(s):

Wendy Bickmore

Keyword(s):

Chromatin Structure ◽

Histone H1 ◽

Higher Order

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Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽

10.1017/s0967199401001356 ◽

2001 ◽

Vol 9 (4) ◽

pp. 309-316 ◽

Cited By ~ 21

Author(s):

Carsten Krischek ◽

Burkhard Meinecke

Keyword(s):

Map Kinase ◽

Protein Kinases ◽

Chromatin Condensation ◽

Specific Inhibitor ◽

Histone H1 ◽

Dependent Manner ◽

Free Medium ◽

Concentration Dependent Manner ◽

Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

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ISWI Regulates Higher-Order Chromatin Structure and Histone H1 Assembly In Vivo

PLoS Biology ◽

10.1371/journal.pbio.0050232 ◽

2007 ◽

Vol 5 (9) ◽

pp. e232 ◽

Cited By ~ 109

Author(s):

Davide F. V Corona ◽

Giorgia Siriaco ◽

Jennifer A Armstrong ◽

Natalia Snarskaya ◽

Stephanie A McClymont ◽

...

Keyword(s):

Chromatin Structure ◽

Histone H1 ◽

Higher Order

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In Vivo and In Vitro Evaluation of the Protective Effects of Hesperidin in Lipopolysaccharide-Induced Inflammation and Cytotoxicity of Cell

Molecules ◽

10.3390/molecules25030478 ◽

2020 ◽

Vol 25 (3) ◽

pp. 478 ◽

Cited By ~ 6

Author(s):

Rasha Al-Rikabi ◽

Hanady Al-Shmgani ◽

Yaser Hassan Dewir ◽

Salah El-Hendawy

Keyword(s):

Breast Cancer ◽

Chromatin Condensation ◽

Control Group ◽

Potential Candidate ◽

Dependent Manner ◽

Protective Effects ◽

Mcf7 Cells ◽

Tnf Α

(1) Background: Plant flavonoids are efficient in preventing and treating various diseases. This study aimed to evaluate the ability of hesperidin, a flavonoid found in citrus fruits, in inhibiting lipopolysaccharide (LPS) induced inflammation, which induced lethal toxicity in vivo, and to evaluate its importance as an antitumor agent in breast cancer. The in vivo experiments revealed the protective effects of hesperidin against the negative LPS effects on the liver and spleen of male mice. (2) Methods: In the liver, the antioxidant activity was measured by estimating the concentration of glutathione (GSH) and catalase (CAT), whereas in spleen, the concentration of cytokines including IL-33 and TNF-α was measured. The in vitro experiments including MTT assay, clonogenity test, and sulforhodamine 101 stain with DAPI (4′, 6-diamidino-2-phenylindole) were used to assess the morphological apoptosis in breast cancer cells. (3) Results: The results of this study revealed a significant increase in the IL-33 and TNF-α cytokine levels in LPS challenged mice along with a considerable elevation in glutathione (GSH); moreover, the catalase (CAT) level was higher compared to that of the control group. Cytotoxicity of the MCF-7 cell line revealed significant differences among the groups treated with different concentrations when compared to the control groups, in a concentration-dependent manner. Hesperidin significantly inhibited the colony formation of MCF7 cells when compared to that of control. Clear changes were observed in the cell shape, including cell shrinkage and chromatin condensation, which were associated with a later apoptotic stage. (4) Conclusion: The results indicate that hesperidin might be a potential candidate in preventing diseases.

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Rho5p Is Involved in Mediating the Osmotic Stress Response in Saccharomyces cerevisiae, and Its Activity Is Regulated via Msi1p and Npr1p by Phosphorylation and Ubiquitination

Eukaryotic Cell ◽

10.1128/ec.00120-08 ◽

2008 ◽

Vol 7 (9) ◽

pp. 1441-1449 ◽

Cited By ~ 17

Author(s):

Robert B. Annan ◽

Cunle Wu ◽

Daniel D. Waller ◽

Malcolm Whiteway ◽

David Y. Thomas

Keyword(s):

Saccharomyces Cerevisiae ◽

Osmotic Stress ◽

Posttranslational Modifications ◽

Bound State ◽

Small Gtpases ◽

Rho Proteins ◽

Vitro Assay ◽

Regulatory Circuit

ABSTRACT Small GTPases of the Rho family act as molecular switches, and modulation of the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, the regulation of Rho-type GTPases by posttranslational modifications is poorly understood. Here, we present evidence of the control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5(Q91H) allele in an Δste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as being high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as being a possible Rho5p GTPase-activating protein based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 was previously identified as being a suppressor of hyperactive Ras/cyclic AMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here, we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated, and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as being a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.

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Natural chromatin is heterogeneous and self-associates in vitro

Molecular Biology of the Cell ◽

10.1091/mbc.e17-07-0449 ◽

2018 ◽

Vol 29 (13) ◽

pp. 1652-1663 ◽

Cited By ~ 12

Author(s):

Shujun Cai ◽

Yajiao Song ◽

Chen Chen ◽

Jian Shi ◽

Lu Gan

Keyword(s):

Divalent Cations ◽

Large Mass ◽

Higher Order ◽

Linker Histone ◽

Order Structure ◽

Face To Face ◽

Chromatin Packing ◽

Electron Cryotomography

The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

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Regulation of the higher-order structure of chromatin by histones H1 and H5.

The Journal of Cell Biology ◽

10.1083/jcb.90.2.279 ◽

1981 ◽

Vol 90 (2) ◽

pp. 279-288 ◽

Cited By ~ 80

Author(s):

J Allan ◽

G J Cowling ◽

N Harborne ◽

P Cattini ◽

R Craigie ◽

...

Keyword(s):

Spatial Organization ◽

Single Species ◽

Histone H1 ◽

Higher Order ◽

Linker Histone ◽

Micrococcal Nuclease ◽

Order Structure ◽

Chicken Erythrocyte ◽

Base Pairs ◽

Native Chicken

Chicken erythrocyte chromatins containing a single species of linker histone, H1 or H5, have been prepared, using reassembly techniques developed previously. The reconstituted complexes possess the conformation of native chicken erythrocyte chromatin, as judged by chemical and structural criteria; saturation is reached when two molecules of linker histone are bound per nucleosome, as in native erythrocyte chromatin, which the resulting material resembles in its appearance in the electron microscope and quantitatively in its linear condensation factor relative to free DNA. The periodicity of micrococcal nuclease-sensitive sites in the linker regions associated with histone H1 or H5 is 10.4 base pairs, suggesting that the spatial organization of the linker region in the higher-order structure of chromatin is similar to that in isolated nucleosomes. The susceptible sites are cut at differing frequencies, as previously found for the nucleosome cores, leading to a characteristic distribution of intensities in the digests. The scission frequency of sites in the linker DNA depends additionally on the identity of the linker histone, suggesting that the higher-order structure is subject to secondary modulation by the associated histones.

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Histidine-rich Ca2+-binding protein stimulates the transport cycle of SERCA through a conformation-dependent fuzzy complex

10.1101/2020.09.17.302869 ◽

2020 ◽

Author(s):

Temitope I. Ayeotan ◽

Line Cecilie Hansen ◽

Thomas Boesen ◽

Claus Olesen ◽

Jesper V. Møller ◽

...

Keyword(s):

Electrostatic Interactions ◽

Binding Protein ◽

Intrinsically Disordered Protein ◽

Bound State ◽

Disordered Proteins ◽

Dependent Manner ◽

Intrinsically Disordered ◽

Intracellular Ca ◽

P Type ◽

Rate Limiting

AbstractThe histidine-rich Ca2+-binding protein (HRC) stimulates the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) to increase Ca2+-uptake into the lumen. HRC also binds the triadin scaffold in a Ca2+-dependent manner, and HRC tunes both the uptake and release of Ca2+ depending on the concentration in the intracellular Ca2+-stores. We investigated how HRC stimulates SERCA pumping using biochemical and biophysical assays, and show that HRC is an intrinsically disordered protein that binds directly to SERCA via electrostatic interactions. The affinity of the interaction depends on the conformation of SERCA, and HRC binds most tightly in the calcium-released E2P state. This state marks the end of the rate-limiting [Ca2]E1P to E2P transition of SERCA, and suggests that HRC stimulates SERCA by preferentially stabilizing the end point of this transition. HRC remains disordered in the bound state and thus binds in a dynamic, fuzzy complex. The binding of HRC to SERCA shows that fuzzy complexes formed by disordered proteins may be conformation-specific, and use this specificity to modulate the functional cycle of complex molecular machines such as a P-type ATPase.

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