Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide

Protein phosphorylation is a key regulatory mechanism in eukaryotic cells.
In the intrinsically disordered histone tails, phosphorylation is often a part of combinatorial post-translational modifications and an integral part of the “histone code” that regulates gene expression.
Here, we study the association between two histone H3 tail peptides modified to different degrees, using fully atomistic molecular dynamics simulations.
Assuming that the initial conformations are either α-helical or fully extended, we compare the propensity of the two peptides to associate with one another when both are unmodified, one modified and the other unmodified, or both modified.
The simulations lead to the identification of distinct inter-and intramolecular interactions in the peptide dimer, highlighting a prominent role of a fine-tuned phosphorylation rheostat in peptide association.
Progressive phosphorylation appears to modulate peptide charge, inducing strong and specific intermolecular interactions between the monomers, which do not result in the formation of amorphous or ordered aggregates, as documented by experimental evidence derived from Circular Dichroism and NMR spectroscopy.
However, upon complete saturation of positive charges by phosphate groups, this effect is reversed: intramolecular interactions prevail and dimerization of zero-charge peptides is markedly reduced.
These findings underscore the role of phosphorylation thresholds in the dynamics of intrinsically disordered proteins.
Phosphorylation rheostats might account for the divergent effects of histone modifications on the modulation of chromatin structure.

PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC

High expression of programmed death-ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) cells usually inhibits the proliferation and functions of T cells, leading to immune suppression in tumor microenvironment.
However, very little has been described regarding the mechanism of PD-L1 overexpression in HCC cells. In the present study, we found epidermal growth factor (EGF) stimulation promoted the expression of PD-L1 mRNA and protein in HCC cells.
Inhibition of epidermal growth factor receptor (EGFR) could reverse EGF-induced the expression of PD-L1 mRNA and protein.
Subsequently, we also observed that the phosphorylation level of Pyruvate kinase isoform M2 (PKM2) at Ser37 site was also increased in response to EGF stimulation.
Expression of a phosphorylation-mimic PKM2 S37D mutant stimulated PD-L1 expression as well as H3-Thr11 phosphorylation in HCC cells, while inhibition of PKM2 significantly blocked EGF-induced PD-L1 expression and H3-Thr11 phosphorylation.
Furthermore, mutation of Thr11 of histone H3 into alanine abrogated EGF-induced mRNA and protein expression of PD-L1, Chromatin immunoprecipitation (ChIP) assay also suggested that EGF treatment resulted in enhanced H3-Thr11 phosphorylation at the PD-L1 promoter.
In a diethylnitrosamine (DEN)-induced rat model of HCC, we found that the expression of phosphorylated EGFR, PKM2 nuclear expression, H3-Thr11 phosphorylation, as well as PD-L1 mRNA and protein, was higher in the livers than that in normal rat livers.
Taken together, our study suggested that PKM2-dependent histone H3-Thr11 phosphorylation was crucial for EGF-induced PD-L1 expression at the transcriptional level in HCC. These findings may provide an alternative target for the treatment of hepatocellular carcinoma.

EGF promotes DKK1 transcription in hepatocellular carcinoma by enhancing the phosphorylation and acetylation of histone H3

The protein Dickkopf-1 (DKK1) is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC) and promotes metastatic progression through the induction of β-catenin, a Wnt signaling effector.
We investigated how DKK1 expression is induced in HCC and found that activation of the epidermal growth factor receptor (EGFR) promoted parallel MEK-ERK and PI3K-Akt pathway signaling that converged to epigenetically stimulate DKK1 transcription.
In HCC cell lines stimulated with EGF, EGFR-activated ERK phosphorylated the kinase PKM2 at Ser37, which promoted its nuclear translocation.
Also in these cells, EGFR-activated Akt phosphorylated the acetyltransferase p300 at Ser1834 Subsequently, PKM2 and p300 mediated the phosphorylation and acetylation, respectively, of histone H3 at the DKK1 promoter, which synergistically enhanced DKK1 transcription.
The mechanism was supported with mutational analyses in cells and in a chemically induced HCC model in rats.
The findings suggest that dual inhibition of the MEK and PI3K pathways might suppress the expression of DKK1 and, consequently, tumor metastasis in patients with HCC.

Phosphorylation of histone H3 by Haspin regulates chromosome alignment and segregation in maize mitosis

In human cells, Haspin-mediated histone H3 threonine 3 phosphorylation (H3T3ph) promotes centromeric localization of the Chromosome Passenger Complex, thereby ensuring proper kinetochore-microtubule attachment. Haspin also binds to PDS5 cohesin-associated factor B (Pds5B), antagonizing the Wings apart-like protein homolog (Wapl) -Pds5B interaction and thus preventing Wapl from releasing centromeric cohesion during mitosis.
However, the role of Haspin in plant chromosome segregation is not well understood.
Here, we show that in maize (Zea mays) mitotic cells, ZmHaspin localized to the centromere during metaphase and anaphase while localizing to the telomeres during meiosis.
These results suggest that ZmHaspin plays different roles during mitosis and meiosis.
ZmHaspin knockout led to decreased H3T3ph and H3S10ph and defects in chromosome alignment and segregation in mitosis.
These lines of evidence suggest that Haspin regulates chromosome segregation in plants via the mechanism described for humans, namely, H3T3ph.
Plant Haspin proteins lack the RTYGA and PxVxL motifs needed to bind Pds5B and HP1 and no obvious cohesion defects were detected in ZmHaspin knockout plants.
Taken together, these results highlight the conserved but slightly different roles of Haspin proteins in cell division in plants vs. animals.

MicroRNA-941 regulates the proliferation of breast cancer cells by altering histone H3 Ser 10 phosphorylation

Breast cancer including triple-negative breast cancer (TNBC) represents an important clinical challenge, as these tumors often develop resistance to conventional chemotherapeutics.
MicroRNAs play a crucial role in cell-cycle regulation, differentiation, apoptosis, and migration.
Herein, we performed Affymetrix Gene Chip miRNA 4.0 microarray and observed differential regulation of miRNAs (75 upregulated and 199 downregulated) in metastatic MDA-MB-231 cells as compared to immortalized human non-tumorigenic breast epithelial (MCF-10A) cells. MicroRNA-941 was significantly upregulated in MDA-MB-231 cells (almost nine-fold increase) in comparison to MCF-10A cells.
Transfection of MiRNA-941 inhibitor significantly decreased the proliferation and migration of MDA-MB-231 cells by altering the expressions of p21, Cyclin D1, PP2B-B1, E-cadherin and MMP-13.
Interestingly, we provide first evidence that inhibiting miR-941 prevents cell proliferation and phosphorylation of histone H3 at Ser10 residue.
Xenograft model of breast cancer was developed by subcutaneous injection of MDA-MB-231 cells into the mammary fat pad of female athymic nude mice (Crl:NU-Foxn1nu).

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EpiQuik Global Histone H3 Phosphorylation (Ser10) Assay Kit (Colorimetric) 

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EpiQuik Global Histone H3 Phosphorylation (Ser10) Assay Kit (Fluorometric) 

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EpiQuik Global Histone H3 Phosphorylation (Ser28) Assay Kit (Colorimetric) 

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TYR phosphorylation Assay Kit

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The tumours were allowed to grow to around 60 mm3, thereafter which we divided the animals into seven groups (n = 5).
Notably, intratumoral injection of miR-941 inhibitor significantly abolished the tumour growth in MDA-MB-231 xenograft model. 5-Fluorouracil (10 mg/kg, i.p.) was used as positive control in our study.
To the best of our knowledge, we report for the first time that targeting miR-941 improves the sensitivity of MDA-MB-231 cells to 5-fluorouracil.
This can be of profound clinical significance, as it provides novel therapeutic approach for treating variety of cancers (overexpressing miRNA-941) in general and breast cancers in particular.

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