Tyrosine phosphorylation of HoxA10 decreases DNA binding and transcriptional repression during interferon gamma -induced differentiation of myeloid leukemia cell lines

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. The CYBB gene, which encodes the respiratory burst oxidase component gp91(phox), is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon gamma (IFN-gamma)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-gamma treatment. However, IFN-gamma-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreases in vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation.     

Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.     

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.     

REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.     

Sphingosine 1-phosphate dynamically regulates myosin light chain phosphatase activity in human endothelial cells

Sphingosine 1-phosphate (S1P) is known to induce reorganization of the actin cytoskeleton through activation of the GTPase Rho. We have investigated the dynamic behavior of Rho/Rho kinase-regulated myosin light chain (MLC) phosphatase activity and MLC phosphorylation in Human Umbilical Vein Endothelial Cells (HUVEC) stimulated with S1P. Immediately (30-60 s) after S1P stimulation, MLC phosphatase activity dropped and MLC phosphorylation increased in a Rho/Rho kinase-dependent manner. Shortly thereafter (2 min), MLC phosphatase increased above baseline and MLC phosphorylation correspondingly decreased to near control values. At this time point, formation of actin ruffles and Rac activity assays indicated activation of Rac. Finally, between 5 and 15 min, MLC phosphatase dropped to a plateau below baseline. In parallel, MLC phosphorylation became constantly elevated above control values. These findings indicate that S1P is able to induce dynamic cycles of MLC phosphatase deactivation and activation. This novel feature of S1P could contribute to its chemotactic and angiogenic activity.     

Holocarboxylase synthetase synergizes with methyl CpG binding protein 2 and DNA methyltransferase 1 in the transcriptional repression of long-terminal repeats

Holocarboxylase synthetase (HLCS) is a chromatin protein that facilitates the creation of histone H3 lysine 9-methylation (H3K9me) gene repression marks through physical interactions with the histone methyltransferase EHMT-1. HLCS knockdown causes a depletion of H3K9me marks in mammalian cell cultures and severe phenotypes such as short lifespan and low stress resistance in Drosophila melanogaster. HLCS displays a punctuate distribution pattern in chromatin despite lacking a strong DNA-binding domain. Previous studies suggest that the binding of HLCS to chromatin depends on DNA methylation. We tested the hypothesis that HLCS interacts physically with the DNA methyltransferase DNMT1 and the methyl CpG binding protein MeCP2 to facilitate the binding of HLCS to chromatin, and that these interactions contribute toward the repression of long-terminal repeats (LTRs) by H3K9me marks. Co-immunoprecipitation and limited proteolysis assays provided evidence suggesting that HLCS interacts physically with both DNMT1 and MeCP2. The abundance of H3K9me marks was 207% greater in the LTR15 locus in HLCS overexpression human embryonic kidney HEK293 cells compared with controls. This gain in H3K9me was inversely linked with a 87% decrease in mRNA coding for LTRs. Effects of HLCS abundance on LTR expression were abolished when DNA methylation marks were erased by treating cells with 5-azacytidine. We conclude that interactions between DNA methylation and HLCS are crucial for mediating gene repression by H3K9me, thereby providing evidence for epigenetic synergies between the protein biotin ligase HLCS and dietary methyl donors.