The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex

The Arabidopsis Flowering Locus C (FLC) protein is a repressor of flowering regulated by genes in the autonomous and vernalization pathways. Previous genetic and transgenic data have suggested that FLC acts by repressing expression of the floral integrator genes SOC1 and FT. We have taken an in vivo approach to determine whether the FLC protein interacts directly with potential DNA targets. Using chromatin immunoprecipitation, we have shown that FLC binds to a region of the first intron of FT that contains a putative CArG box, and have confirmed that FLC binds to a CArG box in the promoter of the SOC1 gene. MADS box proteins are thought to bind their DNA targets as dimers or higher-order multimers. We have shown that FLC is a component of a multimeric protein complex in vivo and that more than one FLC polypeptides can be present in the complex.     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms

FLOWERING LOCUS C (FLC), a repressor of flowering, is a major determinant of flowering time in Arabidopsis. FLC expression is repressed by vernalization and in plants with low levels of DNA methylation, resulting in early flowering. This repression is not associated with changes of DNA methylation within the FLC locus in either vernalized plants or plants with low levels of DNA methylation. In both cases, there is a reduction of histone H3 trimethyl-lysine 4 (K4) and acetylation of both histones H3 and H4 around the promoter-translation start of FLC. The expression of the two genes flanking FLC is also repressed in both conditions and repression is associated with decreased histone H3 acetylation. The changes in histone modifications at the FLC gene cluster, which are similar in vernalized plants and in plants with reduced DNA methylation, must arise by different mechanisms. VERNALIZATION 1, VERNALIZATION 2 and VERNALIZATION INSENSITIVE 3 modulate FLC expression in vernalized plants; these proteins play no role in the downregulation of FLC in plants with low levels of DNA methylation. Chimeric FLC::GUS transgenes respond to vernalization but these same transgenes show a position-dependent response to low levels of DNA methylation. In plants with reduced DNA methylation, expression of the five MADS AFFECTING FLOWERING (MAF) genes is repressed, suggesting that DNA methylation alters the expression of a trans-acting regulator common to FLC and members of the related MAF gene family. Our observations suggest that DNA methylation is not part of the vernalization pathway.     

Dynamic and differential in vivo modifications of the isoform HMGA1a and HMGA1b chromatin proteins

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between non-malignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from non-metastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.     

Reciprocal control of flowering time by OsSOC1 in transgenic Arabidopsis and by FLC in transgenic rice

In a screen for MADS box genes which activate and/or repress flowering in rice, we identified a gene encoding a MADS domain protein (OsSOC1) related to the Arabidopsis gene AtSOC1. AtSOC1 and OsSOC1 show a 97% amino acid similarity in their MADS domain. The rice gene contains a large first intron of 27.6 kb compared to the 1 kb intron in Arabidopsis. OsSOC1 is located on top of the short arm of chromosome 3, tightly linked to the heading date locus, Hd9. OsSOC1 is expressed in vegetative tissues, and expression is elevated at the time of floral initiation, 40-50 days after sowing, and remains uniformly high thereafter, similar to the expression pattern of AtSOC1. The constitutive expression of OsSOC1 in Arabidopsis results in early flowering, suggesting that the rice gene is a functional equivalent of AtSOC1. We were not able to identify FLC-like sequences in the rice genome; however, we show that ectopic expression of the Arabidopsis FLC delays flowering in rice, and the up-regulation of OsSOC1 at the onset of flowering initiation is delayed in the AtFLC transgenic lines. The reciprocal recognition and flowering time effects of genes introduced into either Arabidopsis or rice suggest that some components of the flowering pathways may be shared. This points to a potential application in the manipulation of flowering time in cereals using well characterized Arabidopsis genes.     

Phosphorylation of Y14 modulates its interaction with proteins involved in mRNA metabolism and influences its methylation

The multicomponent exon junction complex (EJC) is deposited on the spliced mRNA during pre-mRNA splicing and is implicated in several post-splicing events, including mRNA export, nonsense-mediated mRNA decay (NMD), and translation control. This report is the first to identify potential post-translational modifications of the EJC core component Y14. We demonstrate that Y14 is phosphorylated at its repeated arginine/serine (RS) dipeptides, likely by SR protein-specific kinases. Phosphorylation of Y14 abolished its interaction with EJC components as well as factors that function downstream of the EJC. A non-phosphorylatable Y14 mutant was equivalent to the wild-type protein with respect to its association with spliced mRNA and its ability in NMD activation, but the mutant sequestered EJC and NMD factors on ribosome-containing mRNA ribonucleoproteins (mRNPs). We therefore hypothesize that phosphorylation of Y14 occurs upon completion of mRNA surveillance, leading to dissociation of Y14 from ribosome-containing mRNPs. Moreover, we found that Y14 is possibly methylated at multiple arginine residues in the carboxyl-terminal domain and that methylation of Y14 was antagonized by phosphorylation of RS dipeptides. This study reveals antagonistic post-translational modifications of Y14 that may be involved in the remodeling of Y14-containing mRNPs.     

Application of chemical selective cleavage methods to analyze post-translational modification in proteins

Three chemical specific cleavage reactions, one for the carboxyl side of aspartyl peptide bonds, one for the carboxyl side of asparaginyl peptide bonds and another for the amino side of seryl/threonyl peptide bonds have been recently established. Additionally, these reactions simultaneously react on several post-translationally modified groups in peptides or proteins. The modified groups cover the external modifications N-formyl, N-acetyl, N-pyroglutamyi residues and C-terminal-alpha amide, as well as the internal modifications such as O-acetyl serine, phosphorylated serine/tyrosine, sulfonylated tyrosine, glycosylated serine/threonine and glycosylated asparagine. These three cleavage reactions relate to key amino acids for modifications, deamidation for asparagine, phosphorylation and acetylation for serine, and glycosylation for asparagine, serine and threonine. The chemical reactions on these modifications change the peptide mapping pattern, and information from these reactions may contribute characterization and location of post-translational modified groups in the protein.     

Regulation of flowering time by Arabidopsis MSI1

The transition to flowering is tightly controlled by endogenous programs and environmental signals. We found that MSI1 is a novel flowering-time gene in Arabidopsis. Both partially complemented msi1 mutants and MSI1 antisense plants were late flowering, whereas ectopic expression of MSI1 accelerated flowering. Physiological experiments revealed that MSI1 is similar to genes from the autonomous promotion of flowering pathway. Expression of most known flowering-time genes did not depend on MSI1, but the induction of SOC1 was delayed in partially complemented msi1 mutants. Delayed activation of SOC1 is often caused by increased expression of the floral repressor FLC. However, MSI1 function is independent of FLC. MSI1 is needed to establish epigenetic H3K4 di-methylation and H3K9 acetylation marks in SOC1 chromatin. The presence of these modifications correlates with the high levels of SOC1 expression that induce flowering in Arabidopsis. Together, the control of flowering time depends on epigenetic mechanisms for the correct expression of not only the floral repressor FLC, but also the floral activator SOC1.     

Phosphorylation of serine residues in histidine-tag sequences attached to recombinant protein kinases: a cause of heterogeneity in mass and complications in function

High-level recombinant expression of protein kinases in eukaryotic cells or Escherichia coli commonly gives products that are phosphorylated by autocatalysis or by the action of endogenous kinases. Here, we report that phosphorylation occurred on serine residues adjacent to hexahistidine affinity tags (His-tags) derived from several commercial expression vectors and fused to overexpressed kinases. The result was observed with a variety of recombinant kinases expressed in either insect cells or E. coli. Multiple phosphorylations of His-tagged full-length Aurora A, a protein serine/threonine kinase, were detected by mass spectrometry when it was expressed in insect cells in the presence of okadaic acid, a protein phosphatase inhibitor. Peptide mapping by liquid chromatography-mass spectrometry detected phosphorylations on all three serine residues in an N-terminal tag, alpha-N-acetyl-MHHHHHHSSGLPRGS. The same sequence was also phosphorylated, but only at a low level, when a His-tagged protein tyrosine kinase, Pyk2 was expressed in insect cells and activated in vitro. When catalytic domains of Aurora A and several other protein serine/threonine kinases were expressed in E. coli, serines in the affinity tag sequence GSSHHHHHHSSGLVPRGS were also variably phosphorylated. His-Aurora A with hyperphosphorylation of the serine residues in the tag aggregated and resisted thrombin-catalyzed removal of the tag. Treatment with alkaline phosphatase partly restored sensitivity to thrombin. The same His-tag sequence was also detected bearing alpha-N-d-gluconoylation in addition to multiple phosphorylations. The results show that histidine-tag sequences can receive complicated posttranslational modification, and that the hyperphosphorylation and resulting heterogeneity of the recombinant fusion proteins can interfere with downstream applications.     

Establishment of the vernalization-responsive, winter-annual habit in Arabidopsis requires a putative histone H3 methyl transferase

Winter-annual accessions of Arabidopsis thaliana are often characterized by a requirement for exposure to the cold of winter to initiate flowering in the spring. The block to flowering prior to cold exposure is due to high levels of the flowering repressor FLOWERING LOCUS C (FLC). Exposure to cold promotes flowering through a process known as vernalization that epigenetically represses FLC expression. Rapid-cycling accessions typically have low levels of FLC expression and therefore do not require vernalization. A screen for mutants in which a winter-annual Arabidopsis is converted to a rapid-cycling type has identified a putative histone H3 methyl transferase that is required for FLC expression. Lesions in this methyl transferase, EARLY FLOWERING IN SHORT DAYS (EFS), result in reduced levels of histone H3 Lys 4 trimethylation in FLC chromatin. EFS is also required for expression of other genes in the FLC clade, such as MADS AFFECTING FLOWERING2 and FLOWERING LOCUS M. The requirement for EFS to permit expression of several FLC clade genes accounts for the ability of efs lesions to suppress delayed flowering due to the presence of FRIGIDA, autonomous pathway mutations, or growth in noninductive photoperiods. efs mutants exhibit pleiotropic phenotypes, indicating that the role of EFS is not limited to the regulation of flowering time.