Histone lysine methylation can have positive or negative effects on transcription, depending on the precise methylation site. According to the "histone code" hypothesis these methylation marks can be read by proteins that bind them specifically and then regulate downstream events. Hetero-chromatin protein 1 (HP1), an essential component of heterochromatin, binds specifically to methylated Lys 9 of histone H3 (K9/H3). The linker histone H1.4 is methylated on Lys 26 (K26/H1.4), but the role of this methylation in downstream events remains unknown. Here we identify HP1 as a protein specifically recognizing and binding to methylated K26/ H1.4. We demonstrate that the Chromo domain of HP1 is mediating this binding and that phosphorylation of Ser 27 on H1.4 (S27/ H1.4) prevents HP1 from binding. We suggest that methylation of K26/H1.4 could have a role in tethering HP1 to chromatin and that this could also explain how HP1 is targeted to those regions of chromatin where it does not colocalize with methylated K9/H3. Our results provide the first experimental evidence for a "phospho switch" model in which neighboring phosphorylation reverts the effect of histone lysine methylation.In eukaryotic cells the DNA is packaged into chromatin. The building block of chromatin is the nucleosomal core particle containing a histone octamer (two each of the core histones H2A, H2B, H3, and H4) around which 147 bp of DNA are wrapped (1). Linker histone H1 binds to the DNA between the nucleosomal core particles and stabilizes higher order chromatin structure (2).The N-terminal tails of the core histones protrude from the nucleosomal surface and are subject to multiple covalent modifications including methylation, phosphorylation, and acetylation. These modifications have the potential to regulate chromatin architecture and thereby can affect all aspects of DNA processing. According to the so called "histone code" hypothesis these modifications could be read by proteins that bind to specific modifications and then can regulate downstream events (3, 4).Methylation of lysines has positive as well as negative effects on transcription, depending on the methylation site. The methylation of lysine 9 of histone H3 (K9/H3) and lysine 27 of histone H3 (K27/H3) has generally been implicated in transcriptional repression. Methylated K9/H3 is specifically recognized and bound by Heterochromatin protein 1 (HP1) 3 (5-8). HP1 has a role in heterochromatin organization, maintenance, and in gene repression. In mammals three HP1 isoforms HP1␣, HP1␤(⌴31), and HP1␥(M32) have been identified. The HP1 proteins are very similar in their amino acid sequence, they contain a conserved N-terminal Chromo domain (CD), a more variable hinge region and a conserved C-terminal Chromoshadow domain (CSD). This modular organization of HP1 allows several proteins to bind simultaneously to HP1. The CD binds specifically to methylated K9/H3; the hinge region can bind to RNA, DNA, and chromatin, whereas the CSD is involved in self-association and interacts, e.g. with the DNA m...
SummaryIn receptor-mediated transport pathways in mammalian cells, clathrin-coated vesicle (CCV) m-adaptins are the main binding partners for the tyrosine sorting/internalization motif (YXXé). We have analyzed the function of the mA-adaptin, one of the ®ve m-adaptins from Arabidopsis thaliana, by pull-down assays and plasmon resonance measurements using its receptor-binding domain (RBD) fused to a histidine tag. We show that this adaptin is able to bind the consensus tyrosine motif YXXé from the pea vacuolar sorting receptor (VSR)-PS1, as well as from the mammalian trans-Golgi network (TGN)38 protein. Moreover, the tyrosine residue was revealed to be crucial for binding of the complete cytoplasmic tail of VSR-PS1 to the plant mA-adaptin. The trans-Golgi localization of the mA-adaptin strongly suggests its involvement in Golgito vacuole-traf®cking events.
Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors.
Intracellular protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus and within the Golgi apparatus is facilitated by COP (coat protein)-coated vesicles. Their existence in plant cellshas not yet been demonstrated, although the GTPbinding proteins required for coat formation have been identified. We have generated antisera against glutathione-S-transferase-fusion proteins prepared with cDNAs encoding the Arabidopsis Sec21p and Sec23p homologs (AtSec21p and AtSec23p, respectively). The former is a constituent of the COPI vesicle coatomer, and the latter is part of the Sec23/24p dimeric complex of the COPII vesicle coat. Cauliflower (Brassica oleracea) inflorescence homogenates were probed with these antibodies and demonstrated the presence of AtSec21p and AtSec23p antigens in both the cytosol and membrane fractions of the cell. The membrane-associated forms of both antigens can be solubilized by treatments typical for extrinsic proteins. The amounts of the cytosolic antigens relative to the membranebound forms increase after cold treatment, and the two antigens belong to different protein complexes with molecular sizes comparable to the corresponding nonplant coat proteins. Sucrose-densitygradient centrifugation of microsomal cell membranes from cauliflower suggests that, although AtSec23p seems to be preferentially associated with ER membranes, AtSec21p appears to be bound to both the ER and the Golgi membranes. This could be in agreement with the notion that COPII vesicles are formed at the ER, whereas COPI vesicles can be made by both Golgi and ER membranes. Both AtSec21p and AtSec23p antigens were detected on membranes equilibrating at sucrose densities equivalent to those typical for in vitro-induced COP vesicles from animal and yeast systems. Therefore, a further purification of the putative plant COP vesicles was undertaken.
Neoadjuvant imatinib therapy successfully reduces tumour size in GISTs. Since resistance relevant secondary mutations of the activation loop of KIT may be observed after neoadjuvant imatinib therapy, the time elapse with preoperative imatinib therapy should be chosen as short as curative tumour resection or function sparing surgery can be carried out. The determination of the optimal time point for surgery is therefore a critical event and will be discussed.
The nuclear receptor binding SET [su(var) 3-9, enhancer of zeste, trithorax] domain-containing protein 1 (NSD1) protein lysine methyltransferase (PKMT) was known to methylate histone H3 lysine 36 (H3K36). We show here that NSD1 prefers aromatic, hydrophobic, and basic residues at the -2, -1 and +2, and +1 sites of its substrate peptide, respectively. We show methylation of 25 nonhistone peptide substrates by NSD1, two of which were (weakly) methylated at the protein level, suggesting that unstructured protein regions are preferred NSD1 substrates. Methylation of H4K20 and p65 was not observed. We discovered strong methylation of H1.5 K168, which represents the best NSD1 substrate protein identified so far, and methylation of H4K44 which was weaker than H3K36. Furthermore, we show that Sotos mutations in the SET domain of NSD1 inactivate the enzyme. Our results illustrate the importance of specificity analyses of PKMTs for understanding protein lysine methylation signaling pathways.
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