SU(VAR)3-9 like histone methyltransferases control heterochromatic domains in eukaryotes. In Arabidopsis, 10 SUVH genes encode SU(VAR)3-9 homologues where SUVH1, SUVH2 and SUVH4 (KRYPTONITE) represent distinct subgroups of SUVH genes. Loss of SUVH1 and SUVH4 causes weak reduction of heterochromatic histone H3K9 dimethylation, whereas in SUVH2 null plants monoand dimethyl H3K9, mono-and dimethyl H3K27, and monomethyl H4K20, the histone methylation marks of Arabidopsis heterochromatin are significantly reduced. Like animal SU(VAR)3-9 proteins SUVH2 displays strong dosage-dependent effects. Loss of function suppresses, whereas overexpression enhances, gene silencing, causes ectopic heterochromatization and significant growth defects. Furthermore, modification of transgene silencing by SUVH2 is partially transmitted to the offspring plants. This epigenetic stability correlates with heritable changes in DNA methylation. Mutational dissection of SUVH2 indicates an implication of its N-terminus and YDG domain in directing DNA methylation to target sequences, a prerequisite for consecutive histone methylation. Gene silencing by SUVH2 depends on MET1 and DDM1, but not CMT3. In Arabidopsis, SUVH2 with its histone H3K9 and H4K20 methylation activity has a central role in heterochromatic gene silencing.

Leaf senescence, the final step of leaf development, involves extensive reprogramming of gene expression. Here, we show that these processes include discrete changes of epigenetic indexing, as well as global alterations in chromatin organization. During leaf senescence, the interphase nuclei show a decondensation of chromocenter heterochromatin, and changes in the nuclear distribution of the H3K4me2, H3K4me3, and the H3K27me2 and H3K27me3 histone modification marks that index active and inactive chromatin, respectively. Locus-specific epigenetic indexing was studied at the WRKY53 key regulator of leaf senescence. During senescence, when the locus becomes activated, H3K4me2 and H3K4me3 are significantly increased at the 5¢ end and at coding regions. Impairment of these processes is observed in plants overexpressing the SUVH2 histone methyltransferase, which causes ectopic heterochromatization. In these plants the transcriptional initiation of WRKY53 and of the senescence-associated genes SIRK, SAG101, ANAC083, SAG12 and SAG24 is inhibited, resulting in a delay of leaf senescence. In SUVH2 overexpression plants, significant levels of H3K27me2 and H3K27me3 are detected at the 5¢-end region of WRKY53, resulting in its transcriptional repression. Furthermore, SUVH2 overexpression inhibits senescence-associated global changes in chromatin organization. Our data suggest that complex epigenetic processes control the senescence-specific gene expression pattern.

We have developed a new rapid cell-free assay for endocytic clathrin-coated vesicle formation using highly purified rat liver plasma membrane sheets. After incubation in the presence of cytosol and nucleotides, released vesicles were collected by high-speed centrifugation and incorporated cargo receptors were detected by Western blotting. Three different cargo receptors were internalized into vesicles while a receptor, known to be excluded from coated pits, was not. The recruitment of cargo receptors into the vesicle fraction was cytosol, ATP and temperature-dependent and was enhanced by addition of GTP. Vesicle formation in this assay was confirmed by subcellular fractionation and EM analysis. Plasma membranes stripped of their endogenous coat proteins with 0.5 M Tris retained vesicle formation activity, which was highly dependent on clathrin and dynamin. Coat proteins and dynamin were not sufficient for clathrin-coated vesicle formation, and other peripheral membrane proteins recruited from the cytosol are required. The nonhydrolyzable ATP analogue, AMPPNP did not support clathrincoated vesicle formation; however, surprisingly, GTPg S was as effective as GTP. This assay will provide a powerful tool to dissect the minimum machinery and to probe the hierarchy of events involved in cargo selection and endocytic clathrin-coated vesicle formation.

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