Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response (DDR), detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 minutes) and transiently induced at DNA damage sites in response to UV. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL31 and the demethylase FTO2. In the absence of METTL3 catalytic activity, cells showed delayed repair of UV-induced cyclobutane pyrimidine (CPD) adducts and elevated sensitivity to UV, demonstrating the importance of m6A in the UV-responsive DDR. Multiple DNA polymerases are involved in the UV response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair (NER) pathway3, while others participate in trans-lesion synthesis (TLS) to allow replication past damaged lesions in S phase4. DNA polymerase κ (Pol κ), which has been implicated in both NER and TLS5,6, required the catalytic activity of METTL3 for immediate localization to UV-induced DNA damage sites. Importantly, Pol κ over-expression qualitatively suppressed the CPD removal defect associated with METTL3 loss. Taken together, we have uncovered a novel function for RNA m6A modification in the UV-induced DDR, and our findings collectively support a model whereby m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Posttranslational modifications of proteins by small ubiquitin-like modifiers (SUMOs) regulate protein degradation and localization, protein-protein interaction, and transcriptional activity. SUMO E3 ligase functions are executed by SIZ1/SIZ2 and Mms21 in yeast, the PIAS family members RanBP2, and Pc2 in human. The Arabidopsis thaliana genome contains only one gene, SIZ1, that is orthologous to the yeast SIZ1/SIZ2. Here, we show that Arabidopsis SIZ1 is expressed in all plant tissues. Compared with the wild type, the null mutant siz1-3 is smaller in stature because of reduced expression of genes involved in brassinosteroid biosynthesis and signaling. Drought stress induces the accumulation of SUMO-protein conjugates, which is in part dependent on SIZ1 but not on abscisic acid (ABA). Mutant plants of siz1-3 have significantly lower tolerance to drought stress. A genome-wide expression analysis identified ;1700 Arabidopsis genes that are induced by drought, with SIZ1 mediating the expression of 300 of them by a pathway independent of DREB2A and ABA. SIZ1-dependent, drought-responsive genes include those encoding enzymes of the anthocyanin synthesis pathway and jasmonate response. From these results, we conclude that SIZ1 regulates Arabidopsis growth and that this SUMO E3 ligase plays a role in drought stress response likely through the regulation of gene expression.

Highlights d An assay to visualize ALT telomere DNA synthesis in APBs d APBs are functionally important for ALT DNA synthesis d RAD52 promotes telomere D-loops in vitro and ALT DNA synthesis in APBs d A RAD52-independent BIR pathway is responsible for C-circle formation

Summary Epigenetic regulation of gene expression by histone-modifying co-repressor complexes is central to normal animal development. The NAD+-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 co-repressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

Post-translational modification of transcription factors by the small ubiquitin related modifier SUMO is associated with transcriptional repression, but the underlying mechanisms remain incompletely described. We have identified binding of the LSD1/CoREST1/HDAC co-repressor complex to SUMO-2. Here we show that CoREST1 binds directly and non-covalently to SUMO-2, but not SUMO-1, and CoREST1 bridges binding of the histone demethylase LSD1 to SUMO-2. Depletion of SUMO-2/3 conjugates led to transcriptional de-repression, reduced occupancy of CoREST1 and LSD1 and changes in histone methylation and acetylation at some, but not all, LSD1/CoREST1/HDAC target genes. We have identified a non-consensus SUMO-interaction motif (SIM) in CoREST1 required for SUMO-2 binding and we show that mutation of the CoREST1 SIM disrupted SUMO-2 binding and transcriptional repression of some neuronal specific genes in non-neuronal cells. Our results reveal that direct interactions between CoREST1 and SUMO-2 mediate SUMO-dependent changes in chromatin structure and transcription important for cell type-specific gene expression.

Posttranslational modification of substrates by the small ubiquitin-like modifier, SUMO, regulates diverse biological processes, including transcription, DNA repair, nucleocytoplasmic trafficking, and chromosome segregation. SUMOylation is reversible, and several mammalian homologs of the yeast SUMO-specific protease Ulp1, termed SENPs, have been identified. We demonstrate here that SENP5, a previously uncharacterized Ulp1 homolog, has SUMO C-terminal hydrolase and SUMO isopeptidase activities. In contrast to other SENPs, the C-terminal catalytic domain of SENP5 preferentially processed SUMO-3 compared to SUMO-1 precursors and preferentially removed SUMO-2 and SUMO-3 from SUMO-modified RanGAP1 in vitro. In cotransfection assays, SENP5 preferentially reduced high-molecular-weight conjugates of SUMO-2 compared to SUMO-1 in vivo. Full-length SENP5 localized to the nucleolus. Deletion of the noncatalytic N-terminal domain led to loss of nucleolar localization and increased de-SUMOylation activity in vivo. Knockdown of SENP5 by RNA interference resulted in increased levels of SUMO-1 and SUMO-2/3 conjugates, inhibition of cell proliferation, defects in nuclear morphology, and appearance of binucleate cells, revealing an essential role for SENP5 in mitosis and/or cytokinesis. These findings establish SENP5 as a SUMO-specific protease required for cell division and suggest that mechanisms involving both the catalytic and noncatalytic domains determine the distinct substrate specificities of the mammalian SUMO-specific proteases.Posttranslational modification of proteins by the small ubiquitin-like modifier SUMO is an important mechanism to regulate numerous biological processes, including nucleocytoplasmic trafficking, transcription, and DNA repair and replication, as well as mitotic and meiotic chromosome behavior (13,16,20). SUMO is covalently attached to lysine residues in substrate proteins in a process similar to ubiquitination (for review, see reference 19). SUMO conjugation requires an E1-activating enzyme (Aos1/Uba2) and an E2-conjugating enzyme (Ubc9), and SUMOylation of specific substrates may be stimulated by the action of diverse E3 ligases (8,9,34). Covalent modification of proteins by SUMO is reversible, and a number of SUMO-specific proteases have been predicted based on homology to yeast Ulp1, the first identified SUMO-specific protease (23). The SUMO-specific proteases have dual roles in the SUMOylation pathway. First, they are responsible for the initial processing of SUMO precursors to generate a C-terminal diglycine motif required for conjugation. Second, these proteases execute the deconjugation reaction that removes SUMO from high-molecular-weight SUMO conjugates. At least seven Ulp1 homologs have been identified in mammals , which share a conserved cysteine protease domain at their C termini (12,14,22,30,46). Four of these, SENP1, -2, -3, and -6, have been reported to have SUMO-specific C-terminal hydrolase and/or isopeptidase activity. In contrast, the Ulp homolog SENP8 has deconjugation activity...

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.

S -Adenosyl-L -methionine:phosphoethanolamine N -methyltransferase (PEAMT; EC 2.1.1.103) catalyzes the key step in choline (Cho) biosynthesis, the N -methylation of phosphoethanolamine. Cho is a vital precursor of the membrane phospholipid phosphatidylcholine, which accounts for 40 to 60% of lipids in nonplastid plant membranes. Certain plants use Cho to produce the osmoprotectant glycine betaine, which confers resistance to salinity, drought, and other stresses. An Arabidopsis mutant, t365 , in which the PEAMT gene is silenced, was identified using a new sense/antisense RNA expression system. t365 mutant plants displayed multiple morphological phenotypes, including pale-green leaves, early senescence, and temperature-sensitive male sterility. Moreover, t365 mutant plants produced much less Cho and were hypersensitive to salinity. These results demonstrate that Cho biosynthesis not only plays an important role in plant growth and development but also contributes to tolerance to environmental stresses. The temperature-sensitive male sterility caused by PEAMT silencing may have a potential application in agriculture for engineering temperature-sensitive male sterility in important crop plants.