Project description:These methylation data generated using EM-seq for all the NAM lines (as a part of the genome assembly project of NAM by the NAM Consortium Group). B73=project ID PRJEB32225/ERP114875; B73Ab10=project ID PRJEB35367/ERP118403; the rest of the NAMs=project ID PRJEB31061/ERP113571
Project description:Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome re-assembly in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. We find that Rtt106 recruitment to a number of promoters depends on Elg1. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin assembly defects of an elg1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.
Project description:Ribosome assembly in eukaryotes involves the activity of hundreds of assembly factors that direct the hierarchical assembly of ribosomal proteins and numerous ribosomal RNA folding steps. However, detailed insights into the function of assembly factors and ribosomal RNA folding events are lacking. To address this, we have developed ChemModSeq, a method that combines structure probing, high throughput sequencing and statistical modeling, to quantitatively measure RNA structural rearrangements during the assembly of macromolecular complexes. By applying ChemModSeq to purified 40S assembly intermediates we obtained nucleotide-resolution maps of ribosomal RNA flexibility revealing structurally distinct assembly intermediates and mechanistic insights into assembly dynamics not readily observed in cryo-electron microscopy reconstructions. We show that RNA restructuring events coincide with the release of assembly factors and predict that completion of the head domain is required before the Rio1 kinase enters the assembly pathway. Collectively, our results suggest that 40S assembly factors regulate the timely incorporation of ribosomal proteins by delaying specific folding steps in the 3’ major domain of the 20S pre-ribosomal RNA. Three datasets of yeast ribosomal samples subjected to different chemical modifications; 1M7 dataset contains 8 different modified samples and 2 control samples; NAI dataset contains 3 different modified samples and 2 control samples; DMS dataset contains 1 modified sample and 1 control sample. Each sample consists of at least two replicates.
Project description:In the ciliated protozoan Tetrahymena, de novo heterochromatin body formation is accompanied by programmed DNA elimination. We previously reported that dephosphorylation of the HP1-like protein Pdd1p is required for the formation of heterochromatin bodies during the process of programmed DNA elimination in the ciliated protozoan Tetrahymena. Here, we show that the heterochromatin body component Jub4p is required for Pdd1p phosphorylation, heterochromatin body formation and DNA elimination. Moreover, our analyses of unphosphorylatable Pdd1p mutants demonstrate that Pdd1p phosphorylation is required for heterochromatin body formation and DNA elimination, while it is dispensable for local heterochromatin assembly. Therefore, both phosphorylation and the following dephosphorylation of Pdd1p are necessary to facilitate the formation of heterochromatin bodies. We suggest that Jub4p-mediated phosphorylation of Pdd1p creates a chromatin environment that is a prerequisite for subsequent heterochromatin body assembly and DNA elimination. New macronuclei (MACs) of exconjugants were isolated from wild-type and various mutant cells at 12 hpm (hours post-mixing), sheared chromatin was immunoprecipitated andprecipitated DNA was analyzed by high-throughput sequencing
Project description:Vesicle associated membrane protein 2 (VAMP2/synaptobrevin2), a core SNARE protein residing on synaptic vesicles (SVs), forms helix bundles with syntaxin-1 and SNAP25 for the SNARE assembly. Prior to the SNARE assembly, the structure of VAMP2 is unclear. Here, by using in-cell NMR spectroscopy, we describe the dynamic membrane association of VAMP2 SNARE motif in mammalian cells, and the structural change of VAMP2 upon the change of intracellular lipid environment. We analyze the lipid compositions of the SV membrane by mass-spectrometry-based lipidomic profiling, and further reveal that VAMP2 forms distinctive conformations in different membrane regions. In contrast to the non-raft region, the membrane region of cholesterol-rich lipid raft markedly weakens the membrane association of VAMP2 SNARE motif, which releases the SNARE motif and facilitates the SNARE assembly. Our work reveals the regulation of different membrane regions on VAMP2 structure and sheds light on the spatial regulation of SNARE assembly.