Project description:Math2 (NEX-1/NeuroD6) is a member of the bHLH transcription factor family and is involved in neuronal differentiation and maturation. In the present study, we identified the genes targeted by Math2 using DNA microarrays and cultured rat cortical cells transfected with Math2. Of the genes regulated by Math2, we focused on plasticity-related gene 1 (Prg1). Prg1 expression induced by Math2 was confirmed in cultured rat cortical cells and PC12 cells analyzed by real-time quantitative PCR. Examining the promoter region of rat Prg1, we found four E-boxes designated -E1 to -E4 (CANNTG) which were recognized by the bHLH transcription factor. Using chromatin immunoprecipitation (ChIP) assays, we found that Math2 directly bound to the E-box(es) in the Prg1 promoter. The reporter assay of Prg1 showed that -E1 was critical for the regulation of the Prg1 expression by Math2. Then, the functional role of Math2 and Prg1 was investigated in PC12 cells. Seventy-two hours after transfection of Math2 or Prg1, neurite length and number was significantly induced in PC12 cells. Co-transfection with Prg1-siRNA completely inhibited Math2-mediated morphological changes. Our results suggest that Math2 directly regulates Prg1 expression and Math2-Prg1 cascade plays an important role in neurite outgrowth in PC12 cells.
Project description:Cells organize their actions partly through tightly controlled protein-protein interactions – collectively termed the interactome. Here we use crosslinking mass spectrometry (XL-MS) to chart the interactome of intact human nuclei. We overall identified ~8700 crosslinks, of which 2/3 represent links connecting distinct proteins. From this data we constructed an overview of the nuclear interactome. We observed that the histone proteins on the nucleosomes expose well-defined crosslinking hot-spots. For several nucleosome-interacting proteins, such as USF3 and Ran GTPase, the data allowed us to build models of their binding mode to the nucleosome. For HMGN2 the data guided the construction of a refined model of the interaction with the nucleosome, based on complementary NMR, XL-MS and modeling. Excitingly, several isoform-specific interactors seem to exist for distinct histone H1 variants and the analysis of crosslinks carrying post-translational modifications allowed us to extract how specific modifications influence the nucleosome interactome. Overall, our data depository will support future structural and functional analysis of cell nuclei, including the nucleoprotein assemblies they harbor.
Project description:To investigate the genome-wide occupancies of INCREASED LEAF INCLINATION1 (ILI1)-BINDING bHLH-1 (IBH1) (At2g43060), IBH1 ChIP analysis followed by direct sequencing (ChIP-Seq) on the p35S::IBH1-GFP (IBH1OE) line and a control line p35S::GFP (GFP OE) were conducted. Antibodies against GFP in the GFP-tagged IBH1 were utilized for immunoprecipitation of the endogenous protein-DNA complex, and the obtained DNA was subjected to next generation sequencing. Observation of genome-wide occupancies of atypical bHLH, INCREASED LEAF INCLINATION1 (ILI1)-BINDING bHLH-1 (IBH1) (At2g43060)
Project description:To identify the molecular mechanism of Mist1 during hepatoblasts differentiation, we performed gene expression analysis in Mist1 over-expressed cells. Dlk+ hepatoblasts derived from E13 fetal livers were purified. Purification of Dlk+ cells were performed using anti-Dlk antibody after hematopoietic cells were removed. Cells were infected with mock or Mist1-overexpressing retroviruses and cultured for 3days with oncostatin M. Virus infection was performed at the day primary culture was began (day0). Oncostatin M was added at day 1 and the cells were collected at day3. Two independent experiments were performed.
Project description:To understand how atypical bHLH, INCREASED LEAF INCLINATION1 (ILI1)-BINDING bHLH-1 (IBH1) (At2g43060), and close homologue, IBH1-like1 (IBL1) (At4g30410), interact to regulate cell elongation, genome-wide RNA-Seq expression analyses of IBH1 and IBL1 gain-(IBH1OE, IBL1OE) and loss-of-function (ibh1 (SALK 049177), ibl1(SALK 119457)) mutants were conducted. For loss-of-function mutant, homozygous ibh1(SALK 049177) and ibl1(SALK 119457) were compared to wild type (Col). For gain-of-function mutant, homozygous 35Spro:IBH1-GFP and 35Spro:IBL1-GFP were compared to wild type (Col). Total RNAs were extracted from seedling of each genotypes. For each genotype two biological replicates were sequenced.