Project description:Eukaryotic genes often generate a variety of RNA isoforms that can lead to functionally distinct protein variants. The synthesis and stability of RNA isoforms is however poorly characterized. The reason for this is that current methods to quantify RNA metabolism use short-read sequencing that cannot detect RNA isoforms. Here we present nanopore sequencing-based Isoform Dynamics (nano-ID), a method that detects newly synthesized RNA isoforms and monitors isoform metabolism. nano-ID combines metabolic RNA labeling, long-read nanopore sequencing of native RNA molecules and machine learning. nano-ID derived RNA stability estimates enable a distinctive evaluation of stability determining factors such as sequence, poly(A)-tail length, RNA secondary structure, translation efficiency and RNA binding proteins. Application of nano-ID to the heat shock response in human cells reveals that many RNA isoforms change their stability. nano-ID also shows that the metabolism of individual RNA isoforms differs strongly from that estimated for the combined RNA signal at a specific gene locus. nano-ID enables studies of RNA metabolism on the level of single RNA molecules and isoforms in different cell states and conditions.
Project description:Stem cell functions require activation of stem cell-intrinsic transcriptional programs as well as intimate extracellular interactions with a niche microenvironment. How the core pluripotency transcriptional machinery controls residency of stem cells in the niche microenvironment is unknown. Here we show that the helix loop helix transcriptional regulators Id (Inhibitors of DNA binding) are the master regulators that coordinate stem cell activities with anchorage of neural stem cells (NSCs) to the embryonic and postnatal niche. Conditional inactivation of Id genes (Id1, Id2 and Id3) in the mouse NSC compartment triggered detachment of embryonic and post-natal NSCs from the ventricular and vascular niche respectively, followed by premature differentiation. Through an unbiased interrogation of the gene modules directly targeted by deletion of Id genes in NSCs, we discovered that Id proteins repress the bHLH-mediated activation of Rap1GAP, thus serving to maintain the GTPase activity of RAP1, a key mediator of cell adhesion. Preventing the elevation of Rap1GAP efficiently countered the consequences of Id loss on NSC-niche interaction and stem cell identity. Thus, by preserving anchorage to the extracellular environment of NSCs, Id activity synchronizes NSC functions to residency in the specialized niche.
Project description:Calcium ions serve as key intracellular signals. Local, transient increases in calcium concentrations can activate calcium sensor proteins that in turn trigger downstream effectors. In neurons, such calcium transients trigger pre-synaptic vesicle release and mediate post-synaptic plasticity. It is challenging to capture the molecular events associated with these localized and ephemeral calcium signals, however. Here we report the development of an engineered biotin ligase that combines the power of genetically encoded calcium indicators with protein proximity labeling. The enzyme, Cal-ID, biotinylates nearby proteins in response to elevated local calcium levels. The biotinylated proteins can be visualized by microscopy and identified via mass spectrometry. Cal-ID mass spectrometry applied to HEK293T cells identified cell cycle-dependent calcium signaling microdomains at centrosomes. Our results from mouse primary neurons indicated active calcium signaling near calcium extrusion sites on the plasma membrane. Therefore, we propose Cal-ID as a biochemical recorder of calcium signaling in living cells.
Project description:We developed and performed LIMe-ID to simultaneously measure lamina association and DNA methylation in one experimental workflow. We then conducted LIMe-ID following perturbations of PRC2 to better understand the influence of H3K27me3 on lamina association.
Project description:Stem cell functions require activation of stem cell-intrinsic transcriptional programs as well as intimate extracellular interactions with a niche microenvironment. How the core pluripotency transcriptional machinery controls residency of stem cells in the niche microenvironment is unknown. Here we show that the helix loop helix transcriptional regulators Id (Inhibitors of DNA binding) are the master regulators that coordinate stem cell activities with anchorage of neural stem cells (NSCs) to the embryonic and postnatal niche. Conditional inactivation of Id genes (Id1, Id2 and Id3) in the mouse NSC compartment triggered detachment of embryonic and post-natal NSCs from the ventricular and vascular niche respectively, followed by premature differentiation. Through an unbiased interrogation of the gene modules directly targeted by deletion of Id genes in NSCs, we discovered that Id proteins repress the bHLH-mediated activation of Rap1GAP, thus serving to maintain the GTPase activity of RAP1, a key mediator of cell adhesion. Preventing the elevation of Rap1GAP efficiently countered the consequences of Id loss on NSC-niche interaction and stem cell identity. Thus, by preserving anchorage to the extracellular environment of NSCs, Id activity synchronizes NSC functions to residency in the specialized niche. We generated Id-cTKO mice carrying a Cre-recombinase-oestrogen-receptor-T2 (Cre-ER) allele targeted to the ubiquitously expressed ROSA26 locus (Id-cTKO-Rosa-Cre-ER). In this system, 4-hydroxytamoxifen (4-OHT) releases the Cre recombinase inhibition and allows recombination of genomic loxP sites. Indeed, efficient deletion of Id1 and Id2 with complete loss of Id protein expression was detectable after treatment of NSCs with 4-OHT for 72 h. Total RNA was extracted from triplicate samples of Id-cTKO-Rosa-Cre-ER NSCs treated for different times (6 h, 12 h, 18 h, 24 h, 48 h, 96 h, 144 h) with 4-OHT or control vehicle and used for analysis on Illumina MouseWG-6 expression BeadChip. The raw array data was normalized using the Bioconductor package Lumi using quantile normalization.
Project description:To study the function of SUMOylation and SUMO-SIM interactions for particular substrates poses several challenges. SUMOylation occurs transiently and often in a small percentage of the total copy numbers of a given substrate. Modified proteins can be readily deSUMOylated and SUMO can be recycled and passed to other substrates. SUMO-SIM interactions are also difficult to analyze due to their weak affinity (Kd 1-100 µM). To overcome these technical issues, we developed SUMO-ID, a new strategy based on Split-TurboID to identify SUMO- interactors of specific substrates dependent on SUMO conjugation or interaction. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors. , the high sensitivity of SUMO-ID allowed to identify novel interactors of SUMOylated SALL1, a less characterized SUMO substrate.