Project description:Lineage tracing of individual cells during directed differentiation human iPSC into alveolospheres was performed using a lentiviral barcode labeling system (Weinreb et al., 2020) as described in Hurley et al., 2020.
Project description:We examined all transcriptome-level expressions in three initial cell-population densities (862, 1724 and 5172 cells/cm2) in the first two days of differentiation in N2B27. We collected cells in 10-mL tubes and centrifuged them using a pre-cooled centrifuge. We then extracted RNA from each cell-pellet using the PureLink RNA Mini Kit (Ambion, Life Technologies) according to its protocol. We next prepared the cDNA library with the 3′ mRNASeq library preparation kit (Quant-Seq, Lexogen) according to its protocol. We then loaded the cDNA library onto an Illumina MiSeq system using the MiSeq Reagent Kit v3 (Illumina) according to its protocol. We analyzed the resulting RNA-seq data as previously described (Trapnell et al., Nat Protoc 2012). We performed the read alignment using TopHat, read assembly using Cufflinks and analyses of differential gene expression data using Cuffdiff. We used the reference genome for Mus musculus from UCSC (mm10). We performed enrichment analysis of genes based on their FPKM values (e.g., more than 2-fold expressed when two initial population densities are compared) by using GO-terms from PANTHER (Mi et al., Nucl Acids Res 2019) and custom MATLAB script (MathWorks). We visualized results of pre-sorted, Yap1-related genes (LeBlanc et al., Elife 2018; Mugahid et al., Elife 2020; Yu et al., Oncogene 2018; Huh et al., Cells 2019; Zhu et al., Nature Sci Rep 2018; Zhou et al., Int J Mol Sci 2016; Vigneron & Vousden, EMBO J 2012; Kim et al., Cell 2015) into heat maps that displays the normalized expression value (row Z-score) for each gene and each condition.
Project description:While DNA methylation is an important gene regulatory mechanism in mammals (Razin and Riggs 1980; Moore, Le, and Fan 2013), its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its role in caste development by regulating gene expression and splicing (Elango et al. 2009; Lyko et al. 2010; Bonasio et al. 2012; Flores et al. 2012; Foret et al. 2012; Li-Byarlay et al. 2013; Marshall, Lonsdale, and Mallon 2019; Shi et al. 2013)(Alvarado et al. 2015; Kucharski et al. 2008). However, such findings are not always consistent across studies, and have therefore remained controversial (Arsenault, Hunt, and Rehan 2018; Cardoso-Junior et al. 2021; Harris et al. 2019; Herb et al. 2012; Libbrecht et al. 2016; Oldroyd and Yagound 2021b; Patalano et al. 2015). Here we use CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, Ooceraea biroi. Mutants have greatly reduced DNA methylation but no obvious developmental phenotypes, demonstrating that, unlike mammals (Brown and Robertson 2007; En Li, Bestor, and Jaenisch 1992; Jackson-Grusby et al. 2001; Panning and Jaenisch 1996), ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste development. However, mutants are sterile, while in wildtypes, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1 plays a crucial but unknown role in the insect germline (Amukamara et al. 2020; Arsala et al. 2021; Bewick et al. 2019; Schulz et al. 2018; Ventós-Alfonso et al. 2020; Washington et al. 2020).
Project description:VSMCs expressing SCA1 have increased proliferative capacity (Dobnikar et al, 2018; Worssam et al, 2022; Pan et al, 2020). We therefore, mapped chromatin accessibility changes using bulk ATAC-seq for SCA1+ and SCA1- lineage traced VSMCs.
Project description:CD47 is a transmembrane glycoprotein that is ubiquitously expressed in different organs and tissues (Barclay and Van den Berg 2014; Liu, et al. 2017). In the human immune system, CD47 interacts with some integrins, two counter-receptor signal regulator protein (SIRP) family members, and the secreted thrombospondin-1 (TSP1) (Barclay and Van den Berg 2014; Gao, et al. 2016; Kaur, et al. 2013; Oldenborg, et al. 2000). CD47 has two established roles in the immune system. The CD47-SIRPα interaction was identified as a critical innate immune checkpoint, which delivers an antiphagocytic signal to macrophages and inhibits neutrophil cytotoxicity (Martínez- Sanz, et al. 2021). Its interaction with inhibitory SIRPα is a physiological anti-phagocytic “don’t eat me” signal on circulating red blood cells that is co-opted by cancer cells (Matlung, et al. 2017). Many malignant cells overexpress CD47 (Betancur, et al. 2017; Chao, et al. 2011; Jaiswal, et al. 2009; Majeti, et al. 2009; Oronsky, et al. 2020; Petrova, et al. 2017). CD47/SIRPα-targeted therapeutics have been developed to overcome this immune checkpoint for cancer treatment (Kaur, et al. 2020; Matlung, et al. 2017). Secondly, engagement of CD47 on T cells by TSP1 regulates their differentiation and survival (Grimbert, et al. 2006; Lamy, et al. 2007) and inhibits T cell receptor signaling and antigen presentation by dendritic cells (DCs) (Kaur, et al. 2014; Li, et al. 2002; Liu, et al. 2015; Miller, et al. 2013; Soto-Pantoja, et al. 2014; Weng, et al. 2014). TSP1/CD47 signaling has similar inhibitory functions to limit NK cell activation (Kim, et al. 2008; Nath, et al. 2018; Nath, et al. 2019; Schwartz, et al. 2019) and IL1β production by macrophages (Stein, et al. 2016). CD47 is therefore a checkpoint that regulates both innate and adaptive immunity. The recent understanding of CD47 antagonism associated with increased antigen presentation by DCs (Liu, et al. 2016) and natural killer cell cytotoxicity (Nath, et al. 2019) contributes to the heightened interest in CD47 as a therapeutic target (Kaur, et al. 2020).
Project description:Chemical cross-linking coupled to mass spectrometry was used to study binary and ternary complexes involving cyclin-dependent kinase 19 (CDK19), cyclin-C, and an N-terminal fragment of subunit 12 of the Mediator complex (MED12 1-100). Cross-linking was performed using disuccinimidyl suberate (DSS). These results were generated in the context of the study published as Klatt et al., A precisely positioned MED12 activation helix stimulates CDK8 kinase activity, Proc. Natl. Acad. Sci. USA 2020 (DOI: 10.1073/pnas.1917635117) with the associated data set PXD015394, but were not included in the article.
Project description:AML patient GMPs or CMPs were FACS sorted as described in Supplementary Information (Reckzeh, K et al. 2020) TET2 deficiency cooperates with CBFB-MYH11 to induce acute myeloid leukemia and represents an early leukemogenic event. Reckzeh, K et al
Project description:In this study, we profiled for LINE1 binding loci in RSeT+DT naïve hES cells with the Chromatin Isolation by RNA Purification (ChIRP)-seq strategy. We followed a previously published ChIRP protocol described (Percharde et al. 2018; Lu et al. 2020).
Project description:inv(16)/Tet2-/- GMPs (Lin- Sca1- ckit+ CD41- FcgR+ CD150-) were FACS sorted as described in Supplementary Information (Reckzeh, K et al 2020) TET2 deficiency cooperates with CBFB-MYH11 to induce acute myeloid leukemia and represents an early leukemogenic event. Reckzeh, K et al.
Project description:Eukaryotic cells maintain homeostasis of their outer membrane by controlled internalization of lipid and protein constituents by endocytosis (Kaksonen & Roux, Nat Rev Mol Cell Biol. 2018). Endocytosis is evolutionary conserved and utilizes similar structural folds. How these structural folds are combined into proteins and protein complexes however differs between eukaryotic kingdoms (Kraus, Pleskot & Van Damme, Ann review 2024). The TPLATE complex in plants is an evolutionary ancient protein module that combines several endocytic folds into a single octameric protein complex (Gadeyne et al., Cell 2014, Hirst et al., eLife 2014, More et al., Curr Biol. 2020). Its molecular architecture, lipid-nucleated condensate formation, and its requirement for clathrin cage curvature revealed its function in endocytosis initiation in plants (Yperman & Wang et al., Sci Adv. 2021, Dragwidge et al., Nat Cell Biol. 2024, Johnson et al., PNAS 2021). Mechanistic understanding of how this complex drives membrane deformation during plant endocytosis is, however, lacking. Here, we used an integrative structural approach to obtain a precise molecular structure of the TPLATE complex. In addition, our approach also allowed visualizing the structural flexibility that hallmarks this enigmatic complex. We prove that the intrinsic structural flexibility is required for its functionality and membrane recruitment. We map different lipid-binding preferences onto specific domains located at the curved face of the complex. Finally, we show that the crescent shape of the structured part of the complex is sufficient for membrane curvature generation. Our mechanistic insight answers the longstanding question of how plants can execute endocytosis without cytoskeletal-based force generation.