Project description:Males and females often differ on propensity for a substance use disorder (SUD), etiology and clinical manifestation of SUD, and response to SUD treatment strategies (Riley et al 2018). The fluctuation of sex-related hormones in the brain and/or circulation may contribute to some of these differences. For example, estradiol activity has been associated with sensitivity to drugs of abuse (Tonn Eisinger et al 2018) and vulnerability to SUD (Anker and Carroll 2011). In particular, estrogen can affect the dopamine system in brain and this may contribute to differences in the etiology and the clinical manifestation of SUD (Bobzean et al 2014). Without direct association with SUD, others have shown that stage of the estrous cycle influences RNA transcription levels and splicing in particular brain regions (Duclot and Kabbaj 2015; DiCarlo et al 2017). What has not been thoroughly explored is whether genetic factors can modify the effect of estrous cycle on RNA transcript and ultimately, whether the interaction of these two factors can influence the sex-related differences in SUD vulnerability.
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:Adult neural stem cells (aNSCs) show multilineage differentiation potential influenced by intrinsic and extrinsic signaling cues. We and others have shown that stimulation of aNSCs with bone marrow mesenchymal stem cell (MSC) secreted factors substantially enhances in vitro oligodendrogenesis at an expense of astrogenesis by yet unknown mechanisms (Rivera et al. 2006, Jadasz et al. 2013; 2018, Rivera et al., 2019). In the present study, we demonstrate that aNSCs pre-treated with MSC secretomes for different periods in vitro preferentially differentiate to oligodendrocytic cells in vivo after transplantation into the adult rat spinal cord. Analysis of different time points after transplantation revealed a stable survival rate of transplanted aNSCs and an emphasized pro-oligodendroglial differentiation in response to MSC secreted factors. MSC derived secretomes were then analyzed by mass spectrometry-based proteomics and label-free quantification to identify secreted proteins contributing to oligodendroglial lineage fate determination. To exclude possible contaminants derived from dead cells or serum, our approach includes a comparison of the abundances of proteins present in MSC derived secretomes with corresponding proteins in cell lysates (Grube et al., 2018, Schira-Heinen et al., 2019).
Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) causes the B cell malignancy primary effusion lymphoma (PEL). We have previously shown that cultured PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival (Manzano et al., Nat Comm 2018), although KSHV encodes a viral homolog of this protein (vFLIP). Here we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout/knockdown.
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: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:DNA damage can promote altered RNA splicing and decreased gene expression (Gregersen and Svejstrup, 2018; Milek et al., 2017; Munoz et al., 2009; Shkreta and Chabot, 2015), and aberrant splicing is implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), Fragile X syndrome and spinal muscular atrophy (SMA) (Conlon et al., 2016; Jia et al., 2012; Loomis et al., 2014; Qiu et al., 2014; Scotti and Swanson, 2016). Therefore, we used RNA-seq data to assess RNA-splicing in double-mutant brain tissue using multivariate analysis of transcriptional splicing (rMATS) (Shen et al., 2014) and a splicing deficiency score algorithm (Bai et al., 2013) to assess intron retention.
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.