Transcriptome sequencing of skeletal muscle for PRMT7 knockout mouse
ABSTRACT: We report that whole body PRMT7-/- adult mice display a significant reduction in in muscle mass. RNA sequencing was performed to identify potential PRMT7 targets. We found that top canonical pathways affected by the loss of PRMT7 includes cell cycle and senescence. RNA was extracted from tibialis anterior muscles harvested from 3 WT and 3 PRMT7 null mice at 8months. RNA sequencing was performed to compare mRNA in skeletal muscles between WT and KO mice.
Project description:The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. We determined in vivo binding sites of 39 human C2H2-ZF proteins, and correlated them with potential functions for these proteins. We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Chromatin immunoprecipitation was performed as described before (Schmidt et al., Methods, 2009), and ChIP samples along with several control samples from different experimental batches were sequenced on Illumina HiSeq 2000. Reads were mapped to hg19 (GRCh37) assembly, and peaks were identified by MACS using an experiment-specific background that controls for various biases, such as the Sono-Seq effect as well as potential co-purification of targets of other (interacting) proteins.
Project description:In response to infection, antigen-specific CD8+ T cells are primed in the T cell zone of secondary lymphoid organs and differentiate into cytotoxic effector T (TC) cells. Concurrently, CD4+ T cells differentiate into follicular helper T (TFH) cells that localize to B cell follicles and promote protective antibody responses. During unresolved infections, however, some viruses including human immunodeficiency virus (HIV) or Epstein–Barr virus (EBV) escape immune control and persist in TFH cells and B cells, respectively. Exclusion of Tc cells from B cell follicles is thought to be a major mechanism of immune evasion. New strategies are therefore needed to eradicate infected cells in follicles for a permanent cure. Using mouse infection models and human samples, we here identify a specialized group of TC cells expressing the chemokine receptor CXCR5 that can selectively enter B cell follicles and eradicate infected TFH and B cells. We demonstrate that differentiation of these cells, which we term follicular cytotoxic T (TFC) cells, requires the transcription factors Bcl6, E2A and Tcf1, whereas the transcriptional regulators Blimp1, Id3 and Id2 inhibit their development. We demonstrate that Blimp1 and E2A directly regulate Cxcr5 expression, and together with Bcl6 and Tcf1 form a transcriptional circuit that guides the TFC differentiation. The identification of a follicular subset of TC cells has far reaching implications for developing better strategies for the control of infections that target B cells and TFH cells and for the eradication of B cell derived malignancies. There is no associated input. The E2A Bio-ChIP-seq was performed with total thymocytes from Tcf3Bio/Bio Rosa26BirA/BirA mice
Project description:The goal of the study was to identify genes and pathways that were altered in the absence of the gene Interferon-Stimulated Gene 16 (ISG15) in human pancreatic ductal adenocarcinoma (PDAC) cancer stem cells. Primary adherent cultures established from patient-derived xenograft passaged in mice were established (Panc185). The ISG15 gene was edited using CRISPR-CAS9 technology and 3 commercially available guide RNAs introduced via lentiviral infection. Control cultures were also established using control guide RNAs. Once a polyclonal population was established and ISG15 loss confirmed, cultures were grown as 3D spheres in ultralow attachment plates to achieve a CSC-enriched culture. Following 5 days in culture as spheres, Total RNA was isolated by the guanidine thiocyanate (GTC) method using standard protocols. PolyA+ RNA fraction was processed as in Illumina’s ‘‘TruSeq RNA Sample Preparation v2 Protocol’’. The resulting purified cDNA library was applied to an Illumina flow cell for cluster generation (TruSeq cluster generation kit v5) and sequenced on the Genome Analyzer IIx with SBS TruSeq v5 reagents by following manufacturer’s protocols. RNA-seq data sets were analyzed using the tool Nextpresso.
Project description:A three-dimensional chromatin state underpins the structural and functional basis of the genome by bringing regulatory elements and genes into close spatial proximity to ensure proper, cell-type specific gene expression profiles. Here, we perform Hi-C chromosome conformation to investigate how the three-dimensional organization of the cancer genome is disrupted in the context of epigenetic remodelling and atypical gene expression programs. Hi-C, ChIP-seq and RNA-seq were conducted in three human prostate cell lines: normal prostate epithelial cells (PrEC) and prostate cancer cells (PC3 and LNCaP).
Project description:Versatile roles of REVOLUTA (REV), a Class III homeodomain-leucine zipper (HD-ZIP III) transcription factor, have been mainly depicted in Arabidopsis and Populus. In this study, we investigated the functions of its tomato homolog, namely SlREV. Over-expression of a microRNA166-resistant version of SlREV (35S::REVRis) not only resulted in vegetative abnormities such as curly leaves and fasciated stems, but also caused dramatic reproductive alterations including continuous production of flowers at pedicel abscission zone (AZ) and ectopic fruit formation on receptacles. Microscopic analysis showed that meristem-like structures continuously emerged out from the exodermises of pedicel AZs and ectopic carpels formed between the first and the second whorl of floral buds in 35S::REVRis plants. Therefore, we performed Illumina’s digital gene expression (DGE) system, a tag-based transcriptome sequencing methodTranscriptional data to dicover differential expressed genes in early buds (1-2 mm floral buds at stage 6-8) of overexpression line SlREVRis-1. The result suggests that SlREV may regulate genes related to meristem maintenance and cell differentiation in the development of flower pedicel abscission zone, and modulate genes in homodomain and MADS-box families and hormone pathways during fruit formation. These results reveal important roles of SlREV in tomato. 1-2 mm floral buds at stage 6-8 were sampled from three individual plants of 35S::REVRis-1 and corresponding WT control. Three aliquots of RNA from transgenic or WT plants were pooled. Then, the digital expression profile were generated by Illumina Cluster Station and Illumina HiSeq™ 2000 System (BGI Inc.).
Project description:Specification and propagation of the centromeres of eukaryotic chromosomes is determined by epigenetic mechanisms. Unfortunately, the epigenetic characteristics of centromeric DNA and chromatin are difficult to define because the centromeres are composed of highly repetitive DNA sequences in most eukaryotic species. Several rice centromeres have been fully sequenced, making rice an excellent model for centromere research. We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The DNA sequences in the core domains of rice Cen4, Cen5, and Cen8 showed elevated methylation levels compared to the sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains compared to the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared to the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin. mCIP-seq of one sample of rice seedling
Project description:Glioblastoma (GBM) is thought to be driven by a sub-population of cancer stem cells (CSCs) that self-renew and recapitulate tumor heterogeneity, yet remain poorly understood. Here we present a comparative epigenomic analysis of GBM CSCs that reveals widespread activation of genes normally held in check by Polycomb repressors. These activated targets include a large set of developmental transcription factors (TFs) whose coordinated activation is unique to the CSCs. We demonstrate that a critical factor in the set, ASCL1, activates Wnt signaling by repressing the negative regulator DKK1. We show that ASCL1 is essential for maintenance and in vivo tumorigenicity of GBM CSCs. Genomewide binding profiles for ASCL1 and the Wnt effector LEF1 provide mechanistic insight and suggest widespread interactions between the TF module and the signaling pathway. Our findings demonstrate regulatory connections between ASCL1, Wnt signaling and collaborating TFs that are essential for the maintenance and tumorigenicity of GBM CSCs. Histone modification profiling for multiple marks by ChIP-Seq in untreated glioblastoma cancer stem cells, glioblastoma non-stem cells and neural stem cells
Project description:Msgn1 is a bHLH transcription factor and is a direct target gene of the Wnt/b-catenin signaling pathway. During mouse embryogenesis, Msgn1 is expressed in the mesodermal compartment of the primitive streak and is required for the differentiation of presomitic mesoderm. Msgn1-/- mutants show defects in somitogenesis leading to a lack of trunk skeletal muscles, vertebra and ribs. The goal of this study is to dissect the molecular and cellular function of Msgn1 in Embryonic Stem Cells (ESC) and mouse development. In order to identify direct Msgn1 targets, we performed transcriptional profiling and CHIP-seq of Msgn1 expressing differentiating ES cells. Integration of these data sets, we found that Msgn1 is a master regulator of PSM differentiation regulating gene expression programs of PSM identity, EMT, motility and Notch segmentation clock. Inducible Flag Msgn1 ES cells were differentiated to form Embryoid bodies (EBs) for 2 days. Flag-Msgn1 was induced on day 2 with doxycycline and samples were collected 36h later. Here Input DNA is used as control.
Project description:∆FosB epigenetically regulates target gene expression; however, whether ∆FosB targets the same genes when induced by recurrent seizures in different neurological conditions like Alzheimer's disease (AD) is unclear. We performed hippocampal ChIP-sequencing to identify ∆FosB target genes in APP mice, a model of AD, and in pilocarpine-treated wildtype mice (Pilo mice), a model of epilepsy. Functional domains modulated by ∆FosB target genes are expanded and diversified in APP mice and in Pilo mice (vs respective controls), and primarily involve neuronal excitability and neurotransmission, neurogenesis, chromatin remodeling, and cellular stress and immunity. To identify genes bound by ∆FosB regardless of seizure etiology, we focused on the 442 ∆FosB target genes in both APP and Pilo mice (not respective controls), which related to pathways including membrane potential regulation, glutamatergic signaling, complement activation, neuron-glia population maintenance, and chromatin dynamics. RNA-sequencing and RT-qPCR in independent mice detected altered expression of several ∆FosB targets shared in APP and Pilo mice. Our findings indicate that seizure-induced ∆FosB can bind genes in seizure etiology-dependent patterns, but can also bind genes in patterns regardless of seizure etiology. Understanding factors that influence ∆FosB binding patterns could reveal novel insights into control of gene expression in disorders with recurrent seizures.