Transcriptome profiles of hematopoietic lineages from BAP1 WT and KO mice
Ontology highlight
ABSTRACT: BAP1 has been studied as a tumor suppressor. Our aim was to characterize genes that were altered in various hematopoietic cell types upon deletion of BAP1. BAP1 WT versus KO cells.
Project description:Baseline gene expression of mouse Cardiac Progenitor Cells (CPC) We used microarrays to analyze the global gene expression of mouse CPCs. Compare the gloable gene expression of mouse CPCs and Cardiomyocytes. Although the chip is two color, we only used Cy5 as the data channel.
Project description:Unilateral naris occlusion was performed the day after birth in three FVB strain mice. At 25 days of age olfactory mucosa was collected from open nasal fossa, the occluded nasal fossa and from three untreated mice. Total RNA was extracted and gene profiles among these three treatment conditions were compared. The goal of the study was to determine the effect of stimulus deprivation on the genetic profile of olfactory mucosa. There were three treatment conditions: open mucosa, occluded mucosa, and untreated mucosa. Each of these conditions had three biological replicates. Open and occluded however were repeated measures i.e. from the same individuals.
Project description:The purpose of this study is to search for aberrant genes in HaCaT keratinocytes after chronic exposure to arsenic trioxide. The objective of the investigation was to discover the mechanism of arsenic carcinogenicity in human epidermal keratinocytes. We hypothesize that a combined strategy of DNA microarray, qRT-PCR and gene function annotation will identify aberrantly expressed genes in HaCaT keratinocyte cell line after chronic treatment with arsenic trioxide. HaCaT cells were chronically exposed to 0.5M-BM-5g/mL arsenic trioxide (As2O3) up to 22 passages and RNA was extracted. Microarray data analysis identified 14 up-regulated genes and 21 down-regulated genes in response to arsenic trioxide Two experimental groups: 1. The treatment group was sub-cultured up to passage 22 to establish a chronic exposure state. 2. The passage control group was also sub-cultured up to 22 passages but with no exposure to arsenic trioxide. 4 technical replicates with 3 replicates making a total of 8X3 =24 samples HaCat Cell untreated (passage control): 1. H1_H001, H1_H002, H1_H003 2. H2_ H004, H2_H005, H2_H006 3. H3_ H007, H3_H008, H3_H009 4. H4_ H010, H4_H011, H4_H012 HaCat Cell treated with 0.5M-BM-5g/ml of arsenic trioxide: 5. A1_H013, A1_H014, A1_H015 6. A2_H016, A2_H017, A2_H018 7. A3_H019, A3_H020, A3_H021 8. A4_H022, A4_H023, A4_H024 Cell Type: Human Skin Keratinocyte: 1.5 M-CM-^W105 HaCaT cells were cultured in 7.5 ml of complete DMEM containing 10% Fetal Bovine Serum (FBS) and 1% penicillin, streptomycin in T-25 culture plate. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 M-BM-:C. The treatment groups were exposed to 0.5M-BM-5g/mL As2O3 (equivalent to LC 0.5), and passaged at 90% confluent. Total RNA was extracted from 4 technical replicates of unexposed HaCaT cells and HaCaT cells chronically exposed to arsenic trioxide up to passage 22 using RNA STAT-60 (TEL-TEST, INC, Friendswood, TX, USA).
Project description:BRCA1-associated protein 1 (BAP1) is a multidomain deubiquitinase (DUB) with a ubiquitin carboxyl-terminal hydrolase (UCH) domain at its N-terminus. BAP1 mainly localizes in the nucleus and primarily functions as a transcriptional regulator in diverse cellular pathways, including cell proliferation, cell cycle progression, cell death and DNA repair. However, a comprehensive understanding of the mechanism by which the nucleocytoplasmic trafficking of BAP1 is regulated remains lacking. To identify protein factors that may be responsible for the nuclear import of BAP1, we used transiently expressed a N-terminally FLAG-tagged BAP1 in HEK293 freestyle (HEK293F) cells as a bait to pull down BAP1-interacting proteins for mass spectrometry-based proteomic analysis.
Project description:Analysis of the effect that reduced BAP1 levels have on global gene expression.The hypothesis tested was that reduction in BAP1 levels would produce changes in gene expression similar to changes observed in class 2 uveal melanomas. Data provided insight into genes that are disrupted with reduced BAP1 levels. Total RNA was isolated from 92.1 cells transfected with either control or BAP1 knockdown constructs for global expression analysis.
Project description:The deubiquitinase BAP1 is a candidate tumor suppressor regulating cell proliferation in human and is required for development in Drosophila. BAP1 is assembled into high molecular weight transcriptional multi-protein complexes. In order to identify potential BAP1 target genes, global mRNA expression profiling using microarrays was conducted. U2OS cells, transfected with a non-target control shRNA or shRNAs targeting BAP1, were selected with puromycin containing medium and then synchronized at the G1/S border to allow comparative analysis of gene expression.
Project description:Uveal melanoma is a highly aggressive cancer with a strong propensity for metastasis, yet little is known about the biological mechanisms underlying this metastatic potential. We recently showed that most metastasizing uveal melanomas, which exhibit a class 2 gene expression profile, contain inactivating mutations in the tumor suppressor BAP1. The aim of this study was to investigate the role of BAP1 in uveal melanoma progression. To that end, uveal melanoma cells were studied following stable shRNA-mediated depletion of BAP1. RNA was isolated from three independent uveal melanoma cell lines each stably depleted using shRNA for either BAP1 or the control gene GFP. Two biological replicates were performed for each cell line.
Project description:Nontypeable Haemophilus influenzae (NTHi) is a common causative organism of acute otitis media (AOM) in children. A human cDNA microarray comprising 30,968 human genome probes was used to evaluate the transcriptional changes that occur in peripheral blood mononuclear cells (PBMC) at the onset of clinical AOM caused by NTHi infection in children after comparison of microarray results with the pre-infection healthy stage of the same children. Four to ten milliliters of heparinized peripheral venous blood was collected from children at 6 to 30 months of age when they were in acute otitis media (AOM) stage and pre-infection healthy stage. The diagnosis of AOM was based on symptoms and signs as well as Nontypeable Haemophilus influenzae culture positive in the middle ear fluid. Patients with polymicrobial infections, history of immunodeficiency, history of chronic or recurrent AOM, chronic disease, or receiving steroids or other immunomodulatory agents were excluded. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll gradient and total RNA was extracted from PBMCs using the QIAamp RNA blood Mini Kit (Qiagen, Maryland, USA) according to manufacturerM-bM-^@M-^Ys instructions. Double-stranded cDNA generated from total RNA was labeled with Cyanine-5 and subsequently hybridized to Human OneArray glass slides according to the manufacturer's standard protocols (PhalanxBio Inc, CA, USA). Microarrays were scanned at 5 M-NM-<m resolution using an Agilent scanner. Raw intensity signals for each microarray were captured using a Molecular Dynamics Axon 4100A scanner, measured using GenePixProM-bM-^DM-" Software. The data from all microarrays in each experimental set was then analyzed using Omicsoft Array Studio software; control and missing features were removed, and the remaining signals were quantile normalized.
Project description:ASXL1 is the obligate regulatory subunit of a deubiquitinase complex whose catalytic subunit is BAP1. Heterozygous mutations of ASXL1 that result in premature truncations are frequent in myeloid leukemias and Bohring-Opitz syndrome. Here, we demonstrate that truncated ASXL1 proteins confer enhanced activity on the ASXL1-BAP1 complex. Stable expression of truncated, hyperactive ASXL1-BAP1 complexes in a hematopoietic precursor cell line resulted in global erasure of H2AK119Ub, striking depletion of H3K27me3, selective upregulation of a subset of genes whose promoters bore both H2AK119Ub and H3K4me3, and spontaneous differentiation to the mast cell lineage. These outcomes required the catalytic activity of BAP1, indicating these events were downstream consequences of H2AK119Ub erasure. In bone marrow precursors, truncated ASXL1-BAP1 expression cooperated with TET2 loss-of-function to increase differentiation to the myeloid lineage in vivo. We propose that pathological ASXL1 mutations confer gain-of-function on the ASXL-BAP1 complex. ChIP-Seq for H2AK119Ub, H3K4me3, H3K27me3 on EML cells. RNA-Seq on EML cells expressing ASXL1(1-479)+BAP1 and control.
Project description:The tumor suppressor and deubiquitinase (DUB) BAP1 regulates chromatin-associated processes and is frequently mutated in various malignancies. BAP1 and its drosophila orthologue Calypso assemble DUB complexes with ASXL-1, -2, -3 paralogues and ASX respectively, and these cofactors are required for stimulating their DUB activity. However how the DUB activity of BAP1 is regulated remains largely unknown. Here we show that BAP1 promotes monoubiquitination of ASXLs on the ASXM/DEUBAD domain. ASXL2 monoubiquitination promotes its stability or proteasomal degradation, stimulates BAP1 DUB activity and is required for mammalian cell proliferation. Monoubiquitination of ASXL2 is directly catalyzed by UBE2E family of ubiquitin conjugating enzymes and is regulated by deubiquitination. Monoubiquitination of ASX is regulated by Calypso and is required for drosophila development. We further revealed a switch mechanism that tightly regulate BAP1 function, as a monoubiquitination of BAP1 UCH domain is mutually exclusive with ASXL2 monoubiquitination, thus ensuring highly coordinated DUB-mediated signaling.