Expression and FoxA1 binding in asynchronous and mitotic HUH7 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Gregory Donahue
PROVIDER: E-GEOD-39243 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:During mitosis, RNA polymerase and most transcription factors are excluded from the chromosomes and transcription ceases. The transcriptional re-activation of the genome, following mitosis, requires the re-setting of cell-type specific programs that were initially established during development. However, only about one-fifth of transcription factors are retained on chromosomes throughout mitosis and a subset of these have been shown to facilitate target gene reactivation during mitotic exit. How such “bookmarking” factors bind to chromatin in mitosis and re-activate transcription is central to the stability of transcriptional programs across multiple cell cycles. We compared a diverse set of transcription factors involved in liver differentiation and found different modes of mitotic chromosome binding. The pioneer transcription factor FoxA1, which is among the first to bind liver genes in development, exhibits virtually complete mitotic chromosome binding, whereas other liver factors bind with a range of efficiencies. Yet genome-wide analysis shows that only about 15% of the FoxA1 interphase target sites are bound in mitosis; the latter include sites at genes for maintaining cell differentiation. FoxA1 mutants that perturb specific and nonspecific DNA binding reveal a significant contribution of nonspecific binding events in mitotic chromatin. Such nonspecific binding appears to spread from interphase FoxA1 targets and may serve as storage sites. The hierarchy of specific binding, nonspecific binding, partial chromatin binding, and failure to bind mitotic chromosomes reflects the temporal sequence of the factors’ developmental roles in gene activation. Gene expression was assessed in asynchronously cycling cells in order to determine whether FoxA1 is disposed to bind to high- or low-expressed genes during mitosis. Heatmap analysis shows that FoxA1's mitotic targets are among the highest-expressed genes in asynchronous cells. Total RNA was collected from three different plates with asynchronous HUH7 cells using the RNeasy mini kit (Qiagen Valencia CA). Expression microarrays were performed with a Human Gene 1.OST array (Affymetrix) at the UPenn Microarray Core Facility and assessed using Partek.
2013-03-31 | E-GEOD-39208 | biostudies-arrayexpress
Project description:During mitosis, RNA polymerase and most transcription factors are excluded from the chromosomes and transcription ceases. The transcriptional re-activation of the genome, following mitosis, requires the re-setting of cell-type specific programs that were initially established during development. However, only about one-fifth of transcription factors are retained on chromosomes throughout mitosis and a subset of these have been shown to facilitate target gene reactivation during mitotic exit. How such M-bM-^@M-^\bookmarkingM-bM-^@M-^] factors bind to chromatin in mitosis and re-activate transcription is central to the stability of transcriptional programs across multiple cell cycles. We compared a diverse set of transcription factors involved in liver differentiation and found different modes of mitotic chromosome binding. The pioneer transcription factor FoxA1, which is among the first to bind liver genes in development, exhibits virtually complete mitotic chromosome binding, whereas other liver factors bind with a range of efficiencies. Yet genome-wide analysis shows that only about 15% of the FoxA1 interphase target sites are bound in mitosis; the latter include sites at genes for maintaining cell differentiation. FoxA1 mutants that perturb specific and nonspecific DNA binding reveal a significant contribution of nonspecific binding events in mitotic chromatin. Such nonspecific binding appears to spread from interphase FoxA1 targets and may serve as storage sites. The hierarchy of specific binding, nonspecific binding, partial chromatin binding, and failure to bind mitotic chromosomes reflects the temporal sequence of the factorsM-bM-^@M-^Y developmental roles in gene activation. Three replicate chIP-seq data sets each are included for mitotic and asynchronously cycling cells; a single input lane from each condition is also included.
2013-03-31 | E-GEOD-39241 | biostudies-arrayexpress
Project description:To examine the effects of SRp20 on genome-wide RNA splicing in tumor cells, we utilized Exonhit SpliceArray to compare genome-wide changes of RNA splicing and transcription in U2OS cells with or without knockdown of SRp20 by RNAi. U2OS cells were treated by SRp20 siRNA or non-specific siRNA, each performed in triplicate. A human genome-wide splice-array assay was performed by using a service provider ExonHit Therapeutics, Inc (Gaithersburg, MD). Fifty ng of total RNA isolated from U2OS cells with or without knockdown of SRp20 was quality controlled and analyzed by using Affymetrix GeneChip platform to profile the expression of 20,649 genes and splicing events of 19,066 genes.
2012-12-30 | E-GEOD-22149 | biostudies-arrayexpress
Project description:To identify low abundance autocrine growth factors in CHO cell conditioned media, we utilized a label-free shotgun proteomics approach. CHO cell conditioned media were harvested from fed batch bioreactors and concentrated using methanol/chlorofrom precipitation. Proteins in the samples were then subjected to proteolysis with trypsin, and then subjected to primary fractionation using a SCX column, followed by RP liquid chromatography MS (LC-MS) with a LTQ Orbitrap Velos instrument using the data dependent acquisition (DDA) method. The MS system was set up and run with a method which enabled fast acquisitions of high quality peptide precursor and fragment ion data, with the desired precursor mass accuracy of ±5 ppm. For the LTQ Orbitrap Velos MS, the data-dependent MS/MS analytical workflow in positive ion mode was used. Each precursor survey scan (m/z: 300 to 1800) by the Orbitrap mass analyzer (resolution = 60,000 FWHM) was linked to 10 MS/MS events using the 2D ion trap CID approach, with dynamic ion exclusion set at 60 s. This value was determined based on the observed mean peptide chromatographic peak width. All other instrument parameters were set up according to the manufacturer’s suggested values for complex peptide samples. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. Raw data files from the LTQ Orbitrap Velos MS were processed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific). The LC-MS data were searched against the human (Homo sapiens; UniProtKb, updated in August 2012, 45 848 entries), mouse (Mus musculus; UniProtKb, updated in August 2012, 31 528 entries) and chinese hamster (Cricetulus griceus; UniprotKb, updated in August 2012, 24 609 entries) protein databases using the Sequest search engine for the LTQ Orbitrap Velos LC-MS data, assuming tryptic digestion with precursor ions to fall within 10 ppm of projected m/z values and a fragment ion mass tolerance of 0.5 m/z. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and a maximum of two missed cleavage events. Reverse database searching resulted in a specific false discovery rate (FDR) of 1% at the peptide and protein level.
2020-01-22 | PXD000214 | Pride
Project description:Lysosome-targeting chimeras (LYTACs) have emerged as a revolutionary targeted protein degradation (TPD) technology in modulating the levels of extracellular and membrane proteins. However, lack of lysosome-trafficking receptors (LTRs) limits the development of LYTACs. Here, we firstly confirm that folate receptor α (FRα) is a new generation of lysosome-targeting receptor (LTR) of LYTAC, facilitating the transport of membrane proteins to lysosomes to realize degradation. Moreover, novel FRTACs are constructed by a new “polyvalent crosslinking strategy”, instead of the traditional “one folate conjugating one drug strategy”. Polyvalency creates avidity, allowing FRTACs crosslinking FRα to dramatically improve the degradation efficiency. As a result, the optimized FRTACs, including EGFR-targeting FR-Ctx, PD-L1-targeting FR-Atz, TROP2-targeting FR-Stz, and HER2-targeting FR-Ttz, successfully eliminate cell surface targets with a subnanomole activity. Mechanism investigation reveals that FRTACs trigger targets degradation in a FRα- and lysosomal-dependent manner. Besides, FR-Ctx reduces cancer cell proliferation, and FR-Atz increases the cytotoxicity of T cells toward tumor cells. Furthermore, FR-Atz exhibits potent degradation efficiency of PD-L1 in vivo and elicits tumor-specific immune responses by switching the tumor immune microenvironment from a suppressed state to an activated state in both RM-1 mice model and humanized PD-1/PD-L1 B16F10 mice model. To our knowledge, FRTACs are the most potent protein degrader ever reported. The novel FRTACs will expand the application of FRα and provide a new platform for designing tumor-targeting LYTACs.
2025-05-30 | PXD058539 | Pride
Project description:Lysosome-targeting chimeras (LYTACs) have emerged as a revolutionary targeted protein degradation (TPD) technology in modulating the levels of extracellular and membrane proteins. However, lack of lysosome-trafficking receptors (LTRs) limits the development of LYTACs. Here, we firstly confirm that folate receptor α (FRα) is a new generation of lysosome-targeting receptor (LTR) of LYTAC, facilitating the transport of membrane proteins to lysosomes to realize degradation. Moreover, novel FRTACs are constructed by a new “polyvalent crosslinking strategy”, instead of the traditional “one folate conjugating one drug strategy”. Polyvalency creates avidity, allowing FRTACs crosslinking FRα to dramatically improve the degradation efficiency. As a result, the optimized FRTACs, including EGFR-targeting FR-Ctx, PD-L1-targeting FR-Atz, TROP2-targeting FR-Stz, and HER2-targeting FR-Ttz, successfully eliminate cell surface targets with a subnanomole activity. Mechanism investigation reveals that FRTACs trigger targets degradation in a FRα- and lysosomal-dependent manner. Besides, FR-Ctx reduces cancer cell proliferation, and FR-Atz increases the cytotoxicity of T cells toward tumor cells. Furthermore, FR-Atz exhibits potent degradation efficiency of PD-L1 in vivo and elicits tumor-specific immune responses by switching the tumor immune microenvironment from a suppressed state to an activated state in both RM-1 mice model and humanized PD-1/PD-L1 B16F10 mice model. To our knowledge, FRTACs are the most potent protein degrader ever reported. The novel FRTACs will expand the application of FRα and provide a new platform for designing tumor-targeting LYTACs.
2025-05-30 | PXD058523 | Pride
Project description:Lysosome-targeting chimeras (LYTACs) have emerged as a revolutionary targeted protein degradation (TPD) technology in modulating the levels of extracellular and membrane proteins. However, lack of lysosome-trafficking receptors (LTRs) limits the development of LYTACs. Here, we firstly confirm that folate receptor α (FRα) is a new generation of lysosome-targeting receptor (LTR) of LYTAC, facilitating the transport of membrane proteins to lysosomes to realize degradation. Moreover, novel FRTACs are constructed by a new “polyvalent crosslinking strategy”, instead of the traditional “one folate conjugating one drug strategy”. Polyvalency creates avidity, allowing FRTACs crosslinking FRα to dramatically improve the degradation efficiency. As a result, the optimized FRTACs, including EGFR-targeting FR-Ctx, PD-L1-targeting FR-Atz, TROP2-targeting FR-Stz, and HER2-targeting FR-Ttz, successfully eliminate cell surface targets with a subnanomole activity. Mechanism investigation reveals that FRTACs trigger targets degradation in a FRα- and lysosomal-dependent manner. Besides, FR-Ctx reduces cancer cell proliferation, and FR-Atz increases the cytotoxicity of T cells toward tumor cells. Furthermore, FR-Atz exhibits potent degradation efficiency of PD-L1 in vivo and elicits tumor-specific immune responses by switching the tumor immune microenvironment from a suppressed state to an activated state in both RM-1 mice model and humanized PD-1/PD-L1 B16F10 mice model. To our knowledge, FRTACs are the most potent protein degrader ever reported. The novel FRTACs will expand the application of FRα and provide a new platform for designing tumor-targeting LYTACs.
2025-05-30 | PXD058514 | Pride
Project description:Five NPC cell lines (HONE1, CNE1, CNE2, 5-8F, and 6-10B) and an immortalized nasopharyngeal epithelial cell line NP69 were evaluated with 250K SNP arrays (Affymetrix) to detect the genome-wide DNA copy number alterations. The same type of data of 45 Chinese samples from the HapMap project was used as normal control. Data was analyzed using GCOS (GeneChip operating software, Ver.1.4), GTYPE (GeneChip Genotyping Analysis Software, Ver.4.1), and CNAT (chromosome copy number analysis tool, Ver.4.0, BRLMM algorithm).
2009-03-04 | E-GEOD-15051 | biostudies-arrayexpress
Project description:Analysis of gene expression changes associated with lentiviral vector mediated expression of pre-microRNA-150 expression in the AML cell lines PL21 and HL60 cells. To identify genes, direct putative miRNA-150 targets, biological and molecular functions and pathways that distinguish cells that express miR-150 from those that do not. Total RNA isolated from HL60 or PL21 cells transduced with pre-microRNA-150 or empty vector control and assayed by gene expresssion analysis 20 days post transduction. Two replicates were performed for each condition (miR-150 and control) in each of two cell lines (HL60 and PL21).
2013-01-15 | E-GEOD-40147 | biostudies-arrayexpress
Project description:The phenotypically characterized hTERT immortalized porcine olfactory bulb neuroblast cell line (OBGF400) was subjected to an extensive whole genome-scaled expression profile for establishing their use as an in vitro neuronal disease model system. Microarrays were used to provide a comprehensive knowledge underlying the genomic complexity and overall gene expression capacity of the immortalized OBGF400 cells. The analysis revealed the elaborate signaling mechanisms of this unique subpopulation of porcine neuronally committed progenitors that mirrors the intricate organization of postnatal neurongenic zones. SUBMITTER_CITATION: Transcriptome Profile and Cytogenetic Analysis of Immortalized Neuronally Restricted Progenitor Cells Derived from the Porcine Olfactory Bulb. Animal biotechnology 2009 vol:20 iss:4 page:186-215 Experiment Overall Design: Total cellular RNA extracts from independent OBGF400 (neuroblasts) and PK15 (non-neuronal, epithelial origin) cell cultures (9 each) were pooled into a total of 3 biological replicates per cell line. The concentrations and purity of the pooled RNA preparations were determined using the BioAgilent RNA assay prior to hybridization on Affymetrix GeneChip® Porcine Genome Expression Arrays. To ascertain the genes that were preferentially expressed by the OBGF400 neuroblasts, we used the PK15 cellular array in an effort to exclude somatic cell background.
2009-11-10 | E-GEOD-16855 | biostudies-arrayexpress