Next Generation Sequencing confirmed exon alternative splicing and lineage switching in one patient
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA589082 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10435249_1.fastq.gz | Fastqsanger.gz | |||
| SRR10435249_2.fastq.gz | Fastqsanger.gz | |||
| SRR10435250_1.fastq.gz | Fastqsanger.gz | |||
| SRR10435250_2.fastq.gz | Fastqsanger.gz |
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Project description:Purpose: To analyze the expression of two different CD19 isoforms during BiTE treatment, and to further determine cell lineage specific molecules before and after BiTE treatment Methods: For transcriptome sequencing, we isolated patient's bone marrow samples, quality of total RNA was evaluated by Agilent 2100. RNA libraries were prepared for sequencing using NEBNext® UltraTM RNA Library Prep Kit for Illumina (NEB,USA). RNA sequencing process and the data acquisition were finished on Novogene Experimental Department according to the manufacturer’s protocol. Reads were aligned to hg38 reference genome. Relative abundance of CD19 and other gene transcripts were mapped and estimated at both gene and transcript level (FPKM) by feature Counts. Results: The expression of CD19 isoform with exon 2 deletion was found at diagnosis before BiTE therapy. The patient did not achieve remission after BiTE treatment, and the expression of CD19 antigen turned negative by flow cytometry detection. But the expression ratio of exon 2 deleted CD19 was not increased, and the flow cytometry phenotype and transcriptome sequencing confirmed that no linage switching occurred, which suggested the expression of CD19 isoform caused by exon alternative splicing and lineage switching was not the driving mechanism of CD19 epitope loss in this patient. Immune escape could not be prevented by targeting alternative exons. This patient achieved complete remission by sequential infusions of our own developed CD22 CAR-T and CD19 CAR-T after disease progression Conclusions: transcriptome sequencing confirmed that no linage switching occurred in this patient, which suggested the expression of CD19 isoform caused by exon alternative splicing and lineage switching was not the driving mechanism of CD19 epitope loss in this patient.
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Project description:'Non-coding transcripts originating upstream of the immunoglobulin constant region (I-transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of I and I1 transcription in response to interleukin-4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. Here we combine novel mouse reporters and single-cell RNA-seq to reveal the heterogeneity in IL-4 induced I-transcription. Transgenic mice were generated with one allele labelled with an eGFP fluorescent reporter gene in the first exon of the gamma-1 non-coding RNA and one allele with a tdTom fluorescent reporter gene in the first exon of the epsilon non-coding RNA. Ex vivo splenic B cells from these mice were stimulated with IL-4, lipopolysaccharide and B cell activating factor. After 24 hours culture, cell were sorted according to their fluorescence and used for single-cell mRNA-sequencing. Two independent experiments were performed (labelled as ''batch 1'' and ''batch 2'').'
2016-12-08 | E-MTAB-4825 | biostudies-arrayexpress
Project description:Although splicing occurs in most multi-exon genes, the generation of distinct isoforms through the alternate use of mutually exclusive exons is less prevalent. As exon-switching events have the potential to give rise to isoforms with different cellular functions, we have explored the role of the muscle-specific (Mef2Da2) and ubiquitously expressed (Mef2Da1) isoforms of the transcription factor Mef2D in myogenesis. Here we show that both isoforms of Mef2D bind a largely overlapping subset of genomic loci, yet only the muscle-specific Mef2Da2 isoform can activate the late myogenic gene expression program. This differential ability to activate transcription is modulated by PKA signaling where Mef2Da1 is efficiently phosphorylated by the kinase to enhance its association with repressive HDAC-deacetylase complexes. In contrast, alternate exon usage in Mef2Da2 renders the protein resistant to PKA phosphorylation, allowing it to interact with transcriptionally permissive Ash2L-trithorax complex. Our findings support a model wherein alternative exon usage allows Mef2D to transition from a repressor to activator in a myogenic environment rich in PKA activity. Thus we have identified a novel paradigm in which a ubiquitously expressed transcription factor has evolved to undergo tissue-specific alternative exon usage to permit the proper temporal activation of a gene expression program during differentiation. ChIP-Seq profiling of Mef2Da1 and Mef2Da2 isoforms generated by alternate splicing
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Project description:Although splicing occurs in most multi-exon genes, the generation of distinct isoforms through the alternate use of mutually exclusive exons is less prevalent. As exon-switching events have the potential to give rise to isoforms with different cellular functions, we have explored the role of the muscle-specific (Mef2Da2) and ubiquitously expressed (Mef2Da1) isoforms of the transcription factor Mef2D in myogenesis. Here we show that both isoforms of Mef2D bind a largely overlapping subset of genomic loci, yet only the muscle-specific Mef2Da2 isoform can activate the late myogenic gene expression program. This differential ability to activate transcription is modulated by PKA signaling where Mef2Da1 is efficiently phosphorylated by the kinase to enhance its association with repressive HDAC-deacetylase complexes. In contrast, alternate exon usage in Mef2Da2 renders the protein resistant to PKA phosphorylation, allowing it to interact with transcriptionally permissive Ash2L-trithorax complex. Our findings support a model wherein alternative exon usage allows Mef2D to transition from a repressor to activator in a myogenic environment rich in PKA activity. Thus we have identified a novel paradigm in which a ubiquitously expressed transcription factor has evolved to undergo tissue-specific alternative exon usage to permit the proper temporal activation of a gene expression program during differentiation.
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Project description:We describe a method, MADS (Microarray Analysis of Differential Splicing), for discovery of differential alternative splicing from exon tiling microarrays. MADS incorporates a series of low-level analysis algorithms motivated by the “probe-rich” design of exon arrays, including background correction, iterative probe selection, and removal of sequence-specific cross-hybridization to off-target transcripts. We used MADS to analyze Affymetrix Exon 1.0 array data on a mouse neuroblastoma cell line after shRNA-mediated knockdown of the splicing factor PTB. From a list of exons with pre-determined inclusion/exclusion profiles in response to PTB depletion, MADS recognized all exons known to have large changes in transcript inclusion levels, and offered improvement over Affymetrix’s analysis procedure. We also identified numerous novel PTB-dependent splicing events. 30 novel events were tested by RT-PCR, and 27 were confirmed. This work demonstrates that the exon tiling microarray design is an efficient and powerful approach for global, unbiased analysis of pre-mRNA splicing. Keywords: control / knockdown comparison
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