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Investigation of mRNA expression pattern by over-expression of two CEBPB transcription factor isoforms

ABSTRACT: Investigation of mRNA expression pattern by over-expression of two CEBPB transcription factor isoforms

PROVIDER: PRJNA433422 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6685140.fastq.gz	Fastqsanger.gz
	SRR6685141.fastq.gz	Fastqsanger.gz
	SRR6685142.fastq.gz	Fastqsanger.gz
	SRR6685143.fastq.gz	Fastqsanger.gz
	SRR6685144.fastq.gz	Fastqsanger.gz

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Project description:The CCAAT/enhancer-binding proteins (CEBPs) are transcription factors involved in hematopoietic cell development and induction of several inflammatory mediators. Here, we generated CEBP-beta (CEBPB) and CEBP-epsilon (CEBPE) double-knockout (bbee) mice and compared their phenotypes to those of single-deficient (bbEE and BBee) and wild-type (BBEE) mice. The bbee mice were highly susceptible to fatal infections and died within 2-3 months. Morphologically, their neutrophils were blocked at the myelocytes/metamyelocytes stage, and clonogenic assays of bone marrow cells indicated a significant decrease in the number of myeloid colonies of the bbee mice. In addition, the proportion of hematopoietic progenitor cells [Lin(-)Sca1(+)c-Kit(+)] in the bone marrow of the bbee mice was significantly increased, reflecting the defective differentiation of the myeloid compartment. Furthermore, microarray expression analysis of lipopolysaccharide (LPS)- and interferon-gamma (IFN-gamma)-activated bone marrow-derived macrophages from bbee compared to single knockout mice revealed decreased expression of essential immune response-related genes and networks, including some direct CEBP targets such as Marco and Clec4e. Overall, the phenotype of the bbee mice is distinct from either the bbEE or BBee mice, demonstrating that both transcription factors are crucial for the maturation of neutrophils and macrophages, as well as the innate immune system, and can at least in part compensate for each other in the single knockout mice. To rule out the regulatory influence of both CEBPB and CEBPE on macrophage-related genes, expression analysis of bone marrow-derived macrophages was performed. Macrophages were derived from murine bone marrow with the use of murine M-CSF. The macrophages were stimulated with both LPS (100 ng) and IFN-gamma (100 ng) for 24h, and RNA was extracted for array analysis. Overall, RNA was extracted from stimulated macrophages of one WT mouse, one CEBPB-KO mouse, one CEBPE-KO mouse and one double-KO mouse.

Project description:Gene isoforms are mRNAs produced from the same locus, but that differ in their transcription start sites, protein coding DNA sequences, and/or untranslated regions. Consequently, different isoforms of the same gene can have altered gene function or even serve different biological functions. Conventional RNA-Seq strategies cannot accurately distinguish expression levels between distinct isoforms, and differences in gene expression studies almost always report total gene expression. However, increasing evidence indicates that differences in isoform usage may be important for the regulation of biological functions and for development of diseases such as cancer. Here, we aimed to define whether gene isoforms are subjected to differential regulation and expression by characterizing 24-hour rhythms in polyadenylation site (PAS) usage over the course of the day in the mouse liver. Conventional RNA-Seq experiments have shown that 15-30% of genes in the mouse liver are rhythmically expressed, and it is assumed that the comprising isoforms are expressed in a similar pattern. By performing 3‟-end mRNA-Seq and using stringent criteria for defining differential rhythmic expression, we show that 15% of the genes with more than one PAS exhibit differential rhythmic expression. In particular, many genes known to be rhythmic in the mouse liver harbor at least one constitutively expressed isoform, while several hundred genes characterized as arrhythmically expressed also exhibit a rhythmic isoform. Analysis of PAS usage in nuclear mRNA and single-cell data reveals that the vast majority of isoform-specific regulation does not involve post-transcriptional regulation (e.g., miRNA targeting, RNA half-life) or cell subtype-specific differences in expression. Finally, characterization of PAS usage in Bmal1-/- mice revealed that co-transcriptional regulation plays a large role in the expression of specific gene isoforms. Taken together, our results indicate that gene isoforms can be differentially regulated, and imply that gene isoforms can behave as distinct transcriptional units. Furthermore, our data suggest that conventional RNA-Seq strategies are not the most appropriate choice for detecting changes in gene isoform expression.

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Investigation of mRNA expression pattern by over-expression of two CEBPB transcription factor isoforms

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