Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage

Abstract

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Figure 1

(A) Comparison of BMM expression profiles during a LPS time-series with the READ mouse tissues expression profiles database on the RIKEN 19,000 element cDNA arrays. The expression profiles of 49-mouse tissue from different developmental stages are compared in hierarchical matter with the expression profiles of BMM from three different mouse strains (BALB/c, C3H/ARC, and C2H/HeJ). The picture shows the experiment trees generated using the unsupervised hierarchical clustering algorithm from GeneSpring4.2 (SiliconGenetics Inc.). (B) Annotation of the 347 macrophage-enriched genes. The ontology pie was based on the RIKEN clone annotation, curated by the Functional Annotation of Mouse Genome consortium (FANTOM). Further information regarding these genes can be found in Supplementary Table 1.

Figure 1

(A) Comparison of BMM expression profiles during a LPS time-series with the READ mouse tissues expression profiles database on the RIKEN 19,000 element cDNA arrays. The expression profiles of 49-mouse tissue from different developmental stages are compared in hierarchical matter with the expression profiles of BMM from three different mouse strains (BALB/c, C3H/ARC, and C2H/HeJ). The picture shows the experiment trees generated using the unsupervised hierarchical clustering algorithm from GeneSpring4.2 (SiliconGenetics Inc.). (B) Annotation of the 347 macrophage-enriched genes. The ontology pie was based on the RIKEN clone annotation, curated by the Functional Annotation of Mouse Genome consortium (FANTOM). Further information regarding these genes can be found in Supplementary Table 1.

Figure 2

(A) Identification of 373 LPS-responsive transcripts conserved between the mouse strains, or known to be involved in TLR4-mediated LPS signaling; 347 transcripts were identified as enriched in BMM compared to nonmacrophage tissues in the READ expression database. Thirteen of these genes (4%) were also induced by LPS across the time course in the LPS-responsive mouse strains C3H/ARC and BALB/c. (B) Annotation of the 373 LPS responsive transcripts. Transcripts were binned into functions based on the Gene Ontology terms ascribed to each clone during the FANTOM2 process.

Figure 2

(A) Identification of 373 LPS-responsive transcripts conserved between the mouse strains, or known to be involved in TLR4-mediated LPS signaling; 347 transcripts were identified as enriched in BMM compared to nonmacrophage tissues in the READ expression database. Thirteen of these genes (4%) were also induced by LPS across the time course in the LPS-responsive mouse strains C3H/ARC and BALB/c. (B) Annotation of the 373 LPS responsive transcripts. Transcripts were binned into functions based on the Gene Ontology terms ascribed to each clone during the FANTOM2 process.

Figure 3

Functional analysis of known transcripts in the immunome libraries; 4284 known gene clusters were represented and analyzed using FANTOM2 GO terms extracted from the TC descriptor. Enzymes were the largest category identified, with 1018 unique members. The cytoskeleton category contained 916 members and included membrane bound proteins (excluding receptors), the actin cytoskeleton, and trafficking molecules. Three hundred ninety transcriptional regulators were identified. Three hundred forty-nine TC annotated as immunity/antigen presentation were specifically involved in MHC processing, antigen presentation, chemokine, leukotriene and cytokine production, components of the complement cascade, and interferon signaling. One hundred seventy-two cell cycle, growth and differentiation TC included a number of macrophage-specific, neutrophil-specific, and T-cell-specific differentiation markers. G-proteins, kinases, and the rab/rac signaling pathways were particularly represented in the 108 signaling TCs. The remaining categories included protein synthesis and modification (433 TC), 243 receptors, oxidative metabolism/stress (139 TC), apoptosis (68 TC), and extracellular matrix (52TC).