Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system

Abstract

Background: In this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells.

Results: Gene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5-6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes.

Conclusion: This study demonstrates a series of analytic approaches that have allowed the implication of genes and regulatory elements that participate in the differentiation, maintenance, and function of the immune system. Polymorphism or mutation of these could adversely impact immune system functions.

Figure 1

Expression profiles of sequences across tissues: Hierarchical tree clustering of genes and tissues was carried out using Pearson correlation and the log of the average of the relative expression ratio for each gene, as measured in replicate arrays. Sequences with similar expression patterns across all tissues are clustered together in the resulting trees, the closeness of the sequences in sub trees is a measure of how closely correlated their expression is. (A) Hierarchical tree clustering of genes across 65 normal adult and fetal tissues. 680 sequences were identified that were highly expressed in thymus, unstimulated and stimulated lymph nodes, spleen, peripheral blood mononuclear cells, and in vitro activated T-cells. To increase specificity, 320 sequences were removed because they were also highly expressed in one or more non-lymphoid tissues, as described in the text. The pattern of expression of the remaining 360 "immune genes" across tissues is shown. (B) Hierarchical tree clustering of 360 immune genes across 18 normal adult and fetal tissues. There are 3 major groups of tissues that show clusters of highly expressed "immune genes" These include the 6 immune tissues, various segments of adult intestine, and fetal day 16.5 lung and intestine. Less prominent clusters are seen in adult lung and liver. Genes in these clusters are described in the text. While the band of high expression extends across all genes for the 6 immune tissues, relative expression of each gene within the immune tissues shows distinct patterning.

Figure 2

Computationally predicted clusters of cis-elements in the promoter region of mouse H2-K and its human ortholog HLA-A: The ATG of human HLA-A is at position 10,001 while that of mouse H2-K is at 10,463. Thus, the region represented relative to ATG is -305 to +97 (human) and -653 to -293 (mouse). Additionally, these regions correspond to chr 6: 30,015,866–30,016,268 (+) of the Human Genome July 2003 Assembly and chr17: 33,638,839–33,639,199 (-) of the Mouse October 2003 Assembly . Families of transcription factor binding sites and the relative positions of the sites in the nucleotide sequences of the two genes are represented as different colored bars stretching across the ortholog gene pair.

Figure 3

Example of a CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules. The genes are those with high expression in thymus. The algorithm used by TraFac and CisMols to display regulatory modules uses a moving 200 bp window to scan regions of DNA for specific sequences characteristic of TF binding sites (cis-elements). Clusters of these cis-elements are not generally distributed evenly across a segment of DNA, but are highly localized to specific segments which are likely to play a role in regulation of gene expression. Because the scanning window is limited to 200 bp and the scan changes the frame of sequences within the window, a regulatory module that contains multiple cis-elements may not be displayed as one list of multiple elements, but rather as a list of several modules of different composition and arrangement within one small segment of DNA. Each colored cube indicates a cluster of 3 or more cis-elements with at least one "lymphoid element". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI's RefSeq database). The legend in the lower left half indicates the composition of each of the modules and the genes that share them. In the lower right hand panel is the Trafac image of one of the cis-element dense region with multiple shared modules of Arid1a gene.

Figure 4

Clusters of TF binding sites immediately upstream of the transcription start site in 3 genes of cluster set 15 (co-expressed genes with high expression in thymus): The upper panels compare the location of TF binding sites surrounding and upstream regions of transcription start site (based on the corresponding mRNA annotations from NCBI's RefSeq database) of human and mouse Arid1a, Abcg1 and Zfp162 genes in GenomeTrafac database . The bottom panels list the gene descriptions and each of the binding sites in their order of occurrence from distal (top) to proximal (bottom) to exon 1 of the human gene, which is on the left within each panel. Binding sites in bold are known "lymphoid elements". The first nucleotide of exon 1 is at bp 40,001. Each of the colored bars represents a class of TF binding sites and connects homologous binding sites in genes of the two species. The orthologous genes may differ in the number and location of specific TF's binding sites. The corresponding coordinates of the regions on human (NCBI Build 35, May 2004) genome assembly are: chr1: 26,706,590–26,706,986 (+), chr21: 42,512,113 42,512,317 (+) and chr11: 64,302,720–64,302,924 (-) for human ARID1A, ABCG1 and SF1 respectively. Coordinates in the mouse genome assemblies (Build 33 Mouse Assembly, May 2004) are: chr4: 132,206,952–132,207,348 (-), chr19: 6,151,958–6,152,162 (+) and chr17: 29,663,342–29,663,546 (+) for Arid1a, Abcg1 and Zfp162 respectively.