Human TRIM gene expression in response to interferons

Abstract

Background: Tripartite motif (TRIM) proteins constitute a family of proteins that share a conserved tripartite architecture. The recent discovery of the anti-HIV activity of TRIM5alpha in primate cells has stimulated much interest in the potential role of TRIM proteins in antiviral activities and innate immunity.

Principal findings: To test if TRIM genes are up-regulated during antiviral immune responses, we performed a systematic analysis of TRIM gene expression in human primary lymphocytes and monocyte-derived macrophages in response to interferons (IFNs, type I and II) or following FcgammaR-mediated activation of macrophages. We found that 27 of the 72 human TRIM genes are sensitive to IFN. Our analysis identifies 9 additional TRIM genes that are up-regulated by IFNs, among which only 3 have previously been found to display an antiviral activity. Also, we found 2 TRIM proteins, TRIM9 and 54, to be specifically up-regulated in FcgammaR-activated macrophages.

Conclusions: Our results present the first comprehensive TRIM gene expression analysis in primary human immune cells, and suggest the involvement of additional TRIM proteins in regulating host antiviral activities.

Figure 1. Human TRIM proteins.

Classification of human TRIM proteins based on the nature of their C-terminal domains(s) as defined by Short and Cox and modified by Ozato et al. . The TRIM protein family is composed of 11 sub-families, from C-I to C-XI, whereas some TRIM proteins remain unclassified (UC), since they do not have a RING finger domain as “true” TRIM proteins. NHL, NHL repeats; COS, COS box motif; FN3, fibronectin type III motif; PHD, plant homeodomain; BROMO, bromodomain; MATH, meprin and TRAF homology domain; TM, transmembrane domain; AR, acid-rich region.

Figure 2. TRIM gene expression in human primary macrophages and lymphocytes.

cDNA was prepared from primary macrophages (MDM) and lymphocytes (PBL) from 3 donors, as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by quantitative RT-PCR array. A. Comparison of the expression of 5 housekeeping genes in untreated MDM and PBL. The mean Ct values for each gene in untreated cells from 3 donors are shown. Error bars show standard deviation. RPL13A presented the smallest standard deviation values and was therefore selected for normalization. B. Constitutive expression of TRIM genes in MDM (M) and PBL (P). Histograms represent mean 2^−ΔCt values for each gene±SD. C. Relative expression of TRIM genes in MDM (M) and PBL (P). Mean 2^−ΔCt values were determined by subtracting RPL13A, and each sample was normalized to the median expression of each gene in both cell types. Resulting 2^−ΔΔCt values were represented as a heat map, using Java TreeView. Green: low relative expression; Yellow: median value (same expression in MDM and PBL); Red: high relative expression.

Figure 3. TRIM gene expression in response to IFN or immune complex.

Primary macrophages (MDM) or lymphocytes (PBL) from 3 donors were left untreated or were treated with type I IFN, type II IFN or preformed immune complexes (IC, in the case of MDM only), as described in the Materials and Methods section. The expression of 86 genes, including 72 TRIM genes and 5 housekeeping genes, was analyzed by PCR array. A. Selection of a housekeeping gene to normalize the expression of TRIM genes in untreated Vs IFN- or IC-treated cells. The diagram shows the mean expression of 5 housekeeping genes in untreated and treated MDM (squares) or PBL (triangles). The mean Ct values for each gene in untreated cells and IFN or IC-treated cells from 3 donors are shown. Error bars show standard deviation. ACTB presented the smallest standard deviation values and was therefore selected for normalization. B. Induction of TRIM genes in primary cells treated with type I IFN (I), type II IFN (II) or IC. Mean 2^−Ct values for each gene in cells from 3 donors were normalized to ACTB expression to calculate 2^−ΔCt values. Normalization to the mean expression of each gene in untreated cells gave the 2^−ΔΔCt values, which were presented as a heat map using Java TreeView. Green: Down-regulation of gene expression; Yellow: No change; Red: Up-regulation of gene expression. A significant modification of gene expression was defined as a >2 down- (dark green) or up-regulation (dark red).

Figure 4. TRIM genes whose expression is regulated by IFN or immune complex.

These diagrams show the TRIM genes whose expression is either up- (>2-fold increase compared to untreated cells) or down-regulated (>2-fold decrease compared to untreated cells) by type I IFN, type II IFN or Immune complex (IC). The effect of each treatment on the expression of non-TRIM genes included in the screen is also shown. Data are represented as fold induction.

Figure 5. Summary of TRIM expression in mouse and human macrophages upon various stimuli and in silico promoter analysis.

TRIM expression in human macrophages upon IFN treatment. This part of the table shows the comparison of TRIM gene expression in mouse macrophages treated with LPS or poly(I:C) , in human macrophages upon IFN-γ and LPS treatment , , and in human macrophages stimulated with either type I or type II (γ) IFN (our study). In silico promoter analysis. Table illustrating potential transcription factor binding sites based on sequence analysis of 1 kb of genomic DNA upstream of each TRIM protein. IRF sites were scored using the PROMO virtual laboratory using matrices specific to selected human transcription factors (TFs). Highest scoring TF binding sites (+++) had dissimilarity values of less than 0.1 and random expectation values (noted RE within the table key) of less than 0.01. Calculated sites meeting only one of the above criteria (++) or neither (+) are indicated. ISRE/IRF sites were further corroborated with MatInspector. Highest scoring TF binding sites (+++) had similarity values above 0.9, (++) values between 0.85–0.89, and (+) values between 0.80–0.84. Numerous positive genomic controls (OAS2, MX1, STAT1, APOBEC3G, APOBEC3F, etc.) and their calculated TF profiles were used to evaluate the stringency of hits. Negative genomic controls GAPDH and ACTB were used to evaluate the stringency of the programs.

Figure 6. Phylogenetic tree of human TRIM proteins.

This joining-neighbor-tree of human TRIM proteins deleted from their Ct domain(s) was constructed using the CLUSTALW and NJplot programs. Numbers indicate bootstrap proportions after 1000 replications. The scale bar represents 0.05 substitutions per amino acid position. Red boxes show TRIM genes which are up-regulated by type I IFN in macrophages, whereas blue boxes show TRIM genes which are up-regulated following activation of MDM with immune complex (IC). TRIM genes belonging to the 11p15.4 cluster are indicated.