Optimized logic rules reveal interferon-γ-induced modes regulated by histone deacetylases and protein tyrosine phosphatases

Abstract

The pro-inflammatory cytokine interferon-γ (IFN-γ) is critical for activating innate and adaptive immunity against tumours and intracellular pathogens. Interferon-γ is secreted at the fetal-maternal interface in pregnant women and mice. The outer layer of the placenta in contact with maternal blood is composed of semi-allogeneic trophoblast cells, which constitute the fetal component of the fetal-maternal interface. The simultaneous presence of pro-inflammatory IFN-γ and trophoblast cells at the fetal-maternal interface appears to represent an immunological paradox, for trophoblastic responses to IFN-γ could potentially lead to activation of maternal immunity and subsequent attack of the placenta. However, our previous studies demonstrate that IFN-γ responsive gene (IRG) expression is negatively regulated in human and mouse trophoblast cells. In human cytotrophoblast and trophoblast-derived choriocarcinoma cells, janus kinase signalling is blocked by protein tyrosine phosphatases (PTPs), whereas in mouse trophoblast, histone deacetylases (HDACs) inhibit IRG expression. Here, we used genome-wide transcriptional profiling to investigate the collective roles of PTPs and HDACs on regulation of IRG expression in human choriocarcinoma cells. Logic-rules were optimized to derive regulatory modes governing gene expression patterns observed upon different combinations of treatment with PTP and HDAC inhibitors. The results demonstrate that IRGs can be divided into several categories in human choriocarcinoma cells, each of which is subject to distinct mechanisms of repression. Hence, the regulatory modes identified in this study suggest that human trophoblast and choriocarcinoma cells may evade the potentially deleterious consequences of exposure to IFN-γ by using several overlapping mechanisms to block IRG expression.

Keywords: choriocarcinoma; interferon-γ signalling; logics; pervanadate; valproic acid.

Figure 1

Jar cells suppress interferon‐γ (IFN‐γ) responsive genes compared with HeLa cells: The number of (a) up‐regulated and (b) down‐regulated genes upon IFN‐γ stimulation of HeLa and Jar cells are represented by a Venn diagram. (c) Heatmap representing mean of Log₂ (Fold change) of differentially expressed genes in IFN‐γ stimulated HeLa and Jar cells relative to the unstimulated cells. Colourmap ranging from blue to red represents low to high mean of the fold‐change from four independent experiments. Differential expression compared four IFN‐γ‐stimulated samples with four unstimulated samples and was defined as a mean of the absolute fold change of at least two relative to the unstimulated cells and a significant (q < 0·05) change in expression by DESeq2.

Figure 2

The transcription response of Jar cells upon inhibition of protein tyrosine phosphatases (PTPs) and histone deacetylases (HDACs): (a) Differentially expressed genes (upper panel: absolute fold change, ≥ 2; false discovery rate < 0·05) and their fold changes (lower panel) were measured for each treatment by comparing their levels of expression (x‐axis) with unstimulated cells (horizontal axis). Colour map ranging from blue to red depicts low to high log₂ (fold change). (b) Venn diagram showing overlap between the up‐regulated (upper panel) and down‐regulated (bottom panel) genes upon treatments with V (valproic acid, which is an HDAC inhibitor), I (human interferon‐γ) +V, P (pervanadate, which inhibits PTPs) and I+P.

Figure 3

Regulatory modes induced by interferon‐γ (IFN‐γ) upon treatments with the inhibitors: Rules (second column) describing differential expression patterns of 399 HeLa IFN‐γ responsive gene (IRGs) upon stimulation of Jar cells with human interferon‐γ (I), valproic acid (V), pervanadate (P), I+V and I+P, and binding sites of the transcription factors (third column) enriched in the gene clusters. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

System‐wide optimization of logic rules and promoter analysis: (a) All 59 rules (second column) describing differential expression patterns (blue/1 = up‐regulation, white/0 = no significant difference and yellow/−1 = down‐regulation) of 5635 genes upon stimulation with human interferon‐γ (I), valproic acid (V), pervanadate (P), I+V and I+P (horizontal axis). (b) f ₁₃, f ₃₃, f ₃₅ and f ₃₉ governed 50% of the differentially expressed genes. The promoter analysis reveals enrichment of several transcription factor binding sites. The distinct patterns described by the f ₁₃, f ₃₃, f ₃₅ and f ₃₉ rules are expected to be governed by a subset of transcription factors unique to those gene clusters, and are depicted by grey, purple, green and yellow colours look different, not the ones described colours, respectively. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Gene‐regulatory network: interactions between gene modules is represented by a hierarchical network where node names with prefix f represent rules described in Fig. 3 and prefix A represent ‘AND’ logic. Edges with directional arrows represent : up‐regulation, : inhibition and : down‐regulation.

Figure 6

Transcription factors regulating master rules: (a) Twelve master rules were identified that could replace all the 59 rules. (b) Four of these 12 rules showed enrichment of transcription factors, indicating their critical roles in signal processing.