The identification of many sequence-specific PDIs for both conventional TFs and uDBPs raises an interesting question; that is whether these uDBPs bind to different target sequences than do annotated TFs. While some proteins in the same functional class were found to have preferred DNA-binding profiles selective to that protein family, the overlap in the DNA motifs recognized by the TFs and uDBPs is remarkable and substantial (Figure S18
), which suggests a complex landscape for human PDI networks and possible crosstalk between TFs and uDBPs. As an example, we found that MAPK1 regulates expression of IFNγ-induced genes via
binding to GATE element, which has also been shown to be bound by C/EBP-β (Roy et al., 2000
Our study suggests that a crosstalk between C/EBP-β and the DNA-binding and kinase activities of MAPK1 results in a negative feedback loop to tightly control the temporal expression pattern of IRF9
upon IFNγ induction. Previously, Kalvakolanu and colleagues showed that upon IFNγ induction C/EBP-β is phosphorylated by MAPK1/2 to activate expression of the GATE-driven genes (Roy et al., 2002
). However, this model does not explain up-regulation of the GATE-driven genes when only MAPK1 is knocked down in cells (Huang et al., 2008
) or the suppression of IRF9
8 hours post IFNγ-treatment (). Based on the newly discovered DNA-binding activity of MAPK1, a plausible explanation is that expression of the GATE-driven genes is dictated by competitive binding of C/EBP-β and MAPK1 to GATE element. In untreated cells, GATE is directly bound by MAPK1 via
its DNA-binding domain and transcription of the downstream genes is inhibited, which explains the up-regulation of those IFN-response genes when MAPK1 is knocked down (Huang et al., 2008
). When cells are treated with IFNγ, C/EBP-β is rapidly induced and phosphorylated by MAPK1/2, which are activated by the MEKK1/MEK1 pathway (Roy et al., 2002
). The activated C/EBP-β in the nucleus then rapidly competes off MAPK1 bound to GATE, resulting in a rapid activation of the GATE-driven genes and a sharp decline of MAPK1 occupancy at GATE (). As this proceeds, the concentration of nuclear MAPK1 gradually increases to a level that it starts to compete off bound C/EBP-β and therefore posts a negative feedback to eventually shut down expression of these genes. Taken together, we believe that the crosstalk between the two independent MAPK1 activities and C/EBP-β partially explains the dynamics of IFNγ-induced gene expression.
A significant advantage of the presented protein-centered approach is that the binding specificity of a given DNA motif can be simultaneously measured for thousands of proteins in a single assay. In our studies, we made careful choice for the biologically meaningful DNA motifs that are either highly conserved during evolution or highly enriched in the regulatory regions of co-expressed genes. Therefore, by exploring the DNA space predicted to be enriched for cis-regulatory elements, we have established possible connections to their upstream effectors. Indeed, the fact that virtually all of the DNA motifs tested in this study bound selectively to proteins on the array supports this notion. Furthermore, our approach can examine a large variety of protein families, providing an opportunity to discover novel DNA-binding proteins. It is expected that combined with DNA-centered approaches, such as protein-binding DNA microarrays and one-hybrid analysis, we will be able to precisely determine DNA binding consensus sequences for many uDBPs.