IRF8 and IRF3 cooperatively regulate rapid interferon-ß induction in human blood monocytes.

Robust and rapid induction of interferon-ß (IFN-ß) in monocytes after pathogenic stimulation is a hallmark of innate immune responses. Here, we reveal the molecular mechanism underlying this key property that is exclusive to human blood monocytes. We found that IFN-ß was produced rapidly in primary human monocytes as a result of cooperation between the myeloid-specific transcription factor IRF8 and the ubiquitous transcription factor IRF3. Knockdown of IRF8 in monocytes abrogated IFN-ß transcription, whereas reintroduction of IRF8 into the IRF8(-/-) 32Dcl3 murine myeloid cell line reinstated IFN-ß transcription. Moreover, we provide evidence that IRF8 constitutively binds to the ETS/IRF composite element of the IFN-ß promoter region together with PU.1 in vivo. Furthermore we uncovered a requirement for IRF3, a master regulator of IFN-ß production, as a previously un-indentified interaction partner of IRF8. We mapped the protein-protein interacting regions of IRF3 and IRF8, and found that their interaction was independent of the DNA-binding domain and the IRF association domain of IRF8 and IRF3, respectively. Therefore, we propose a model for the rapid induction of IFN-ß in monocytes, whereby IRF8 and PU.1 form a scaffold complex on the IFN-ß promoter to facilitate the recruitment of IRF3, thus enabling rapid IFN-ß transcription.

Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited by NF-kappaB upon TLR4 activation.

NF-kappaB is a key mediator of inflammation. Here, we mapped the genome-wide loci bound by the RELA subunit of NF-kappaB in lipopolysaccharide (LPS)-stimulated human monocytic cells, and together with global gene expression profiling, found an overrepresentation of the E2F1-binding motif among RELA-bound loci associated with NF-kappaB target genes. Knockdown of endogenous E2F1 impaired the LPS inducibility of the proinflammatory cytokines CCL3(MIP-1alpha), IL23A(p19), TNF-alpha, and IL1-beta. Upon LPS stimulation, E2F1 is rapidly recruited to the promoters of these genes along with p50/RELA heterodimer via a mechanism that is dependent on NF-kappaB activation. Together with the observation that E2F1 physically interacts with p50/RELA in LPS-stimulated cells, our findings suggest that NF-kappaB recruits E2F1 to fully activate the transcription of NF-kappaB target genes. Global gene expression profiling subsequently revealed a spectrum of NF-kappaB target genes that are positively regulated by E2F1, further demonstrating the critical role of E2F1 in the Toll-like receptor 4 pathway.

HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets.

Type I IFNs induce the expression of IFN-stimulated gene 15 (ISG15) and its conjugation to cellular targets. ISGylation is a multistep process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be identified E3 ligase. Here we report the identification of an IFN-induced HECT-type E3 protein ligase, HERC5/Ceb1, which mediates ISGylation. We also defined a number of proteins modified by ISG15 after IFN triggering or HERC5 overexpression. A reduction in endogenous HERC5 by small interfering RNA inhibition blocks the IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression with Ube1L and UbcH8 induces the ISG15 conjugation in vivo independent of IFN stimulation. A targeted substitution of Cys-994 to Ala in the HECT domain of HERC5 completely abrogates its E3 protein ligase activity. Therefore, this study demonstrates that HERC5/Ceb1 is involved in the conjugation of ISG15 to cellular proteins.