Abstract Toll-like receptor (TLR) pathways have been intensively investigated for almost two decades. However, since most studies have focused on the initial signal transduction and transcription events, the mechanisms of late-sustained gene expression are poorly understood. To address this issue, we conducted an RNAi screen for regulators of TLR-induced IL-6 secretion in RAW264.7 macrophages, and identified a novel putative regulator of this process (Hit4). Hit4 was specifically required to sustain the late expression of genes including Il6, Il1a, Nos2 and Lcn2, but did not affect induction of transiently expressed genes such as Nfkbiz, Egr1, and Egr2. This effect of Hit4 was confirmed by CRISPR/Cas9 gene editing and by RNAi knockdown in primary macrophages. Most interestingly, Hit4 functioned specifically in sustaining rather than initiating transcription, as Hit4 depletion showed no effect on the early mRNA transcription before 2 hr for all gene classes. Further mechanistic investigation revealed that Hit4 functioned at the transcriptional level rather than post-transcriptional level, as Hit4 enhanced the level of nascent transcripts at later time points. Moreover, Hit4 KO RAW264.7 macrophages failed to sustain the response to persistent LPS stimulation, showing a response similar to that observed for transient LPS stimulation. This suggests a role for Hit4 in discriminating brief spurious pulses of infectious signal from a more sustained real infection signal. In summary, we have identified a novel transcriptional regulator that functions to sustain transcription of late inflammatory genes, providing important new insight to the control of TLR-induced gene program.(Supported by the Intramural Research Program of NIAID, NIH)
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