HCF-1 is an essential component of the HSV-1 IE regulatory paradigm. The protein is recruited to viral IE promoter-enhancer domains via complex interactions with multiple factors, including viral IE activators, that contribute to the induced expression of these genes during initiation of viral lytic infection. HCF-1 functions, at least in part, by coordination of chromatin modification complexes, such as the Set1/MLL1 histone methyltransferase and the histone demethylase LSD1, to prevent the accumulation of repressive chromatin and promote the installation of positive chromatin marks.
In addition to its role in lytic infection, chromatin plays a significant role in the regulation of HSV-1 latency and reactivation cycles. Activating marks are evident on the transcribed LAT gene while repressive marks are prevalent on the repressed IE gene promoter regions (1
). Conversely, activating marks accumulate on the viral IE gene promoter domains during the initial stages of reactivation (1
). Strikingly, HCF-1 is sequestered in the cytoplasm of sensory neurons, where the virus establishes latency. The protein is rapidly relocalized to the nucleus upon in vivo and ex vivo stimulation, suggesting that HCF-1 may also play a critical role in reactivation of HSV-1 from latency. However, a direct involvement in viral reactivation has not yet been demonstrated.
In this study, a ChIP approach was used to demonstrate that HCF-1 is recruited to HSV-1 IE gene promoter-enhancer domains upon stimulation of reactivation of HSV-1 from latency in sensory neurons. Recruitment was detected as early as 1 h postinduction of reactivation, and occupancy increased by 4 h postinduction. This time course correlates well with the nuclear transport of HCF-1, where the number of neurons exhibiting nuclear localization increases over the course of 6 h poststimulation. Additionally, HCF-1 occupancy of viral IE promoter domains correlated with occupancy by RNAPII and accumulation of viral IE mRNAs. This study represents the first report of the direct recruitment of a critical component of the IE gene regulatory circuit during viral reactivation.
HCF-1 is not a direct DNA binding component, and its recruitment is dependent upon interactions with site-specific transcription factors. The protein interacts with members of numerous transcription factor families, including several activators (GABP, Sp1, and VP16) that contribute to the induced transcription of viral IE genes during lytic infection. Occupancy of the IE promoter-enhancer domain by HCF-1 during reactivation localizes to the distal promoter region of the enhancer domain. This region contains several defined factor binding sites, including two TAATGARAT enhancer core elements, a consensus GABP, and several Sp1 sites. In contrast, no significant association of HCF-1 with the promoter-proximal enhancer core element or the putative CREB3 site was detected, a factor that was proposed to be involved in HCF-1 recruitment during reactivation (16
). While this suggests that the factor(s) responsible for HCF-1 recruitment is localized distal to the promoter domain, this may also be a reflection of multiple interactions that result in a highly stable association at this region. It is important to note that due to the limitations of the ChIP assay, the data do not exclude the possibility that HCF-1 is transiently or nonstably recruited via factors whose recognition sites are within other regions of the IE enhancer-promoter domains.
It is also possible that the factors that recruit HCF-1 could depend upon the specific reactivation stimuli. Of note, a recent study has indicated that the viral IE activator VP16, a protein whose function is dependent upon HCF-1, may be critical for viral IE transcription and reactivation (27
). The recruitment of HCF-1 to the region containing two enhancer core elements is consistent with this model. However, it remains to be determined which factors are directly responsible for HCF-1 recruitment and whether those factors reflect distinct signaling pathways. Regardless, the demonstration of the direct recruitment of HCF-1 to viral IE genes during the initial stages of reactivation promotes the model that HCF-1 complexes play a critical role in the viral reactivation process.