SERPINB2 is regulated by dynamic interactions with pause-release proteins and enhancer RNAs

Abstract

The SERPINB2 gene is strongly upregulated in inflammatory states. In monocytes, it can constitute up to 1% of total cellular protein. It functions in protection from proteotoxic stress and plays a role in angioedema. The purpose of this study was to define the roles of enhancer RNAs embedded in the SERPIN gene complex. We found that the upstream enhancer RNAs upregulated SERPINB2 and the enhancer RNAs were expressed prior to those of SERPINB2 mRNA. Studies of the SERPINB2 promoter demonstrated the presence of an RNA polymerase II pause-inducing protein, NELF. Stimulation with LPS led to recruitment of the pause-releasing kinase P-TEFb and departure of the pause-inducing protein NELF. RNA immunoprecipitation revealed that NELF and the CDK9 component of P-TEFb bound to the enhancer RNAs after stimulation with distinct kinetics. Knock-down of the enhancer RNAs compromised stimulus induction of promoter and enhancer chromatin changes. Conversely, over-expression was associated with enhanced recruitment of c-JUN and increased expression of SERPINB2 mRNA expression. This study is the first to associate enhancer RNAs with SERPINB2 and is the first demonstration of acquisition of NELF binding by enhancer RNAs on chromatin.

Diversity and Emerging Roles of Enhancer RNA in Regulation of Gene Expression and Cell Fate

“…eRNA knockdown studies have demonstrated important roles for individual eRNAs in the regulation of their target genes ( Supplementary Table 1). Additional studies have shown that exogenous overexpression of eRNAs leads to an increase in their respective mRNA targets (Alvarez-Dominguez et al, 2017;Shii et al, 2017;Jiao et al, 2018). These studies have provided the bulk of evidence implicating a directly causal role for eRNA, rather than the act of transcription, in the association between enhancer and mRNA transcription.…”

“…Extracellular stimuli drive signaling pathways which remodel chromatin and alter eRNA and mRNA expression (Kaikkonen et al, 2013;Heward et al, 2015). eRNA transcription occurs very early in the gene transcription process, preceding mRNA expression from adjacent cis loci (Schaukowitch et al, 2014;Arner et al, 2015;Kim et al, 2015;Baillie et al, 2017;Shii et al, 2017;Tyssowski et al, 2018). Indeed, the ubiquitous and early production of eRNAs makes them an excellent marker for segregating active versus quiescent enhancers, which is discussed in greater detail below (Andersson et al, 2014a;Tyssowski et al, 2018).…”

“…It is generally presumed that such specificity is largely contextdependent and driven by proximity and other factors controlling enhancer-promoter interactions (see van Arensbergen et al (2014) for review of enhancer-promoter specificity). However, ectopically expressed Bloodlinc and SERPINB2 eRNAs have also shown the ability to selectively upregulate their respective mRNAs suggesting that, at least among these eRNAs, mere proximity is insufficient to explain their specificity for their targets (Alvarez-Dominguez et al, 2017;Shii et al, 2017). Several studies utilizing reporter assays to investigate how eRNAs promote transcription at their respective mRNA promoter have demonstrated the importance of the specific eRNA sequence in promoting transcription compared to truncated or "missense" RNA transcripts (Lam et al, 2013;Li et al, 2013;Melo et al, 2013).…”

“…RNAs tethered near enhancer loci were shown to increase the presence of YY1 specifically at those loci, perhaps due to "transcription factor trapping" wherein nascent RNA from enhancers and promoters increase the affinity of otherwise weak DNA-TF interactions creating a kinetic sink that would "trap" escaped transcription factors (Sigova et al, 2015). Such a model may explain observations that eRNA increases the binding of transcription factors such as c-Jun and NF-κB to target loci (Shii et al, 2017;Spurlock et al, 2017;Huang Z. et al, 2018). An emerging idea for how eRNAs may interact with large numbers of proteins is through phase separation.…”

“…Most eRNAs are chromatin-associated, as opposed to free in the nucleoplasm or the cytoplasm (Cinghu et al, 2017;Shii et al, 2017). It may therefore be that eRNAs play important roles through their interactions with chromatinassociated proteins.…”