Too T-ALL for a HAT? Deconstructing genetic regulation at a model enhancer

Enhancers are regions of DNA that act as hubs for protein binding of transcriptional machinery. This machinery can be donated to the promoter of a target gene, upregulating gene expression. Enhancers are bidirectionally transcribed, producing non-coding enhancer RNAs (eRNAs). CREB binding protein (CBP) is a transcriptional coactivator found at most enhancers and upon eRNA binding its histone acetyltransferase (HAT) activity is stimulated (D. A. Bose et al. 2017). In a subset of T-cell acute lymphoblastic leukemia (T-ALL), somatic mutations form a new enhancer that requires the transcription factor (TF) MYB for initiation (Mansour, Abraham, et al. 2014). This enhancer drives oncogenic TAL1 expression by recruiting CBP and a regulatory complex, consisting of proto-oncogenic TFs, including TAL1 and MYB (Sanda et al. 2012; Mansour, Abraham, et al. 2014).

Increasing evidence is emerging for TFs to have RNA binding properties, and despite the growth in eRNA studies, interactions with eRNAs and protein complexes at enhancers have not been well studied. We sought to understand how CBP and TFs found at this enhancer interact with RNA, to determine a role for regulating RNAs at this enhancer.

We showed that CBP is part of this complex; which we term CBP-TAL1, and individually CBP-TAL1 binds to the TAL1 eRNAs. To further investigate CBP-TAL1:RNA interactions, we endogenously tagged MYB and CBP in a single cell line using CRISPR/Cas9. Affinity pull-down experiments followed by RNA sequencing, called tandem affinity cross-linking and analysis of cDNAs (TA-CRAC, Granneman, Petfalski, and Tollervey 2011; Thoms et al. 2015) will shed light on the action of RNAs within this protein complex at enhancers, particularly eRNAs. Additionally we developed a system to deplete initiating MYB at this enhancer, allowing investigation of enhancer formation and assembly.

Overall, we have taken complementary approaches to analyse the TAL1 enhancer, to understand the action of the CBP-TAL1 complex and eRNAs. This will provide deeper insight into eRNA behaviour, regulatory complex action and enhancer function.