Novel Class of Enhancer RNAs Linked to Growth of Cancers

MicroRNAs (miRNAs) and short interfering RNAs (siRNA) comprise a class of about 20 nucleotides-long RNA fragments that block gene expression by attaching to molecules of messenger RNA in a fashion that prevents them from transmitting the protein synthesizing instructions they had received from the DNA. MiRNAs resemble siRNAs of the RNA interference (RNAi) pathway, except miRNAs derive from regions of RNA transcripts that fold back on themselves to form short hairpins, whereas siRNAs derive from longer regions of double-stranded RNA. With their capacity to fine-tune protein expression via sequence-specific interactions, miRNAs help regulate cell maintenance and differentiation.

Augmenting the repertoire of "classical" miRNAs, investigators at the University of California, San Diego (USA) identified several thousand enhancer RNAs (eRNAs) that were robustly produced in colon cancer cells in response to chronic immune signaling. Enhancer RNAs are transcribed from DNA sequences upstream and downstream of extragenic enhancer regions. Depending on the directionality of transcription, enhancer regions generate two different types of non-coding transcripts, unidirectional-eRNAs and bidirectional-eRNAs. The nature of the pre-initiation complex and specific transcription factors recruited to the enhancer may control the type of eRNAs generated.

After transcription, the majority of eRNAs remain in the nucleus, and as they are very unstable, they are actively degraded by the nuclear exosome. Not all enhancers are transcribed, with non-transcribed enhancers greatly outnumbering the transcribed ones in the order of magnitude of dozens of thousands in every given cell type. The theory that not all enhancers are transcribed at the same time and that eRNA transcription correlates with enhancer-specific activity support the idea that individual eRNAs carry distinct and relevant biological functions.

The investigators reported in the August 3, 2018, issue of the journal Nature Structural and Molecular Biology that in human colorectal cancer cells, Bromodomain-containing protein 4 (BRD4) was recruited to enhancers that were co-occupied by mutant p53 and supported the synthesis of enhancer-directed transcripts (eRNAs) in response to chronic immune signaling. BRD4 selectively associated with eRNAs that were produced from BRD4-bound enhancers.

BRD4 is a member of the BET (bromodomain and extra terminal domain) protein family, which also includes BRD2, BRD3, and BRDT. BRD4, similar to other BET family members, contains two bromodomains (BDs) that recognize acetylated lysine residues. BRD4 also has an extended C-terminal domain with little sequence homology to other BET family members.

The investigators used biochemical and biophysical methods to show that BRD4 BDs functioned cooperatively as docking sites for eRNAs and that the BDs of BRD2, BRD3, BRDT, BRG1, and BRD7 directly interacted with eRNAs. BRD4-eRNA interactions increased BRD4 binding to acetylated histones in vitro and augmented BRD4 enhancer recruitment and transcriptional cofactor activities.

"Our findings reveal that eRNAs are key regulators of cancer by acting to reinforce BRD4 binding and keep it anchored on DNA, which keeps the tumor-promoting genes turned on at high levels," said senior author Dr. Shannon Lauberth, assistant professor of biology at the University of California, San Diego. "Interestingly, when we deplete several of these eRNAs, we can significantly reduce the expression of the tumor-promoting genes that the eRNAs and BRD4 are co-regulating. Now that we see that eRNAs impact BRD4 function, we have to rethink the way that we therapeutically target BRD4. Taken together, our findings are consistent with the emerging notion that eRNAs are functional molecules, rather than merely reflections of enhancer activation or simply transcriptional noise."

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