PIRÁMIDE NUTRICIONAL

A recent study has linked HIF2A with the maintenance of super enhancer in ccRCC198_{. In} relation to this, I have demonstrated that KLF6 expression in ccRCC was super enhancer-driven and modulated by HIF2A. Thus, to test whether these present findings were in line with the aforementioned study above, Sakari Vanharanta and I re-examined the H3K27ac ChIP-Seq data of the HA-VHL-reintroduced 786-M1A and OS-LM1 cells, previously generated in the lab by Paulo Rodrigues. We assessed the effect of VHL reintroduction and consequent HIF2A loss to the H3K27ac patterns of the KLF6 super enhancer locus. There was a reduction in H3K27ac signal at one of the enhancer regions downstream of KLF6 locus in both HA-VHL reintroduced- 786-M1A and OS-LM1 cells (Figure 46A). Apart from this, there was no significant difference in the general H3K27ac signals pattern of this KLF6 super enhancer locus between the control and VHL-reintroduced cells. This was consistent with the previous findings that demonstrated the robustness of this particular KLF6-associated super enhancer region (Figure 36A).

Moreover, examination of the previously generated and analysed 786-M1A and OS-LM1 HIF2A ChIP-Seq data revealed that HIF2A bound the same enhancer region that had the H3K27ac signal reduced upon VHL reintroduction (Figure 46B-C). Based on these HIF2A ChIP-Seq data, HIF2A might modulate KLF6 expression in ccRCC by acting through the KLF6 super enhancer locus. In order to test this possibility, the HIF2A binding site, shown in figure 46B, was CRISPRi-inactivated in combination with another enhancer region putatively bound by HIF2A in the 786-M1A cells. The combinatorial enhancers targeting strategy as described in section 4.2.2 above was employed to perform this iHIF2A binding site experiment. The CRISPRi-mediated inactivation of these HIF2A binding sites reduced the KLF6 expression level by 40% (Figure 46D). This finding was relatively similar to the effect of reintroducing HA-VHL

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into these 786-M1A cells (Figure 44A). Overall, these findings confirmed that HIF2A binds to this super enhancer locus to modulate the expression of KLF6 in ccRCC.

Figure 46: HIF2A binds the KLF6 super enhancer locus. (A) H3K27ac ChIP-Seq signal of 786- M1A and OS-LM1 cells transduced with either empty vector or HA-VHL. A region significantly altered in both HA-VHL reintroduced cells when compared to the empty vector control highlighted by grey box. (B and C) A close up of the region highlighted in panel A together with HIF2a ChIP-Seq signals in (B) 786-M1A and (C) OS-LM1 cells. (D) KLF6 expression in the 786-M1A cells in which two putative HIF2a binding sites were inactivated using CRISPRi. Average of three experiments. Error bars, SEM. Two-tailed Student’s t-test. * P < 0.05, ** P < 0.005 and *** P < 0.0005.

D B A

135 4.3 Summary

One of the primary focus of this chapter was to investigate whether KLF6 expression in ccRCC was supported by this nearby super enhancer locus. Using the CRISPRi-based enhancer inactivation approach, it was discovered that this super enhancer region drove the expression of KLF6. In addition, this super enhancer acted in a modular fashion to drive KLF6 expression in ccRCC. Significant reduction in the expression of KLF6 mRNA level was only achieved when either several constituent enhancers were inactivated concurrently or by genetic deletion of the large enhancers cluster, in which in this present study the CRISPR-Cas9 approach was used to delete 113Kb of the enhancers cluster upstream of KLF6 locus.

Moreover, it was observed that this KLF6 super enhancer was robust and insensitive to the perturbations in the activity of its constituent enhancers. There was no interdependency between these constituent enhancers. Inactivating one individual enhancer only resulted in the loss of H3K27ac signals at the specific targeted region without affecting the general H3K27ac signals pattern or the whole super enhancer landscape. This was in contrast to several recent studies that have demonstrated the cancer-associated super enhancers are sensitive to perturbation of its constituent enhancers or regulatory components that establish or maintain the super enhancer landscape. However, such redundancy and robustness are well-aligned with the fact that most critical biological processes and developmental transcriptional programmes are insensitive to environmental and other incoming variations.

Last but not least, the works in this chapter also revealed a link between the ccRCC- initiating VHL-HIF2A pathway and KLF6 expression modulation in ccRCC. Through the previously generated VHL reintroduced-cells H3K27ac ChIP-Seq as well as the HIF2A ChIP-

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Seq data, HIF2A was discovered to bind this KLF6 super enhancer locus, in which the HIF2A binding sites were identified downstream of the KLF6 locus. Collectively, these data suggested that HIF2A supports KLF6 expression by acting through the large KLF6 super enhancer, potentially explaining the relatively high KLF6 levels in ccRCC when compared to other tumour types (Chapter 3, figure 16).

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Chapter 5

Dissecting the transcriptional network