forced linear recruitment of a super-enhancer strongly reactivates the developmentally silenced fetal HBG globin genes
The human genome contains regulatory DNA elements (enhancers, silencers, insulators) that can recruit proteins to activate or repress transcription of distal genes. Previously, it was shown that modifications in these regulatory elements can lead to curative therapies for hemoglobinopathies, such as the destruction of the GATA1 binding site in the BCL11A enhancer that is known as an important HBG repressor.
It is currently unknown if the distance of the regulatory element to the transcriptional target can also exert a function in gene regulation. To test this hypothesis, we aimed to bring the distal super enhancer of the globin genes in closer proximity to the fetal hemoglobin genes (HBG1 and HBG2).
To test our hypothesis, we used spCas9 mediated gene editing using ribonucleoprotein delivery and lentiviral transductions in both HUDEP and hematopoietic stem and progenitor cells (HSPCs). We screened gRNAs to effectively delete 25 kilobases sequence interval bringing the distal super enhancer close to the HBG genes. We used long term cultures of gene edited HSPCs and differentiated these towards enucleated red blood cells (reticulocytes). We analyzed the proliferation, differentiation of these cells using flow cytometry. For the expression of globin genes such as HBG1, HBG2 and HBB we used qRT-PCR, flow cytometry and HPLC.
We found that deletion of a 25kb element, resulting in forced linear recruitment of the super-enhancer bringing it immediately upstream of the otherwise intact HBG gene promoters, significantly reactivates the silenced HBG gene. Additionally, a similar effect can be achieved by inversions that leave the intervening sequence intact and brings parts of the enhancer elements into linear proximity of the HBG genes. We observed HBG reactivation with the 25kb deletion / inversion, reached levels similar to the GATA1 binding site destruction, known as CTX001, that was previously shown to be effective for Sickle cell disease patients. Furthermore, we found no effects on proliferation or differentiation when this 25kb element was removed, indicating that this might potentially be a novel solution as a therapeutic strategy.
We conclude that the HBG genes can be reactivated when the super-enhancer is brought into closer proximity. Overall, this assigns a functional role to seemingly non-regulatory segments in our genome: by providing linear separation, they may support genes to autonomously control their transcriptional response to distal enhancers.
Our strategy might eventually be translated into a clinical trial, as the HBG reactivation levels are exceeding the clinical limit.