The dissection of non-canonical cis-regulatory elements downstream of beta-globin locus in the fetal hemoglobin gene regulation - Project Summary/Abstract In the human genome, only 10% are coding regions responsible for protein coding. Particularly, the non- coding regions that directly regulate the gene expression – cis-regulatory elements (CREs) – are of great importance in normal development and pathogenic disease development. CREs are generally bound by transcriptional factors (TFs). CREs play an important role in the globin switch during fetal to adult erythropoiesis. CREs act as enhancers (LCR region), repressors (BCL11A binding site in γ globin promoter), or insulators (3'HS1 and HS5 CTCF binding sites (CBSs) on canonical β-globin locus border). CREs have been explored as new gene therapy targets for the globin gene expression, which is critical for gene therapy development of hemoglobinopathies and thalassemia. Traditionally, studies of CREs regulating β globin locus gene expression are limited between the border of 5'HS and 3'HS1 CTCF binding sites. Recently, together with others, we revealed a nested multi-loop 3D genomic structure around the β-globin gene cluster. The multi-loop 3D genome structure extends the regulatory landscape of the β-globin gene cluster with an extension of 145kb downstream (forming a 3D genomic structure called d-TAD) and a 110kb sub-TAD upstream (forming a 3D genomic structure called u-TAD). Utilizing genome editing of multiple CBSs (deletions and inversions) bordering the β-globin gene cluster, we found that the deletion of the CBS at 3'HS1 – the β-globin locus downstream boundary frequently disrupted by HPFH deletions – is sufficient to induce γ-globin in adult red blood cells. Importantly, we located an HPFH enhancer that's long speculated for the juxtaposition to activate fetal hemoglobin in HPFHs is responsible for the reactivation of γ-globin. By deleting the 48kb region to bring the HPFH enhancer closer to the beta-globin locus, we also observed an upregulation of fetal hemoglobin, suggesting the HPFH enhancer is a novel conditional enhancer for γ globin expression. Our published work, together with others, strongly suggests the CREs nested in the downstream sub-TAD of the β-globin gene cluster indeed played an important role in regulating globin gene expression. These data lead us to hypothesize that CREs in d-TAD are regulating β globin gene expression through long-range 3D genomic interactions. With the most updated technologies, we propose two research aims. In Aim1, we will use CRISPR/Cas9-based prime editing to introduce TF binding mutant precisely to interrogate the TF binding cooperativity in the TF binding hubs located in d-TAD. Epigenomic editing will also be applied to TF binding hubs at d-TAD in HUDEP-2 cells to examine the histone PTM's role in regulating gene expression. We will also apply genomic and epigenome editing to make CREs more active in promoting γ- globin expression. In Aim2, we propose to study the 3D CRE hubs formed by CREs between d-TAD and canonical β-globin cluster. We will use HiChIP to dissect the interactions mediated by active TFs like GATA1 and repressive TFs like BCL11A. Overall, our proposed research will expand the mechanistic view of the poised expression of γ-globin and provide new gene therapy targets for sickle cell disease or β-thalassemia.
