A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker (ATAC-seq)

Many SNVs associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human alpha-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates alpha-globin expression causing alpha-thalassaemia. Rather than competing for the activity of the enhancers, the new promoter lying between the a-globin genes and their associated super-enhancer acts as an orientation-dependent boundary element. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that promoters may act as boundary elements in mammals as in drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements. Overall design: To characterise the epigenetic landscape and identify nucleosome depleted regions during erythropoiesis, ATAC-seq was carried out at day 21 of hiPSC erythroid differentiation. Lines: LA01, LA06, LA13 (homozygous C-SNV allele); CtoT_Clone1, CtoT_Clone2, CtoT_Clone3, CtoT_Clone4 (edited C-SNV line LA01 to homozygous T allele); TtoC_Clone1, TtoC_Clone2, TtoC_Clone3, TtoC_Clone4 (edited wild type line SBAD02 to homozygous C-SNV allele); Vas_Clone1, Vas_Clone2, Vas_Clone3 (promoter insertion in anti-sense orientation, parental line SBAD02); Vs_Clone1, Vs_Clone2, Vs_Clone3 (promoter insertion in sense orientation, parental line SBAD02); nonINV_Clone1, nonINV_Clone2, nonINV_Clone3 (Lox site inserted non-inverted, parental line LA01); INV_Clone1, INV_Clone2, INV_Clone3 (C-SNV promoter inversion, parental line LA01); AH017 wild type line; SBAD02 wild type line; SBAD03 wild type line; The SNV for both the C-SNV (C/C) lines, and CtoT (T/T) and TtoC (C/C) edited clones is the same and is at position 209,709 of chr16 in genome build hg19.