Discovering Regulatory SNPs by Genome-Wide Analysis of Differential Scl/TAL-1 Occupancy in Human Primary Erythroid Cells,
Authors: De Gobbi Marco , Hughes Jim R. , Lower Karen M. , Derry Chris J. , Marchi Emanuele , Sloane-Stanley Jacqueline , Roberts Nigel , Taylor Stephen , Porcher Catherine , Gibbons Richard J. , Higgs Douglas R. 
Published: October 2019

Abstract
Abstract 3381
It is well established that the level of gene expression can vary significantly between normal individuals, and that the majority of this variation is due to naturally occurring genomic variability caused by single nucleotide polymorphisms (SNPs). Therefore, identifying functional cis-regulatory polymorphisms and understanding how they influence gene expression is an important new task in many areas of medical research, including molecular hematology. We have previously shown that an entirely new form of alpha-thalassemia is caused by a gain of function regulatory SNP in an unremarkable non-coding region in the alpha-globin cluster. This SNP creates a novel, functional GATA site, which recruits a tissue-specific transcription factor (TF) complex. This creates a new promoter-like element, which interferes with activation of the globin genes (De Gobbi et al. Science 2006,312:1215–1257). Here, to investigate the extent and the impact of this class of regulatory SNP, using ChIP-Seq we characterized differences in the occupancy of Scl/TAL-1 (a tissue-specific TF critical for erythroid maturation) in the erythroblasts of two individuals from the same ethnic background (Caucasian 1, C1, and Caucasian 2, C2).
Sequence reads from two biological replicates of each individual were merged and aligned to the human reference genome (NCBI36/hg18) and a total of 2936 Scl/TAL-1 bound regions were identified. Using two de novo motif finding algorithms (MEME and DREME), we identified GATA (WGATAR) and E-box (CAGMTG) sites as the preferred sequences associated with in vivo binding of Scl/TAL-1. In addition, other motifs were enriched at the Scl/TAL-1 targets; among these were binding sites for known TFs (Sp1/Klf, RUNX1 and NFE2).
To identify differentially bound regions between C1 and C2, a two-class paired-test, Rank Product analysis (500 permutations, FDR<0.2) was performed with MeV4.6 TM4 Software. About 1% (25/2936) of these sites showed differential binding. Differences were mostly associated with SNPs directly affecting or lying adjacent to known TF consensus binding sites and deviations from the GATA or E-box consensus motifs corresponded to the inability of the sequence to bind Scl/TAL-1.
Since it has been previously shown that the function of active transcriptional elements can be predicted on the basis of chromatin signatures (e.g. enhancers marked by H3K4me1 and promoters marked by H3K4me3), to further characterize the Scl/TAL-1 differentially occupied sites, we asked which chromatin signatures are associated with these regions. H3K4me1 and H3K4me3 ChIP-Seq experiments, together with analyses of publicly available data sets, showed that the most of the SNPs responsible for variation in the recruitment of Scl/TAL-1 (23/25) lie in DNA sequences that have chromatin signatures predictive of enhancer elements, suggesting a potential long-range function in modulating gene expression.
Finally, Scl/TAL-1 ChIP-Seq analysis of erythroblasts of a third individual from a different ethnic background (African-Caribbean, A3) revealed more distinctive targets including a well known regulatory SNP at the promoter of the DARC gene (encoding the Duffy blood group), which alters a GATA binding motif in A3, conferring the malaria-resistant Duffy-null phenotype.
Given the exponential growth in genome-wide association studies by which numerous SNPs are being either associated with hematological parameters, or implicated in the etiology of hematologic disorders, this study elucidates molecular mechanisms which might account for phenotypic diversity and highlights the importance of carrying out functional characterization of non-coding polymorphisms found to be associated with disease risk.

Disclosures:
No relevant conflicts of interest to declare.