Haemoglobin disorders are amongst the most commonly inherited and debilitating disorders in developing countries. The World Health Organisation estimates that at least 5% of adults are carriers for a haemoglobin disorder, such as sickle cell anaemia or beta-thalassaemia, which result from mutations to the gene encoding adult beta-globin.

Different forms of haemoglobin are produced throughout human development in order to meet the changing oxygen demands of the growing embryo or foetus. Around the time of birth, a developmental switch occurs where the foetal gamma-globin gene is silenced and the adult beta-globin gene is expressed. A rare beneficial condition called Hereditary Persistence of Foetal Haemoglobin (HPFH) has been shown to alleviate the symptoms of sickle cell anaemia and beta-thalassaemia, since the continued expression of the foetal gamma-globin gene during adult life is able to compensate for the lack of functional beta-globin protein. HPFH individuals contain point mutations or small deletions within the promoter region of the gamma-globin gene. The -115 site of the gamma-globin promoter contains four reported HPFH mutations and a small 13 base pair deletion. The -115 HPFH cluster of mutations are thought to disrupt the binding a transcriptional repressor which normally silences the gamma-globin gene around the time of birth. Our aim was to identify this repressor.

We studied a range of potential transcription factors in an effort to identify a DNA-binding domain that could bind to the ‑115 gamma-globin site in vitro using electrophoretic mobility shift assays (EMSA). We compared binding with and without the ‑117 G>A, -114 C>T, -114 C>G and ‑114 C>A HPFH mutations and also the -114 to ‑102 HPFH deletion. This identified one candidate repressor. We also used TALENs to introduce -117 G>A mutation into the gamma-globin promoter of transgenic adult murine erythroid cells (MELs) containing the human beta-globin locus on a BAC. The -117 G>A mutation significantly increased gamma-globin expression and decreased beta-globin expression, a result which is consistent with increased levels of gamma-globin seen in HPFH individuals. We then demonstrated that the candidate repressor bound directly to the gamma globin -115 site using a complex CRISPR mediated epitope tagging procedure. These results identify a promising candidate repressor in silencing gamma-globin through direct binding to the ‑115 site of the gamma-globin promoter.