A Robust Ex-Vivo Erythropoiesis System For Studying Transcriptional Regulation Of Globin Genes In Thalassaemia

Ex-vivo differentiation of hematopoietic stem cells and progenitors to erythroid cells helped tremendously in our understanding of transcriptional and epigenetic regulation of normal erythroid differentiation as large number of nucleated cells can be generated by these protocols. However, to study the disease mechanisms in haematological diseases, there are practical difficulties in obtaining mobilized CD34⁺ cells from the patients. Alternatively, peripheral blood mononuclear cells (PBMNCs), which have a small fraction of haematopoietic progenitors, have also been used to obtain cultured erythroid cells (cErCs). Though PBMNCs is an alternative source, the current protocols using these cells do not generate sufficient number of cErCs, which is a major hurdle for performing transcriptional and epigenetic studies in erythroid cells from patients with β thalassaemia and sickle cell disease. We established a modified two phase culture protocol using a serum free basal medium with IL-3, IL-6, Flt3, SCF in phase I and Epo, SCF, IL-3 and iron compounds in phase II and could generate up to 10⁸ cells from PBMNCs isolated from 5-10ml blood of patients with homozygous β thalassaemia and those who were compound heterozygous for β thalassaemia and HPFH or δβ thalassaemia. Real time quantitative PCR (qPCR) analysis showed steady increase in the expression of α and β globin genes in different stages of erythropoiesis. As we obtained a large number of nucleated cells using this protocol, we could perform chromatin immunoprecipitation (ChIP) to study the binding of RNA Polymerase II (RNAPII), transcription cofactors, CBP and p300, and histone modifications at the regulatory sequences of β globin cluster. High levels of RNAPII was found at the coding sequences of β globin gene in the cErCs from both normal individuals and patients with splice site mutations suggesting that these mutations do not cause significant reduction in the transcription rate. Both CBP and p300 were found to bind at the HS3 and HS2 regions of locus control region in both normal and β thalassaemia cultures though they were not found at the promoter sequences of actively transcribed genes in the β globin cluster. In the patients compound heterozygous for β thalassaemia and HPFH-3 or (Aγδβ)° deletion/Inversion, we found increased γ globin gene expression compared to the normal controls and ChIP also showed high levels of RNAPII occupancy at the coding sequences of γ globin gene. As it has been shown earlier that alternative splicing is a transcriptional regulatory mechanism for cell specific expression of genes, we performed experiments to identify the alternatively spliced transcripts of β globin gene and their co-ordinated expression in human erythropoiesis. RT-PCRs and sequencing of β globin cDNA using various sets of primers that bind at the upstream, downstream and coding sequences showed 3 aberrantly spliced transcripts formed by the cryptic splice sites in exon1 and 4 novel transcripts by the activation of splice sites at the 5’UTR. Interestingly, 5’ splice site mutations in intron 1 did not activate cryptic splice sites in the coding sequences and instead, they activated specific cryptic splice sites upstream b globin gene. Q-PCR with chimeric primers for the exact quantitation of the splice variants showed that the splice site mutations also cause significant reduction (> 90%) in expression of normally spliced transcripts suggesting that nonsense mediated decay is not the major mechanism for the reduced β globin production by these mutations. We found high levels of one of the aberrantly spliced transcripts with 5’ splice site in exon 1 (∼20%) in normal individuals. All the splice variants showed steady increase in their expression from early to late stages of erythropoiesis. Our study shows that ex-vivo erythropoiesis system is an invaluable tool for studying the mechanism of globin gene regulation and switching. Identification of specific splice variants in cErCs of normal individuals and in patients with β thalassaemia could define the molecular mechanisms by which splice site mutations cause β-thalassaemia. The presence of abundant splice variants in normal controls suggests that β globin gene is regulated by multiple promoters and other regulatory elements and identification of these sequences will help in understanding the molecular basis of human globin gene regulation.