Regulation of Fetal Hemoglobin in Erythroid Progenitor Cells

Abstract

Background: Using blood RNA sequencing data on 22,093 Icelanders, deCODE conducted a GWAS study on RNA-based blood fetal globin ratio (FGR). The strongest effect variant rs1281124736[T] (Effect = 1.73SD, P = 8.2 x 10^-25; chr19:4067023C>T) is a rare (Minor Allele Freq=0.05%) variant adjacent to the zinc-finger transcription factor ZBTB7A gene. Although the variant rs1281124736[T] is mapped in the UCSC browser upstream of the ZBTB7A gene, using data from the FANTOM5 database revealed a major erythroid-specific transcription start site, which, if used, the variant maps to the 5′UTR of ZBTB7A and creates a new translational start site generating a larger protein. ZBTB7A is a known repressor of fetal hemoglobin. High fetal hemoglobin (HbF) expression levels are the most important predictor for favorable clinical outcomes in sickle cell disease. Understanding the genetic regulators of HbF expression can reveal novel targets in sickle cell disease.The Aim: This study aimed to assess the effect of the rs1281124736[T] variant on the ZBTB7A's functions and structure and how this variant leads to an increase in fetal hemoglobin levels in an in vitro erythroid differentiation model, using blood-derived cells from heterozygous carriers of rs1281124736[T] and non-carriers. Methods: To evaluate if this larger protein is generated, I overexpressed this long transcript with and without N-terminal extension variant rs1281124736 (ZBTB7A-NTE) in HEK293T cells, I used the overexpression HEK293T lysate for ZBTB7A protein size and levels measurements through Western blot and Simple Western. I next generated an in vitro erythroid differentiation model by isolating CD34+ hematopoietic stem cells from frozen peripheral blood mononuclear cells (PBMCs) from 8 pairs of carriers and non-carriers. After isolating CD34+ cells, they were differentiated towards erythroid cells to analyze the effect of rs1281124736[T] variant in erythroid differentiation. I collected samples at three time points in the differentiation process (Days 7, 11 and 14) and quantified ZBTB7A protein levels by Simple Western and ZBTB7A RNA and adult and fetal globin gene RNA by qPCR. At all-time points, the differentiation process was monitored by FACS analysis. The results were organized in Microsoft Excel and analyzed in R-Studio. Results & Conclusion: From the over-expression of ZBTB7A in HEK293T cells, the cells expressed a transcript with ZBTB7A-NTE, a ZBTB7A protein with a larger molecular weight was measured. However, in erythroid differentiated cells from heterozygous carriers of variant rs1281124736, the protein with a larger molecular weight was not detected, but instead, the cells had lower ZBTB7A protein levels. This reduction in protein levels could not be explained by reduced ZBTB7A RNA expression and is thus more likely due to degradation of the ZBTB7A-NTE protein. The rs1281124736[T] variant clearly affected fetal hemoglobin levels in these cells. I detected a significant increase in fetal hemoglobin levels in carriers’ RNA samples compared to their matched non-carriers. These data indicate that the rs1281124736[T] variant raises HbF levels by decreasing ZBTB7A protein levels, leading to reduced repression of HbF expression.