Identification of a genomic DNA sequence that quantitatively modulates KLF1 transcription factor expression in differentiating human hematopoietic cells

The onset of erythropoiesis is under strict developmental control, with direct and indirect inputs influencing its derivation from the hematopoietic stem cell. A major regulator of this transition is KLF1/EKLF, a zinc finger transcription factor that plays a global role in all aspects of erythropoiesis. Here, we have identified a short, conserved enhancer element in KLF1 intron 1 that is important for establishing optimal levels of KLF1 in mouse and human cells. Chromatin accessibility of this site exhibits cell-type specificity and is under developmental control during the differentiation of human CD34+ cells towards the erythroid lineage. This site binds GATA1, SMAD1, TAL1, and ETV6. In vivo editing of this region in cell lines and primary cells reduces KLF1 expression quantitatively. However, we find that, similar to observations seen in pedigrees of families with KLF1 mutations, downstream effects are variable, suggesting that the global architecture of the site is buffered towards keeping the KLF1 genetic region in an active state. We propose that modification of intron 1 in both alleles is not equivalent to complete loss of function of one allele. Overall design: We performed RNA seq on triplicate samples derived from the larger dual guide-edited or control CD34+ cells isolated at the end of the differentiation protocol.

红细胞生成（erythropoiesis）的启动受到严格的发育调控，其由造血干细胞（hematopoietic stem cell）分化产生的过程受直接与间接因素共同调控。调控这一转变的核心因子为KLF1/EKLF，一种在红细胞生成所有环节中发挥全局调控作用的锌指转录因子（zinc finger transcription factor）。本研究在KLF1内含子1中鉴定出一段短小且保守的增强子元件（enhancer element），该元件对维持小鼠与人类细胞中KLF1的最优表达水平至关重要。该位点的染色质可及性（chromatin accessibility）具有细胞类型特异性，并在人类CD34+细胞向红系谱系（erythroid lineage）分化过程中受发育调控。该位点可结合GATA1、SMAD1、TAL1及ETV6。在细胞系与原代细胞中对该区域进行体内编辑，可定量降低KLF1的表达水平。然而本研究发现，与携带KLF1突变的家族谱系中观察到的现象类似，其下游效应存在异质性，这提示该位点的全局结构存在缓冲机制，以维持KLF1基因区域处于激活状态。我们提出，对两个等位基因的内含子1进行修饰，并不等同于单等位基因功能完全丧失。总体实验设计：本研究对分化方案结束时分离得到的双向导编辑组与对照组CD34+细胞的三份重复样本开展了RNA测序（RNA-seq）。