Transcription factor competition at the ?-globin promoters controls hemoglobin switching [CUT&RUN]

BCL11A, the major regulator of HbF(a2?2) level, represses ?-globin expression through direct promoter binding in adult erythroid cells in a switch to adult adult-type HbA (a2ß2) production. Yet, the mechanism remains unclear. To uncover how BCL11A initiates repression, we used CRISPR/Cas9 and dCas9 screens to dissect the ?-globin promoters and identified an apparent activator element near the BCL11A binding region. Using CUT&RUN and base editing approaches, we demonstrate that this element, the proximal CCAAT box, is the binding site of transcription activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate ?-globin repression, and the distance between the two motifs is critical for direct competition. Occupancy of NF-Y is rapidly established upon BCL11A depletion, and precedes ?-globin derepression and LCR-globin loop formation. Our findings reveal that the critical fetal-to-adult hemoglobin switch is initiated by the competition between transcription factors within a discrete region in the ?-globin promoters. Overall design: Occupany of NFYA or TBP was assessed using CUT&RUN sequencing in erythroid progenitors with BCL11A KO or base editing of TF motifs.

BCL11A作为胎儿血红蛋白（HbF, α2γ2）水平的核心调控因子，在成人红系细胞向成人型血红蛋白HbA（α2β2）生成的转换过程中，可通过直接结合β-珠蛋白启动子来抑制其表达。然而，其具体分子机制仍未明确。为阐明BCL11A启动β-珠蛋白阻遏作用的机制，我们利用CRISPR/Cas9与失活CRISPR/Cas9（dCas9）筛选技术对β-珠蛋白启动子进行了解构分析，并在BCL11A结合区域附近发现了一个疑似转录激活元件。通过CUT&RUN（切割与释放测序）与碱基编辑技术，我们证实该元件——近端CCAAT盒——是转录激活因子NF-Y的结合位点。BCL11A通过空间位阻效应与NF-Y竞争结合该位点，从而启动β-珠蛋白的阻遏，且两个结合基序之间的距离对直接竞争至关重要。在BCL11A被敲除后，NF-Y的结合占有率会快速建立，且这一过程先于β-珠蛋白的去阻遏以及位点控制区（LCR）与β-珠蛋白基因的染色质环形成。我们的研究结果揭示，关键的胎儿-成人血红蛋白转换是由β-珠蛋白启动子内特定区域内的转录因子竞争所启动的。总体实验设计：我们利用CUT&RUN测序技术，在经BCL11A敲除（KO）或转录因子结合基序碱基编辑的红系前体细胞中，检测了NFYA或TBP的结合占有率。