Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Core Facility and Technical Service Center, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China.
Int J Mol Sci. 2022 Mar 30;23(7):3833. doi: 10.3390/ijms23073833.
Gene expression is tightly regulated during hematopoiesis. Recent studies have suggested that RNA polymerase II (Pol II) promoter proximal pausing, a temporary stalling downstream of the promoter region after initiation, plays a critical role in regulating the expression of various genes in metazoans. However, the function of proximal pausing in hematopoietic gene regulation remains largely unknown. The negative elongation factor (NELF) complex is a key factor important for this proximal pausing. Previous studies have suggested that NELF regulates granulocytic differentiation in vitro, but its in vivo function during hematopoiesis remains uncharacterized. Here, we generated the zebrafish mutant for one NELF complex subunit Nelfb using the CRISPR-Cas9 technology. We found that the loss of selectively induced excessive granulocytic development during primitive and definitive hematopoiesis. The loss of reduced hematopoietic progenitor cell formation and did not affect erythroid development. Moreover, the accelerated granulocytic differentiation and reduced progenitor cell development could be reversed by inhibiting Pol II elongation. Further experiments demonstrated that the other NELF complex subunits (Nelfa and Nelfe) played similar roles in controlling granulocytic development. Together, our studies suggested that NELF is critical in controlling the proper granulocytic development in vivo, and that promoter proximal pausing might help maintain the undifferentiated state of hematopoietic progenitor cells.
基因表达在造血过程中受到严格调控。最近的研究表明，RNA 聚合酶 II（Pol II）启动子近端暂停，即在起始后启动子区域下游的暂时停顿，在调节后生动物各种基因的表达中起着关键作用。然而，近端暂停在造血基因调控中的功能在很大程度上仍然未知。负延伸因子（NELF）复合物是该近端暂停的关键因素。先前的研究表明，NELF 调节体外粒细胞分化，但它在造血过程中的体内功能尚未确定。在这里，我们使用 CRISPR-Cas9 技术生成了一个 NELF 复合物亚基 Nelfb 的斑马鱼突变体。我们发现，选择性缺失 导致原始和定型造血过程中过度的粒细胞发育。 缺失减少了造血祖细胞的形成，但不影响红细胞发育。此外，通过抑制 Pol II 延伸可以逆转加速的粒细胞分化和减少祖细胞的发育。进一步的实验表明，其他 NELF 复合物亚基（Nelfa 和 Nelfe）在控制粒细胞发育中也发挥类似的作用。总之，我们的研究表明，NELF 对于控制体内适当的粒细胞发育至关重要，而启动子近端暂停可能有助于维持造血祖细胞的未分化状态。
