A novel role for Lyl1 in primitive erythropoiesis

Stem Cell Leukemia (Scl or Tal1) and Lymphoblastic Leukemia 1 (Lyl1) are highly related members of the basic helix-loop-helix (bHLH) family of transcription factors that are co- expressed in hematopoietic stem cells and the erythro-megakaryocytic lineages. Previous studies suggest that Scl is essential for hematopoietic development including primitive erythropoiesis. However, analysis of single-cell RNA-sequencing data of early embryos showed that primitive erythroid cells express both Scl and Lyl1. Therefore, to determine whether Lyl1 has a functional role in erythropoiesis, we crossed conditional Scl mice with transgenic mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Surprisingly, embryos with markedly reduced expression of Scl from E9.5 survived to adulthood. In contrast, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 due to progressive loss of erythropoiesis. Consistent with a phenocopy of Gata1-null mice, gene expression profiling of DKO yolk sacs prior to the loss of erythrocytes (E9.5) revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Scl and Lyl1 share functional roles in primitive erythropoiesis. RNA-Seq analysis of WT, LYL1 KO, SCL KO, LYL1 SCL DKO e9.5 embryos干细胞白血病因子（Stem Cell Leukemia，简称Scl或Tal1）与淋巴母细胞白血病因子1（Lymphoblastic Leukemia 1，简称Lyl1）是碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）转录因子家族中亲缘关系极为密切的成员，二者在造血干细胞与红系-巨核系谱系中共表达。既往研究表明，Scl对于包括原始红细胞生成在内的造血发育过程至关重要。然而，对早期胚胎的单细胞RNA测序（single-cell RNA-sequencing）数据分析显示，原始红细胞同时表达Scl与Lyl1。因此，为明确Lyl1在红细胞生成中是否发挥功能作用，我们将条件性Scl小鼠与在红系祖细胞中具有活性、受促红细胞生成素受体（Epo receptor）调控表达Cre重组酶的转基因小鼠进行杂交。令人意外的是，自胚胎发育第9.5天（E9.5）起Scl表达显著降低的胚胎可存活至成年。与之相反，Scl表达降低且Lyl1完全缺失（双基因敲除；DKO）的小鼠于胚胎发育第10.5天（E10.5）死亡，死因是红细胞生成进行性丧失。与Gata1基因敲除小鼠的表型模拟结果一致，在红细胞丢失前（E9.5）对DKO小鼠卵黄囊开展基因表达谱分析，发现Gata1以及SCL-GATA1复合物的诸多已知靶基因均出现表达缺失。染色质免疫共沉淀测序（ChIP-seq）分析显示，LYL1仅结合SCL靶基因中的一小部分，其中包括GATA1。综上，本研究首次证实Scl与Lyl1在原始红细胞生成过程中具有共同的功能作用。本研究针对野生型（WT）、LYL1敲除（LYL1 KO）、SCL敲除（SCL KO）以及LYL1与SCL双敲除（DKO）的E9.5胚胎开展了RNA测序（RNA-Seq）分析。