A Newly Identified Gene Ahed Plays Essential Roles in Murine Hematopoiesis through RNA Splicing

[Introduction]

Despite the enormous amount of information available due to the recent advances in DNA sequencing technologies, most genes remain elusive in terms of their biological functions. Given that the responsible gene mutations have not yet been determined in some patients with blood cancers, it is important to search for new essential genes in the development of the hematopoietic system. On the other hand, mammalian cells have diploid genes in the nucleus, and many genetic abnormalities are complemented by normal alleles, making it difficult to screen for recessive genes. To overcome this problem, we previously constructed a homozygous mutant embryonic stem cell (ESC) bank by introducing a mutation in one allele using a gene trap vector with transposon properties, followed by transient termination of the expression of the Bloom syndrome gene that suppresses homologous interchromosomal recombination.

[Methods]

In this study, we extend our method to screen for recessive genetic abnormalities involved in the development of hematopoietic cells, using the established method of in vitro hematopoietic differentiation by co-culture with OP9 stromal cells.

[Results]

To discover uncharacterized genes in the mutant ESC clones, we focused on 21 genes with uncharacterized functions in the hematopoietic system from the approximately 200 homozygous mutant clones in the bank. Of the homozygous mutant ESC clones tested, one clone was severely impaired in terms of differentiation potential toward hematopoietic cells. In this clone, the gene-trap vector was inserted in a previously uncharacterized gene in a biallelic manner. We named this gene “attenuated hematopoietic development (Ahed)”. According to the gene expression database, the expression level of Ahed was higher in tissues of hematopoietic lineages than in other tissues. To understand its specific roles in the hematopoietic system, we generated Ahed conditional knockout (cKO) mice by crossing Ahed-floxed mice with Vav1-cre transgenic mice. We found that Ahed cKO fetuses were normal in gross appearance until E12.5, however, they became anemic from E14.5, characterized by fetal liver (FL) atrophy, leading to prenatal demise. To determine the significance of Ahed in fetal hematopoiesis, we analyzed the E14.5 liver of Ahed cKO fetuses. Flow cytometric analysis revealed that the number of TER119⁺-erythroid cells was significantly decreased in Ahed cKO livers. By contrast, Ahed cKO fetal livers contained a substantial number of lineage⁻Sca1⁺c-kit^high (LSK) cells, representing hematopoietic stem/progenitor cells. We then assessed the hematopoietic function of these LSK cells. Transplantation experiments demonstrate the incapacity of Ahed-deficient hematopoietic cells to reconstitute hematopoiesis in vivo. Employing a tamoxifen-inducible cKO model in adult mice, Ahed cKO mice developed severe hematopoietic failure and all mice died within one week of induction, indicating that Ahed deletion impairs the intrinsic capacity of hematopoietic cells in adult bone marrow. RNA-sequencing analyze revealed that pan-hematopoietic Ahed deletion disrupts critical gene expression such as Gata2 and Runx1. Based on information from a comprehensive database of protein-protein interactions and computer analyses to infer function from protein structure, the Ahed protein was speculated to be involved in the regulation of RNA splicing. In fact, our study confirmed that Ahed protein are localized in the nuclei and mRNAs of genes involved in hematopoiesis including PU.1 are aberrantly spliced in Ahed-deficient cells. Furthermore, published databases present cancer patients with somatic mutations in Ahed human orthologue, c11orf57, especially those with hematologic malignancies such as acute myeloid leukemia.

[Conclusion] We report the first attempt of in vitro hematopoietic differentiation screening of mutant mouse ESC lines and the successful identification of a previously uncharacterized gene, Ahed, whose perturbation disrupts early hematopoiesis both in vitro and in vivo. Our findings underscore the fundamental roles of Ahed in lifelong hematopoiesis, implicating its association with malignancies. Although precise molecular machineries by which Ahed is involved in hematopoiesis have remained to be elucidated, further analyses of the molecular mechanism underlying their function might pave the way for novel cancer therapies.

Nakai:NIPPON SHINYAKU CO., LTD.: Research Funding.