SON Regulates GATA-2 Through Transcriptional Control of the Mir-23a∼27a∼24-2 Cluster

Abstract 110

While characterizing AML1-ETO domains important for leukemia development and identifying proteins interacting with these domains, we discovered SON as a novel AML1-ETO binding protein (PNAS, 2008, 105:17103). SON is a large, poorly characterized serine/arginine rich SR protein localized to nuclear speckles. SON has DNA, single stranded RNA, and double stranded RNA binding domains and long repeats of amino acids. Overexpression of a partial fragment of SON in a transformed cell line decreased the tumorigenic potential of the cell in nude mice and protected yeast from apoptosis. However, partially due to its large size, the SON protein has not been well characterized. Recently, we reported that SON plays an important role in RNA splicing of a specific set of cell cycle related genes: ones that possess weak splice sites (Molecular Cell, 2011, 42:185).

While SON is expressed ubiquitously, its expression level is noticeably higher in hematopoietic organs/tissues and blood cells compared to other tissues, suggesting important roles of SON in the hematopoietic system. To examine whether SON expression is regulated during hematopoietic differentiation, we measured relative mRNA level of mouse Son in different stages of hematopoiesis. Son mRNA level is higher in lineage marker negative (Lin-) bone marrow cells when compared to total bone marrow cells. Macrophages showed less expression of Son, suggesting that Son is down-regulated along the hematopoietic differentiation. We further sorted Lin- cells and measured the Son level in LSK (Lin-, Sca1+, cKit+), CMP (commom myeloid progenitors), GMP (granulocyte/monocyte progenitors) and MEP (megakaryocyte/erythroid progenitors) populations. LSK cells, which precede other progenitors, showed the highest level of Son. In addition, we confirmed that SON is down-regulated during TPA-induced monocytic differentiation of U937 myeloid cells. Taken together, SON is more abundantly expressed in immature hematopoietic cells and down-regulated during differentiation.

Since SON is differentially expressed during hematopoietic differentiation, we examined whether SON is involved in regulation of hematopoietic transcription factors that are key dictators of hematopoietic differentiation. Among the several transcription factors analyzed, we found that Gata-2 mRNA was consistently reduced by two different Son shRNAs in Lin- bone marrow cells. Down regulation of the GATA-2 mRNA level was further confirmed in human K562 leukemic cell line. More interestingly, while having 20∼40% reduction of mRNA level, the GATA-2 protein level is more remarkably down-regulated upon SON knockdown, resulting in 75∼90% reduction. These results indicate that upon SON knockdown, GATA-2 protein level is mainly regulated at the post-transcriptional steps.

Sequence analysis of the 3' UTR of the human GATA-2 gene predicted several candidates of targeting microRNAs. Among them, we confirmed that the mature form of miR-27a and miR-24 are up-regulated upon SON knockdown. Next, we tested the effect of over-expression of miR-27a and miR-24 on GATA-2 expression using a GATA-2 3' UTR-containing luciferase reporter construct and demonstrated that miR-27a indeed inhibits GATA-2 mRNA level. miR-27a is a member of the miR-23a∼27a∼24-2 cluster. RTqPCR showed that primary miRNA of the miR-23a∼27a∼24 cluster is upregulated upon SON knockdown. To test whether the increase of pri-miR of this cluster is due to promoter activation, we used a reporter construct containing the promoter sequence of the miR-23a∼27a∼24-2 cluster fused to the luciferase reporter. The expression of luciferase driven by this promoter is significantly elevated upon SON knockdown, suggesting that that SON functions to repress transcription of the miR-23a-27a-24-2 cluster, thereby relieving GATA-2 mRNA from targeting by miR-27a, and contributes to maintaining the GATA-2 protein level. Taken together, our results reveal a previously unidentified function of SON in microRNA transcription and controlling the GATA-2 protein level in hematopoietic cells.