MicroRNAs That Modulate Hematopoiesis Differentiation

MicroRNAs (miRNAs) are ~22-nt non-coding RNAs that can play important roles in development by targeting the messages of protein-coding genes for cleavage or repression of productive translation. Examples include lin-4 and let-7 miRNAs that control the timing of C. elegans larval development. As shown by the Bartel laboratory and others, humans have between 250 and 300 genes that encode miRNAs, an abundance corresponding to almost one percent of protein-coding genes. Based on the evolutionary conservation of many miRNAs among different animal lineages, it is reasonable to suspect that some mammalian miRNAs might also have important functions during development.

As a first step towards testing the idea that miRNAs might play roles in mammalian development, and more specifically hematopoiesis, Chang-Zheng Chen, in collaboration with Prof. David Bartel, cloned about 100 unique miRNAs from mouse bone marrow. Three, miR-181, miR-223, and miR-142s, were exclusively or preferentially expressed in hematopoietic tissues. miR-181 was very strongly expressed in thymus, the primary lymphoid organ, which mainly contains T-lymphocytes. Mature miR-181 expression in the bone marrow cells was detectable in undifferentiated Lin- progenitor cells and up-regulated in differentiated B-lymphocytes, marked by the B220 surface antigen. In other differentiated lineages, miR-181 expression did not increase over that seen in Lin- cells. Using retrovirus vectors he developed, Chang- Zheng ectopically expressed miR-181 in a population of bone marrow hematopoietic stem and progenitor cells. This led to an increased fraction of B-lineage cells both in tissue-culture differentiation assays and in transplanted adult mice; there was a corresponding decrease in CD-8+ T cells. Expression of miR-142s, in contrast, was most abundant in cells of the granulocyte and macrophage lineages. Overexpression of miR-142s in hematopoietic stem and progenitor cells led to an increase in the numbers of granulocytes and macrophages and a decrease in numbers of both mature CD-8+ and CD-4+ T cells. These results indicate that microRNAs are components of the molecular circuitry controlling mouse hematopoiesis and suggest that other microRNAs have similar regulatory roles during other facets of vertebrate development. Current projects aim to uncover the mRNAs downregulated by miR-181 and miR-142s, and much of this work is being done in Chang-Zheng’s new laboratory at the Stanford University School of Medicine.

Beiyan Zhou has used microRNA microarrays developed in the Bartel laboratory to identify several miRNAs specifically upregulated in populations of thymic and splenic hematopoietic cells she isolated: B cells, immature CD-4- CD-8- and CD-4+ CD-8+ T cells, as well as in more mature thymic CD-4- CD-8+ and CD-4+ CD-8- T cells. She is confirming these results by Northern blotting. Overexpression of these miRNAs in cell lines and animals, as well as “knocking down” their expression in cultured cells, should shed light on the roles of these miRNAs in B- and T- cell development as well as in hematopoiesis more broadly.

Prakash Rao has identified three evolutionarily conserved muscle- specific several miRNAs that are upregulated during differentiation of cultured C2C12 myoblasts into differentiated myotubes. Prakash and Mina Farkhondeh, a UROP student, are collaborating with members of the Bartel laboratory to determine the direct targets of these miRNAs. They are also using several new technologies in an attempt to “knock down” expression of these miRNAs and thus learn more about their specific roles in muscle differentiation. Prakash is also working with Guangtao Ge, a former lab member, to predict the sequences upstream of these muscle- specific miRNA genes that regulate their transcription. They have identified several such sites and they are currently using multiple experimental approaches to determine whether these indeed regulate miRNA expression.

I-hung Shih, a postdoctoral fellow in the Bartel lab who has been working closely with us, has identified several micro RNAs that are upregulated during differentiation in culture of 3T3- L1 preadipocytes to adipocytes. I-hung is currently examining the effects on adipocyte differentiation of overexpressing these micro RNAs in preadipocytes, and knocking down their expression during adipogenesis. Using computational and experimental techniques developed in the Bartel lab she is trying to determine the mRNA target(s) of these miRNAs.