Rbm15 Modulates Notch-Induced Transcriptional Activation and Affects Myeloid Differentiation

Abstract

RBM15 is the fusion partner with MKL in the t(1;22) translocation of acute megakaryoblastic leukemia. To understand the role of the RBM15-MKL1 fusion protein in leukemia, we must understand the normal functions of RBM15 and MKL. Here, we show a role for Rbm15 in myelopoiesis. Rbm15 is expressed at highest levels in hematopoietic stem cells and at more moderate levels during myelopoiesis of murine cell lines and primary murine cells. Decreasing Rbm15 levels with RNA interference enhances differentiation of the 32DWT18 myeloid precursor cell line. Conversely, enforced expression of Rbm15 inhibits 32DWT18 differentiation. We show that Rbm15 alters Notch-induced HES1 promoter activity in a cell type-specific manner. Rbm15 inhibits Notch-induced HES1 transcription in nonhematopoietic cells but stimulates this activity in hematopoietic cell lines, including 32DWT18 and human erythroleukemia cells. Moreover, the N terminus of Rbm15 coimmunoprecipitates with RBPJ, a critical factor in Notch signaling, and the Rbm15 N terminus has a dominant negative effect, impairing activation of HES1 promoter activity by full-length-Rbm15. Thus, Rbm15 is differentially expressed during hematopoiesis and may act to inhibit myeloid differentiation in hematopoietic cells via a mechanism that is mediated by stimulation of Notch signaling via RBPJ.Acute megakaryoblastic leukemia (AML-M7, also referred to as AMKL) comprises approximately 10% of childhood AML, in which it frequently presents in infants with bone marrow fibrosis and progresses rapidly, with a median survival of 8 months. This phenotype is associated with the t(1;22)(p13; q13) translocation, which was first observed in several infants with AML-M7 (5) and subsequently confirmed by others to be associated almost exclusively with this type of AML (2, 30, 31, 34). The t(1;22) translocation has only very rarely been associated with the AML-M7 cases that occur in association with trisomy 21; in general, AML-M7 with trisomy 21 is nearly always associated with mutations in the GATA1 gene (14, 32). In t(1;22), the breakpoint on chromosome 1p13 is within a gene that has been variably named RBM15 for RNA-binding motif protein 15 and OTT (for one twenty-two translocation), and the breakpoint on chromosome 22 is within the MKL1 gene (also known as MAL or BSAC).The MKL1 gene product is a 4.5-kb transcript that is widely expressed in normal tissues (35) and encodes one of three members of the myocardin family. While these three members, i.e., MKL1, MKL2, and myocardin, are only 35% similar to one another at the protein level, they have several highly conserved domains, including RPEL repeats in the N terminus, a region with a B (basic amino acid) box and a glutamine-rich domain that is involved in binding to serum response factor, a leucine zipper-like domain that plays a role in homo-and heterodimerization, and a C-terminal transactivation domain. These proteins also have a SAP domain that, based on its homology to SAF-B, is predicted to associate with matrix attachment regions of transcrip...

Fusion of OTT to BSAC Results in Aberrant Up-regulation of Transcriptional Activity

“…4C). Consistent with a very recent study, in which ectopically expressed OTT localizes in the nucleus (26), endogenous OTT localized in the nucleus (Fig. 4A).…”

“…In addition, knockdown of OTT/RBM15 gene using RNA interference promotes myeloid differentiation (26), suggesting an inhibitory role for OTT in myeloid and megakaryocyte development. These results are consistent with our present study, in which OTT might act as a transcriptional repressor via interaction with HDAC3 (Fig.…”

“…Moreover, OTT interacted with HDAC3, but this interaction was abolished in OTT-BSAC. Given that OTT negatively regulates the myeloid and megakaryocyte expansion (26,27), the loss of suppressor function of OTT along with aberrant up-regulation of BSAC-dependent transactivation caused by the fusion may culminate in the development of leukemia. We and others have previously reported that BSAC activates the promoters containing CArG boxes through association with SRF (9 -11).…”

The Notch signaling pathway: Transcriptional regulation at Notch target genes

“…[85]. Interestingly, there is a SHARP-like protein in mouse and men, Rbm15/OTT1, that is implicated in acute megakaryoblastic leukemia [86,87] and has been shown to interact with RBP-J, although the RBP-J binding site of SHARP is not conserved in Rbm15/ OTT1 [88]. Rbm15/OTT1 deficient mice are characterized by a loss of peripheral B-cells and an increase in stem-myeloid-and megakaryocyte progenitors [89].…”

Canonical Notch signaling in the developing lung is required for determination of arterial smooth muscle cells and selection of Clara versus ciliated cell fate

“…It has been shown that zebrafish Jagged2 expression in presumptive MCCs induced activation of zebrafish Notch3 in neighboring cells, blocking MCC fate and driving the alternative transporting epithelial cell fate. In addition, a Hes1-related protein was involved (Ma et al, 2007). In Xenopus, ciliated cells express Delta ligands to activate Notch signaling (and Hes-related proteins), inhibiting the selection of ciliated cells by neighboring epidermal cells (Deblandre et al, 1999).…”