Ott1(Rbm15) Regulates Thpo Response In HSCs Through Epigenetic Modifications Directing Alternative Splicing Of C-Mpl

Thrombopoietin (Thpo), through its receptor c-Mpl, is essential for Hematopoietic Stem Cell (HSC) function and has a dose-dependent effect in which low concentrations promote quiescence and self-renewal in contrast to high Thpo concentrations which promote proliferation. Thpo production is largely stable in vivo, therefore it is unclear how this dual response is evoked physiologically. HSCs deleted for c-Mpl are unable to tolerate proliferative stress. Ott1(Rbm15), the 5’ fusion partner in t(1;22) acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence during proliferative stress, however the mechanism has not been elucidated. Total c-Mpl expression in Ott1-deleted HSCs does not significantly differ from wild type, however, the existence of a cross-species, conserved isoform, Mpl-TR, with dominant negative activity, suggests a potential mechanism for affecting c-Mpl signaling via alternative splicing. Ott1 is a spliceosome component, is implicated in RNA processing and possesses RNA Recognition Motifs, yet has not been linked with any known physiologic targets. Analysis of c-Mpl isoforms in HSC-containing Lin-Sca1+c-Kit+ fractions and fetal liver megakaryocytes showed a marked increase in the ratio of Mpl-TR transcript. Ott1-deleted HSC populations displayed reduced Stat5 phosphorylation in response to Thpo stimulation consistent with decreased Mpl signaling. Exogenous expression of Mpl-TR in wild type bone marrow dramatically reduced short and long term engraftment into irradiated recipients, confirming in vivo activity of Mpl-TR in HSCs. To determine whether Ott1 complexes with Mpl RNA, RNA-immunoprecipitation was performed using an HA-tagged Ott1 and revealed complex formation with Mpl RNA. Alternative splicing is frequently regulated through a co-transcriptional mechanism utilizing local epigenetic modifications including histone acetylation and H3K4me3 marks. Ott1 was previously shown to bind class I Histone deacetylases (Hdacs) and the histone H3K4 methyl-transferase (HMT), Setd1b. To establish whether Ott1 interacts with the c-Mpl gene, Chromatin-immunoprecipitation (ChIP) using HA-tagged Ott1 was performed and found binding within regions flanking the alternatively spliced exons. ChIP using anti-pan-acetyl-H4 in Ott1 knockout Lin- bone marrow showed increased histone acetylation in the region shown to bind Ott1 compared to wild type. Conversely, ChIP using anti-H3K4me3 in the Ott1 knockout showed decreased H3K4me3 at the site of Ott1 binding consistent with loss of Ott1-associated Hdac and HMT activity. To test the functional consequences on splicing, treatment of wild type cells with either a class I Hdac inhibitor or a HMT inhibitor was able to significantly increase the ratio of Mpl-TR isoform.

In summary, Ott1 regulates the production of the alternatively spliced c-Mpl isoform, Mpl-TR, and consequently Thpo response in HSCs. Mpl-TR expression impairs physiologic HSC function for long and short term engraftment. Ott1 complexes with c-Mpl RNA and chromatin adjacent to the exons alternatively spliced in the Mpl-TR isoform and regulates histone acetylation and methylation marks associated with splice decision. Therefore, Ott1-mediated alternative splicing of Mpl may provide a novel mechanism via chromatin modification for modulating HSC maintenance and proliferation in response to Thpo. Furthermore, the ability to control Mpl alternative splicing through epigenetic inhibitors opens unique possibilities for pharmacologically manipulating HSC function in vitro or in vivo.