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Abstract

Hematopoietic stem cells (HSCs) reside at the top of a tightly regulated and hierarchically organized differentiation cascade. They are multipotent and able to self-renew, thereby ensuring life-long replenishment of mature blood cells. In-depth Omics analyses within the hematopoietic hierarchy, including analysis of HSCs and multipotent progenitor (MPP) cells, revealed not only differential expression of protein-coding genes, but also HSC-specific splicing variants and expression of long non-coding RNAs (lncRNAs) upon HSC commitment (Cabezas-Wallscheid et al., 2014; Klimmeck et al., 2014; Luo et al., 2015). Functional analyses revealed that non-coding RNAs and regulation of RNA biogenesis are crucial for HSC function and potency. In this thesis, the role of the lncRNA maternally expressed gene 3 (Meg3) in hematopoiesis is studied. In addition, I introduce the RNA regulatory mechanism alternative polyadenylation (APA) as a mechanism controlling HSC/MPP biology in homeostasis and during replicative stress response by extensive in silico, in vitro and in vivo analyses. Furthermore, I generated an inducible knockout mouse model, targeting the prominent APA regulator poly(A) binding protein 1 (Pabpn1). Thereby, I aimed to dissect the general role of lncRNAs and RNA regulatory mechanisms in hematopoiesis.

Project 1: The role of the lncRNA Meg3 in HSCs The tumor suppressor lncRNA Meg3 is encoded in the imprinted Dlk1-Meg3 locus and expressed from the maternally inherited allele (Zhou et al., 2012). We and others found Meg3 to be highly and specifically expressed in the HSC compartment compared to MPP cells. In this thesis, I crossed an inducible Meg3 flox mouse model (Klibanski et al., unpublished) to MxCre mice, generating MxCre Meg3 mat flox/pat wt mice. Cre-induction deleted the maternal allele in the hematopoietic compartment and thereby completely abrogated the expression of Meg3 and its associated miRNA cluster in HSCs. Extensive in vivo and in vitro analyses of adult mice harboring a Meg3-deficient blood system surprisingly did not reveal any impairment of hematopoiesis or stem cell function. In addition, I performed serial transplantation assays to investigate the functional capacity of Meg3-deficient HSCs. Again, knockout cells did not exhibit altered blood contribution, even upon tertiary transplantation. Imprinting of the Dlk1- Meg3 locus has recently been reported to regulate fetal liver HSC function (Qian et al., 2016). To analyze effects of the hematopoiesis-specific Meg3 knockout in the developing embryo, we generated VavCre Meg3 mat flox/pat wt mice. Cre+ offspring were born and developed normally. In- depth analysis of adult animals revealed loss of Meg3 expression in HSCs, but again no hematopoietic impairments were detected. Next, I performed interferon-mediated stimulation in MxCre Meg3 mat flox/pat wt mice. During both activation and recovery phase, Meg3-deficient adult HSCs responded highly similar compared to controls. Taken together, my work shows that the highly expressed, imprinted lncRNA Meg3 is dispensable for the function of HSCs during adulthood and embryonic development. In the adult system, loss of Meg3 does not impair the performance in serial reconstitution assays or response to stress mediators. (Sommerkamp et al., 2019)

Project 2: HSC function, differentiation and activation are regulated by APA The majority of mammalian genes have multiple different polyadenylation sites. The RNA editing mechanism APA controls the selection of these sites, thereby altering 3’-UTR length and isoform expression. Thus, APA modulates RNA stability, localization, protein output and even protein localization (Di Giammartino et al., 2011; Tian and Manley, 2017). So far, the role of APA in the regulation of the adult HSC/MPP compartment has not been studied. In this thesis, I show that the APA regulator Pabpn1 is essential for HSCs, as knockdown (KD) of Pabpn1 led to decreased HSC function in vitro and in vivo. To analyze the prevalence of APA at the top of the hematopoietic hierarchy, I established an ultra-low input 3’-Seq approach and performed analysis of HSCs, MPP cells and HSCs activated by inflammation. Bioinformatic analysis revealed dynamic APA patterns in numerous genes between HSCs and MPPs as well as during inflammation-induced HSC stress response. We observed global 3’-UTR shortening both upon HSC differentiation towards MPPs and HSC activation. Further, 3’-Seq analysis of Pabpn1 KD cells revealed that PABPN1 regulates APA in the HSC/MPP compartment. We observed an APA-mediated glutaminase (Gls) isoform switch upon exit of HSCs from quiescence. Gls isoform switching led to enhanced relative expression of the highly active GLS GAC isoform and overall increased GLS protein levels. In line, small molecule-mediated inhibition of glutaminolysis in vitro enhanced HSC maintenance by limiting proliferation. I could show that Gls isoform switching leading to increased glutaminolysis is mediated by the APA regulator NUDT21 and in turn is required for proper HSC function. KD of Nudt21 led to inhibition of Gls isoform switching, impaired HSC function and a partial block in HSC differentiation. In summary, my results install differential employment of APA and associated glutamine metabolism adaptations as novel layers in the regulation of the HSC-controlled hematopoietic hierarchy. (Sommerkamp et al., under revision)

Project 3: Generation of Pabpn1flox mice To enable in-depth in vivo analysis of the role of APA in different tissues and disease settings in the future, we generated an inducible Pabpn1flox mouse model. Here, we used the Easi- CRISPR approach (Miura et al., 2018; Quadros et al., 2017) to generate transgenic animals. I performed extensive in vitro testing of various guide RNAs (gRNAs) to optimize recombination efficiency in vivo. Two different crRNA-tracrRNA:Cas9 complexes targeting upstream and downstream genomic regions, respectively, were injected into one of the pronuclei of zygotes together with a long single-stranded DNA (ssDNA) template. By extensive genotyping, I identified 3 of 17 offspring animals to be correctly targeted. Homozygous offspring mice were successfully bred and can be used in the future to determine functionality of the Pabpn1flox mouse model and to functionally asses the role of APA in different biological settings.