Wendy Dubois

Regulation of microRNA Expression and Abundance during Lymphopoiesis

Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3 or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunitys microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.

Distinct Regulatory Mechanisms and Functions for p53-Activated and p53-Repressed DNA Damage Response Genes in Embryonic Stem Cells

p53 is critical in regulating the differentiation of ES and induced pluripotent stem (iPS) cells. Here, we report a whole-genome study of p53-mediated DNA damage signaling in mouse ES cells. Systems analyses reveal that binding of p53 at the promoter region significantly correlates with gene activation but not with repression. Unexpectedly, we identify a regulatory mode for p53-mediated repression through interfering with distal enhancer activity. Importantly, many ES cell-enriched core transcription factors are p53-repressed genes. Further analyses demonstrate that p53-repressed genes are functionally associated with ES/iPS cell status while p53-activated genes are linked to differentiation. p53-activated genes and -repressed genes also display distinguishable features of expression levels and epigenetic markers. Upon DNA damage, p53 regulates the self-renewal and pluripotency of ES cells. Together, these results support a model where, in response to DNA damage, p53 affects the status of ES cells through activating differentiation-associated genes and repressing ES cell-enriched genes.

Increased mammalian lifespan and a segmental and tissue-specific slowing of aging after genetic reduction of mTOR expression.

We analyzed aging parameters using a mechanistic target of rapamycin (mTOR) hypomorphic mouse model. Mice with two hypomorphic (mTOR(Δ/Δ)) alleles are viable but express mTOR at approximately 25% of wild-type levels. These animals demonstrate reduced mTORC1 and mTORC2 activity and exhibit an approximately 20% increase in median survival. While mTOR(Δ/Δ) mice are smaller than wild-type mice, these animals do not demonstrate any alterations in normalized food intake, glucose homeostasis, or metabolic rate. Consistent with their increased lifespan, mTOR(Δ/Δ) mice exhibited a reduction in a number of aging tissue biomarkers. Functional assessment suggested that, as mTOR(Δ/Δ) mice age, they exhibit a marked functional preservation in many, but not all, organ systems. Thus, in a mammalian model, while reducing mTOR expression markedly increases overall lifespan, it affects the age-dependent decline in tissue and organ function in a segmental fashion.

AID expression levels determine the extent of cMyc oncogenic translocations and the incidence of B cell tumor development

Immunoglobulin (Ig) isotype switching is a recombination event that changes the constant domain of antibody genes and is catalyzed by activation-induced cytidine deaminase (AID). Upon recruitment to Ig genes, AID deaminates cytidines at switch (S) recombination sites, leading to the formation of DNA breaks. In addition to their role in isotype switching, AID-induced lesions promote Igh-cMyc chromosomal translocations and tumor development. However, cMyc translocations are also present in lymphocytes from healthy humans and mice, and thus, it remains unclear whether AID directly contributes to the dynamics of B cell transformation. Using a plasmacytoma mouse model, we show that AID+/− mice have reduced AID expression levels and display haploinsufficiency both in the context of isotype switching and plasmacytomagenesis. At the Ig loci, AID+/− lymphocytes show impaired intra- and inter-switch recombination, and a substantial decrease in the frequency of S mutations and chromosomal breaks. In AID+/− mice, these defects correlate with a marked decrease in the accumulation of B cell clones carrying Igh-cMyc translocations during tumor latency. These results thus provide a causality link between the extent of AID enzymatic activity, the number of emerging Igh-cMyc–translocated cells, and the incidence of B cell transformation.

Constitutive reductions in mTOR alter cell size, immune cell development, and antibody production

Mammalian TOR (mTOR) regulates cell growth, proliferation, and migration. Because mTOR knock-outs are embryonic lethal, we generated a viable hypomorphic mouse by neo-insertion that partially disrupts mTOR transcription and creates a potential physiologic model of mTORC1/TORC2 inhibition. Homozygous knock-in mice exhibited reductions in body, organ, and cell size. Although reductions in most organ sizes were proportional to decreased body weight, spleens were disproportionately smaller. Decreases in the total number of T cells, particularly memory cells, and reduced responses to chemokines suggested alterations in T-cell homing/homeostasis. T-cell receptor-stimulated T cells proliferated less, produced lower cytokine levels, and expressed FoxP3. Decreased neutrophil numbers were also observed in the spleen, despite normal development and migration in the bone marrow. However, B-cell effects were most pronounced, with a partial block in B-cell development in the bone marrow, altered splenic populations, and decreases in proliferation, antibody production, and migration to chemokines. Moreover, increased AKT(Ser473) phosphorylation was observed in activated B cells, reminiscent of cancers treated with rapamycin, and was reduced by a DNA-pk inhibitor. Thus, mTOR is required for the maturation and differentiation of multiple immune cell lineages. These mice provide a novel platform for studying the consequences of constitutively reduced mTORC1/TORC2 activity.

Efficiency alleles of the Pctr1 modifier locus for plasmacytoma susceptibility.

ABSTRACT The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that includedCdkn2a, which encodes p16INK4a and p19ARF, and the coding sequences for the BALB/c p16INK4a and p19ARF alleles were found to be polymorphic with respect to their resistant Pctr1counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, andD4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowingPctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2agene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16INK4a and p19ARF alleles from BALB/c and DBA/2 indicated that the BALB/c p16INK4a allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19ARF alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus,Pctr1, is an “efficiency” allele of the p16INK4a gene.

AID-deficient Bcl-xL transgenic mice develop delayed atypical plasma cell tumors with unusual Ig/Myc chromosomal rearrangements

Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) class switch recombination and somatic hypermutation, and has also been implicated in translocations between Ig switch regions and c-Myc in plasma cell tumors in mice. We asked if AID is required for accelerated tumor development in pristane-treated Bcl-xL transgenic BALB/c mice deficient in AID (pBxAicda−/−). pBxAicda −/− mice developed tumors with a lower frequency (24 vs. 62%) and a longer mean latency (108 vs. 36 d) than AID-sufficient mice. The tumors appeared in oil granuloma tissue and did not form ascites. By interphase fluorescence in situ hybridization, six out of nine pBxAicda −/− primary tumors had T(12;15) and one had T(6;15) chromosomal translocations. Two tumors were transplantable and established as stable cell lines. Molecular and cytogenetic analyses showed that one had an unusual unbalanced T(12;15) translocation, with IgH Cμ and Pvt-1 oriented head to tail at the breakpoint, resulting in an elevated expression of c-Myc. In contrast, the second was T(12;15) negative, but had an elevated N-Myc expression caused by a paracentric inversion of chromosome 12. Thus, novel mechanisms juxtapose Ig and Myc-family genes in AID-deficient plasma cell tumors.

mTORC1 and mTORC2 differentially regulate homeostasis of neoplastic and non-neoplastic human mast cells

Increased mast cell burden is observed in the inflamed tissues and affected organs and tissues of patients with mast cell proliferative disorders. However, normal mast cells participate in host defense, so approaches to preferentially target clonally expanding mast cells are needed. We found that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) are up-regulated in neoplastic and developing immature mast cells compared with their terminally differentiated counterparts. Elevated mTOR mRNA was also observed in bone marrow mononuclear cells of patients exhibiting mast-cell hyperplasia. Selective inhibition of mTORC1 and mTORC2 through genetic and pharmacologic manipulation revealed that, whereas mTORC1 may contribute to mast-cell survival, mTORC2 was only critical for homeostasis of neoplastic and dividing immature mast cells. The cytostatic effect of mTORC2 down-regulation in proliferating mast cells was determined to be via inhibition of cell-cycle progression. Because mTORC2 was observed to play little role in the homeostasis of differentiated, nonproliferating, mature mast cells, these data provide a rationale for adopting a targeted approaching selectively inhibiting mTORC2 to effectively reduce the proliferation of mast cells associated with inflammation and disorders of mast cell proliferation while leaving normal differentiated mast cells largely unaffected.

IL-6 and MYC collaborate in plasma cell tumor formation in mice

Interleukin-6 (IL-6) plays a critical role in the natural history of human plasma cell neoplasms (PCNs), such as plasma cell myeloma and plasmacytoma (PCT). IL-6 is also at the center of neoplastic plasma cell transformation in BALB/c (C) mice carrying a transgene, H2-L(d)-IL6, that encodes human IL-6 under control of the major histocompatibility complex H2-L(d) promoter: strain C.H2-L(d)-IL6. These mice are prone to PCT, but tumor development is incomplete with long latencies ( approximately 40% PCT at 12 months of age). To generate a more robust mouse model of IL-6-dependent PCN, we intercrossed strain C.H2-L(d)-IL6 with strains C.iMyc(Emu) or C.iMyc(Calpha), 2 interrelated gene-insertion models of the chromosomal T(12;15) translocation causing deregulated expression of Myc in mouse PCT. Deregulation of MYC is also a prominent feature of human PCN. We found that double-transgenic C.H2-L(d)-IL6/iMyc(Emu) and C.H2-L(d)-IL6/iMyc(Calpha) mice develop PCT with full penetrance (100% tumor incidence) and short latencies (3-6 months). The mouse tumors mimic molecular hallmarks of their human tumor counterparts, including elevated IL-6/Stat3/Bcl-X(L) signaling. The newly developed mouse strains may provide a good preclinical research tool for the design and testing of new approaches to target IL-6 in treatment and prevention of human PCNs.

Rap2b, a novel p53 target, regulates p53-mediated pro-survival function.

The tumor suppressor p53 is a critical regulator of apoptosis and cell cycle arrest/pro-survival. Upon DNA damage, p53 evokes both cell cycle arrest/pro-survival and apoptosis transcriptional programs. The ultimate cellular outcome depends on the balance of these two programs. However, the p53 downstream targets that mediate this cell fate decision remain to be identified. Using an integrative genomic approach, we identify Rap2b as a conserved p53-activated gene that counters p53-mediated apoptosis after DNA damage. Upon DNA damage, p53 directly binds to the promoter of Rap2b and activates its transcription. The reduction of Rap2b levels by small interference RNA sensitizes cells to DNA damage-induced apoptosis in a p53-dependent manner. Consistent with its pro-survival function, analysis of cancer genomic data reveals that Rap2b is overexpressed in many types of tumors. Anchorage-independent growth assays show that Rap2b has only weak transformation activity, suggesting that it is not an oncogene by itself. Together, our results identify Rap2b as a new player in the pro-survival program conducted by p53 and raise the possibility that targeting Rap2b could sensitize tumor cells to apoptosis in response to DNA damage.