Susana Gonzalez

Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases

Dicer initiates RNA interference by generating small RNAs involved in various silencing pathways. Dicer participates in centromeric silencing, but its role in the epigenetic regulation of other chromatin domains has not been explored. Here we show that Dicer1 deficiency in Mus musculus leads to decreased DNA methylation, concomitant with increased telomere recombination and telomere elongation. These DNA-methylation defects correlate with decreased expression of Dnmt1, Dnmt3a and Dnmt3b DNA methyltransferases (Dnmts), and methylation levels can be recovered by their overexpression. We identify the retinoblastoma-like 2 protein (Rbl2) as responsible for decreased Dnmt expression in Dicer1-null cells, suggesting the existence of Dicer-dependent small RNAs that target Rbl2. We identify the miR-290 cluster as being downregulated in Dicer1-deficient cells and show that it silences Rbl2, thereby controlling Dnmt expression. These results identify a pathway by which miR-290 directly regulates Rbl2-dependent Dnmt expression, indirectly affecting telomere-length homeostasis.

Geriatric muscle stem cells switch reversible quiescence into senescence

Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16INK4a (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16INK4a silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16INK4a is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.

miR-181b negatively regulates activation-induced cytidine deaminase in B cells

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3′ untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

Oncogenic activity of Cdc6 through repression of the INK4/ARF locus

The INK4/ARF locus encodes three tumour suppressors (p15INK4b, ARF and p16INK4a) and is among the most frequently inactivated loci in human cancer. However, little is known about the mechanisms that govern the expression of this locus. Here we have identified a putative DNA replication origin at the INK4/ARF locus that assembles a multiprotein complex containing Cdc6, Orc2 and MCMs, and that coincides with a conserved noncoding DNA element (regulatory domain RDINK4/ARF). Targeted and localized RNA-interference-induced heterochromatinization of RDINK4/ARF results in transcriptional repression of the locus, revealing that RDINK4/ARF is a relevant transcriptional regulatory element. Cdc6 is overexpressed in human cancers, where it might have roles in addition to DNA replication. We have found that high levels of Cdc6 result in RDINK4/ARF-dependent transcriptional repression, recruitment of histone deacetylases and heterochromatinization of the INK4/ARF locus, and a concomitant decrease in the expression of the three tumour suppressors encoded by this locus. This mechanism is reminiscent of the silencing of the mating-type HM loci in yeast by replication factors. Consistent with its ability to repress the INK4/ARF locus, Cdc6 has cellular immortalization activity and neoplastic transformation capacity in cooperation with oncogenic Ras. Furthermore, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16INK4a. We conclude that aberrant expression of Cdc6 is oncogenic by directly repressing the INK4/ARF locus through the RDINK4/ARF element.

Ezh1 Is Required for Hematopoietic Stem Cell Maintenance and Prevents Senescence-like Cell Cycle Arrest

Polycomb group (PcG) proteins are key epigenetic regulators of hematopietic stem cell (HSC) fate. The PcG members Ezh2 and Ezh1 are important determinants of embryonic stem cell identity, and the transcript levels of these histone methyltransferases are inversely correlated during development. However, the role of Ezh1 in somatic stem cells is largely unknown. Here we show that Ezh1 maintains repopulating HSCs in a slow-cycling, undifferentiated state, protecting them from senescence. Ezh1 ablation induces significant loss of adult HSCs, with concomitant impairment of their self-renewal capacity due to a potent senescence response. Epigenomic and gene expression changes induced by Ezh1 deletion in senesced HSCs demonstrated that Ezh1-mediated PRC2 activity catalyzes monomethylation and dimethylation of H3K27. Deletion of Cdkn2a on the Ezh1 null background rescued HSC proliferation and survival. Our results suggest that Ezh1 is an important histone methyltransferase for HSC maintenance.

Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs

Small RNAs can guide chromatin remodeling in mammalian cells, but the mechanisms involved are poorly understood. Previous reports have shown a requirement for overlapping transcription and the involvement of Argonaute (Ago) proteins. Here, we use the Regulatory Domain (RD) of the INK4/ARF locus as an experimental platform susceptible to siRNA-guided chromatin remodeling to interrogate about the mechanisms involved. We show that siRNA-guided chromatin remodeling of RD requires overlapping transcription and targets the transcribed strand, and not to the template strand, supporting an RNA:RNA recognition mechanism between the small RNA and the nascent RNA transcript. We found that heterochromatin formation can be triggered both by perfectly-matched double-stranded RNAs, as well as, by imperfectly-matched double-stranded RNAs. The latter observation, together with the fact that promoters are often subjected to overlapping transcription, suggest that miRNAs could also be able to guide heterochromatin formation at promoters. We proof this possibility using showing that miRNAs miR17-5p and miR20a from the oncomiR cluster miR-17-92 can induce heterochromatic features in promoters that undergo overlapping transcription and possess sequence complementarity to the miRNA seed region. These results unveil a new level of gene regulation by miRNAs.

Ectopic expression of the histone methyltransferase Ezh2 in haematopoietic stem cells causes myeloproliferative disease

Recent evidence shows increased and decreased expression of Ezh2 in cancer, suggesting a dual role as an oncogene or tumour suppressor. To investigate the mechanism by which Ezh2-mediated H3K27 methylation leads to cancer, we generated conditional Ezh2 knock-in (Ezh2-KI) mice. Here we show that induced Ezh2 haematopoietic expression increases the number and proliferation of repopulating haematopoietic stem cells. Ezh2-KI mice develop myeloproliferative disorder, featuring excessive myeloid expansion in bone marrow and spleen, leukocytosis and splenomegaly. Competitive and serial transplantations demonstrate progressive myeloid commitment of Ezh2-KI haematopoietic stem cells. Transplanted self-renewing haematopoietic stem cells from Ezh2-KI mice induce myeloproliferative disorder, suggesting that the Ezh2 gain-of-function arises in the haematopoietic stem cell pool, and not at later stages of myelopoiesis. At the molecular level, Ezh2 regulates haematopoietic stem cell-specific genes such as Evi-1 and Ntrk3, aberrantly found in haematologic malignancies. These results demonstrate a stem cell-specific Ezh2 oncogenic role in myeloid disorders, and suggest possible therapeutic applications in Ezh2-related haematological malignancies.

miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal

Hematopoietic stem cells (HSCs) are defined by their exclusive capacity to both self-renew and to give rise to multipotent progenitors (MPPs) that in turn differentiate into the mature blood cell lineages. The tumor suppressor p53, in addition to its role in the regulation of the cell cycle, plays an importatn role in HSC self-renewal, although it has not fully resolved. Here we report that in super-p53 mice (sp53), which carry one extra gene dose of p53, the miR-33 is down-regulated in HSCs and highly expressed in MPPs. Transplantation assays of miR-33-transduced sp53 HSC results in a significant acquisition of repopulating capacity and a decrease of recipients survival. Moreover, high levels of miR-33 represses the endogenous level of p53 protein in murine embryonic fibroblasts (MEFs), leads both to neoplastic transformation and anchorage independent growth of MEFs, and displays a decrease of apoptotic response using tumor-derived cell lines. Accordingly, we demonstrate that miR-33-mediated down-regulation of p53 is dependent on the binding of miR-33 to two conserved motifs in the 3′UTR of p53. Together, these data show that the miR-33 modifies HSC repopulating efficiency of sp53 mice by impairing the p53 function. Defining the role of miR-33 in controlling the HSC self-renewal through p53 may lead to the prevention and treatment of hematopoietic disorders.

Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch

Genetic data indicate that abrogation of Notch-Rbpj or Wnt-β-catenin pathways results in the loss of the intestinal stem cells (ISCs). However, whether the effect of Notch is direct or due to the aberrant differentiation of the transit-amplifying cells into post-mitotic goblet cells is unknown. To address this issue, we have generated composite tamoxifen-inducible intestine-specific genetic mouse models and analyzed the expression of intestinal differentiation markers. Importantly, we found that activation of β-catenin partially rescues the differentiation phenotype of Rbpj deletion mutants, but not the loss of the ISC compartment. Moreover, we identified Bmi1, which is expressed in the ISC and progenitor compartments, as a gene that is co-regulated by Notch and β-catenin. Loss of Bmi1 resulted in reduced proliferation in the ISC compartment accompanied by p16INK4a and p19ARF (splice variants of Cdkn2a) accumulation, and increased differentiation to the post-mitotic goblet cell lineage that partially mimics Notch loss-of-function defects. Finally, we provide evidence that Bmi1 contributes to ISC self-renewal. Summary: The polycomb complex protein Bmi1 is regulated by Notch and is required to maintain stem cell function in the mouse intestine.