Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion.

Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that hepatocyte growth factor, an activator of MET (an RTK), promotes Rab17- and clathrin-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2's cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence.

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP.

A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

CCCTC-Binding Factor Locks Premature IgH Germline Transcription and Restrains Class Switch Recombination.

In response to antigenic stimulation B cells undergo class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) to replace the primary IgM/IgD isotypes by IgG, IgE, or IgA. CSR is initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues at the switch (S) regions of IgH. B cell stimulation promotes germline transcription (GLT) of specific S regions, a necessary event prior to CSR because it facilitates AID access to S regions. Here, we show that CCCTC-binding factor (CTCF)-deficient mice are severely impaired in the generation of germinal center B cells and plasma cells after immunization in vivo, most likely due to impaired cell survival. Importantly, we find that CTCF-deficient B cells have an increased rate of CSR under various stimulation conditions in vitro. This effect is not secondary to altered cell proliferation or AID expression in CTCF-deficient cells. Instead, we find that CTCF-deficient B cells harbor an increased mutation frequency at switch regions, probably reflecting an increased accessibility of AID to IgH in the absence of CTCF. Moreover, CTCF deficiency triggers premature GLT of S regions in naïve B cells. Our results indicate that CTCF restricts CSR by enforcing GLT silencing and limiting AID access to IgH.

Activation-induced cytidine deaminase targets SUV4-20-mediated histone H4K20 trimethylation to class-switch recombination sites.

Activation-induced cytidine deaminase (AID) triggers antibody diversification in B cells by catalysing deamination and subsequently mutating immunoglobulin (Ig) genes. Association of AID with RNA Pol II and occurrence of epigenetic changes during Ig gene diversification suggest participation of AID in epigenetic regulation. AID is mutated in hyper-IgM type 2 (HIGM2) syndrome. Here, we investigated the potential role of AID in the acquisition of epigenetic changes. We discovered that AID binding to the IgH locus promotes an increase in H4K20me3. In 293F cells, we demonstrate interaction between co-transfected AID and the three SUV4-20 histone H4K20 methyltransferases, and that SUV4-20H1.2, bound to the IgH switch (S) mu site, is replaced by SUV4-20H2 upon AID binding. Analysis of HIGM2 mutants shows that the AID truncated form W68X is impaired to interact with SUV4-20H1.2 and SUV4-20H2 and is unable to bind and target H4K20me3 to the Smu site. We finally show in mouse primary B cells undergoing class-switch recombination (CSR) that AID deficiency associates with decreased H4K20me3 levels at the Smu site. Our results provide a novel link between SUV4-20 enzymes and CSR and offer a new aspect of the interplay between AID and histone modifications in setting the epigenetic status of CSR sites.

CTCF orchestrates the germinal centre transcriptional program and prevents premature plasma cell differentiation.

In germinal centres (GC) mature B cells undergo intense proliferation and immunoglobulin gene modification before they differentiate into memory B cells or long-lived plasma cells (PC). GC B-cell-to-PC transition involves a major transcriptional switch that promotes a halt in cell proliferation and the production of secreted immunoglobulins. Here we show that the CCCTC-binding factor (CTCF) is required for the GC reaction in vivo, whereas in vitro the requirement for CTCF is not universal and instead depends on the pathways used for B-cell activation. CTCF maintains the GC transcriptional programme, allows a high proliferation rate, and represses the expression of Blimp-1, the master regulator of PC differentiation. Restoration of Blimp-1 levels partially rescues the proliferation defect of CTCF-deficient B cells. Thus, our data reveal an essential function of CTCF in maintaining the GC transcriptional programme and preventing premature PC differentiation.

3' Uridylation controls mature microRNA turnover during CD4 T-cell activation.

Activation of T lymphocytes requires a tight regulation of microRNA (miRNA) expression. Terminal uridyltransferases (TUTases) catalyze 3' nontemplated nucleotide addition (3'NTA) to miRNAs, which may influence miRNA stability and function. Here, we investigated 3'NTA to mature miRNA in CD4 T lymphocytes by deep sequencing. Upon T-cell activation, miRNA sequences bearing terminal uridines are specifically decreased, concomitantly with down-regulation of TUT4 and TUT7 enzymes. Analyzing TUT4-deficient T lymphocytes, we proved that this terminal uridyltransferase is essential for the maintenance of miRNA uridylation in the steady state of T lymphocytes. Analysis of synthetic uridylated miRNAs shows that 3' addition of uridine promotes degradation of these uridylated miRNAs after T-cell activation. Our data underline post-transcriptional uridylation as a mechanism to fine-tune miRNA levels during T-cell activation.

AID-expressing epithelium is protected from oncogenic transformation by an NKG2D surveillance pathway.

Activation-induced deaminase (AID) initiates secondary antibody diversification in germinal center B cells, giving rise to higher affinity antibodies through somatic hypermutation (SHM) or to isotype-switched antibodies through class switch recombination (CSR). SHM and CSR are triggered by AID-mediated deamination of cytosines in immunoglobulin genes. Importantly, AID activity in B cells is not restricted to Ig loci and can promote mutations and pro-lymphomagenic translocations, establishing a direct oncogenic mechanism for germinal center-derived neoplasias. AID is also expressed in response to inflammatory cues in epithelial cells, raising the possibility that AID mutagenic activity might drive carcinoma development. We directly tested this hypothesis by generating conditional knock-in mouse models for AID overexpression in colon and pancreas epithelium. AID overexpression alone was not sufficient to promote epithelial cell neoplasia in these tissues, in spite of displaying mutagenic and genotoxic activity. Instead, we found that heterologous AID expression in pancreas promotes the expression of NKG2D ligands, the recruitment of CD8(+) T cells, and the induction of epithelial cell death. Our results indicate that AID oncogenic potential in epithelial cells can be neutralized by immunosurveillance protective mechanisms.

Cancer-initiating enriched cell lines from human glioblastoma: preparing for drug discovery assays.

Glioblastoma multiforme (GBM) is the most lethal type of brain tumour in the adult humans. The cancer-initiating cell (CIC) hypothesis supports the notion that failures in current approaches to GBM treatment might be attributed to the survival of the CIC subpopulation. Recent evidence shows the idea that using CIC-enriched cell lines derived from human GBM as new targets for drug discovery programs, may improve the chance of successfully translating the basic research findings into clinical trials. Although this approach appears promising, many important biological and technical issues (characterization of functional CIC markers, inter- and intra-tumoral CIC heterogeneity, and isolation and maintenance inconsistency) need to be resolved.

Genomic instability of surgical sample and cancer-initiating cell lines from human glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in the adult human, with an average survival of 16 months. A small population of cells within the GBM termed cancer-initiating cells is responsible for the initiation and maintenance of the tumor mass. The traditional glioblastoma cancer cells, grown with serum containing media, display increased rate of genomic instability events, which in turn renders the cell cultures with little resembling to the original tumor, making doubtful their use as preclinical models for screening therapeutic agents. On the contrary, the cancer-initiating cells grown in serum-free media seems to show lower rate of genomic instability processes. However, considering the diversity of genetic and/or epigenetic background, we will need to evaluate the possibility of using different culture conditions to allow for the isolation and culture of such cancer-initiating cells diversity, keeping, at the same time, the genomic instability rate as the original tumor. We summarized the main genetic and epigenetic mechanisms that are driving genomic instability in cancer-initiating cells from human glioblastoma.