A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.

Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine, facilitating DNA demethylation and generating new epigenetic marks. Here we show that concomitant loss of Tet2 and Tet3 in mice at early B cell stage blocked the pro- to pre-B cell transition in the bone marrow, decreased Irf4 expression and impaired the germline transcription and rearrangement of the Igκ locus. Tet2/3-deficient pro-B cells showed increased CpG methylation at the Igκ 3’ and distal enhancers that was mimicked by depletion of E2A or PU.1, as well as a global decrease in chromatin accessibility at enhancers. Importantly, re-expression of the Tet2 catalytic domain in Tet2/3-deficient B cells resulted in demethylation of the Igκ enhancers and restored their chromatin accessibility. Our data suggest that TET proteins and lineage-specific transcription factors cooperate to influence chromatin accessibility and Igκ enhancer function by modulating the modification status of DNA.DOI: http://dx.doi.org/10.7554/eLife.18290.001

Pregnancy loss occurs in about 15% of clinically recognized pregnancies, and defective maternal-fetal immune tolerance contributes to more than 50% of these events. We found that signaling by the type I membrane protein T cell immunoglobulin and mucin-containing protein 3 (Tim-3) in natural killer (NK) cells had an essential protective role during early pregnancy. Tim-3 on peripheral NK (pNK) cells was transiently increased in abundance during the first trimester of pregnancy, which depended on interleukin-4 (IL-4)-signal transducer and activator of transcription 6 (STAT6) and progesterone signaling. Tim-3 pNK cells displayed immunosuppressive activities, including the production of anti-inflammatory cytokines and the induction of regulatory T cells (T) in a transforming growth factor-β1 (TGF-β1)-dependent manner. Tim-3 on pNK cells was stimulated by its ligand galectin-9 (Gal-9), leading to signaling by the kinases c-Jun N-terminal kinase (JNK) and AKT. In recurrent miscarriage (RM) patients, Tim-3 abundance on pNK cells was reduced and the immunosuppressive activity of Tim-3 pNK cells was impaired. Compared to Tim-3 pNK cells from donors with normal pregnancies, RM patient Tim-3 pNK cells exhibited changes in DNA accessibility in certain genetic loci, which were reversed by inhibiting accessible chromatin reader proteins. Furthermore, Tim-3 pNK cells, but not Tim-3 pNK cells, reduced fetal loss in abortion-prone and NK cell-deficient mice. Together, our findings reveal a critical role for Tim-3-Gal-9 signaling-mediated immunoregulation by pNK cells in maternal-fetal immune tolerance and suggest that Tim-3 abundance on pNK cells is a potential biomarker for RM diagnosis.

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Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3' untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3' UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

Intratumoral delivery of chemotherapeutic agents may permit the localization of drugs in tumors, decrease nonspecific targeting and increase efficacy.

Clinical staff should focus on critical time periods during CT, particularly during the third cycle, and provide additional support to patients to ensure that CT is delivered in an optimal fashion.