IgG Immune Complexes Inhibit Naïve T Cell Proliferation and Suppress Effector Function in Cytotoxic T Cells.

Elevated levels of circulating immune complexes are associated with autoimmunity and with worse prognoses in cancer. Here, we examined the effects of well-defined, soluble immune complexes (ICs) on human peripheral T cells. We demonstrate that IgG-ICs inhibit the proliferation and differentiation of a subset of naïve T cells but stimulate the division of another naïve-like T cell subset. Phenotypic analysis by multi-parameter flow cytometry and RNA-Seq were used to characterize the inhibited and stimulated T cells revealing that the inhibited subset presented immature features resembling those of recent thymic emigrants and non-activated naïve T cells, whereas the stimulated subset exhibited transcriptional features indicative of a more differentiated, early memory progenitor with a naïve-like phenotype. Furthermore, we show that while IgG1-ICs do not profoundly inhibit the proliferation of memory T cells, IgG1-ICs suppress the production of granzyme-ß and perforin in cytotoxic memory T cells. Our findings reveal how ICs can link humoral immunity and T cell function.

ELF4 Is a Target of miR-124 and Promotes Neuroblastoma Proliferation and Undifferentiated State.

13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF-miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. IMPLICATIONS: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.

PRC2 activates interferon-stimulated genes indirectly by repressing miRNAs in glioblastoma.

Polycomb repressive complex 2 (PRC2) is a chromatin binding complex that represses gene expression by methylating histone H3 at K27 to establish repressed chromatin domains. PRC2 can either regulate genes directly through the methyltransferase activity of its component EZH2 or indirectly by regulating other gene regulators. Gene expression analysis of glioblastoma (GBM) cells lacking EZH2 showed that PRC2 regulates hundreds of interferon-stimulated genes (ISGs). We found that PRC2 directly represses several ISGs and also indirectly activates a distinct set of ISGs. Assessment of EZH2 binding proximal to miRNAs showed that PRC2 directly represses miRNAs encoded in the chromosome 14 imprinted DLK1-DIO3 locus. We found that repression of this locus by PRC2 occurs in immortalized GBM-derived cell lines as well as in primary bulk tumors from GBM and anaplastic astrocytoma patients. Through repression of these miRNAs and several other miRNAs, PRC2 activates a set of ISGs that are targeted by these miRNAs. This PRC2-miRNA-ISG network is likely to be important in regulating gene expression programs in GBM.

A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus.

Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes.IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.

MicroRNAs reinforce repression of PRC2 transcriptional targets independently and through a feed-forward regulatory network.

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, whereas microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) for the RNA-induced silencing complex (RISC) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that Polycomb repressive complex 2 (PRC2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNA-repressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes corepressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2.

Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies.

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Nonspecific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published data sets. We show that mispriming can occur with as little as two bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned data set. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published data sets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP, and GRO-seq. We further show how RT mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to completely avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

Bivalent Chromatin Domains in Glioblastoma Reveal a Subtype-Specific Signature of Glioma Stem Cells.

Glioblastoma multiforme (GBM) can be clustered by gene expression into four main subtypes associated with prognosis and survival, but enhancers and other gene-regulatory elements have not yet been identified in primary tumors. Here, we profiled six histone modifications and CTCF binding as well as gene expression in primary gliomas and identified chromatin states that define distinct regulatory elements across the tumor genome. Enhancers in mesenchymal and classical tumor subtypes drove gene expression associated with cell migration and invasion, whereas enhancers in proneural tumors controlled genes associated with a less aggressive phenotype in GBM. We identified bivalent domains marked by activating and repressive chromatin modifications. Interestingly, the gene interaction network from common (subtype-independent) bivalent domains was highly enriched for homeobox genes and transcription factors and dominated by SHH and Wnt signaling pathways. This subtype-independent signature of early neural development may be indicative of poised dedifferentiation capacity in glioblastoma and could provide potential targets for therapy.Significance: Enhancers and bivalent domains in glioblastoma are regulated in a subtype-specific manner that resembles gene regulation in glioma stem cells. Cancer Res; 78(10); 2463-74. ©2018 AACR.

Chd1 co-localizes with early transcription elongation factors independently of H3K36 methylation and releases stalled RNA polymerase II at introns.

BACKGROUND: Chromatin consists of ordered nucleosomal arrays that are controlled by highly conserved adenosine triphosphate (ATP)-dependent chromatin remodeling complexes. One such remodeler, chromodomain helicase DNA binding protein 1 (Chd1), is believed to play an integral role in nucleosomal organization, as the loss of Chd1 is known to disrupt chromatin. However, the specificity and basis for the functional and physical localization of Chd1 on chromatin remains largely unknown. RESULTS: Using genome-wide approaches, we found that the loss of Chd1 significantly disrupted nucleosome arrays within the gene bodies of highly transcribed genes. We also found that Chd1 is physically recruited to gene bodies, and that its occupancy specifically corresponds to that of the early elongating form of RNA polymerase, RNAPII Ser 5-P. Conversely, RNAPII Ser 5-P occupancy was affected by the loss of Chd1, suggesting that Chd1 is associated with early transcription elongation. Surprisingly, the occupancy of RNAPII Ser 5-P was affected by the loss of Chd1 specifically at intron-containing genes. Nucleosome turnover was also affected at these sites in the absence of Chd1. We also found that deletion of the histone methyltransferase for H3K36 (SET2) did not affect either Chd1 occupancy or nucleosome organization genome-wide. CONCLUSIONS: Chd1 is specifically recruited onto the gene bodies of highly transcribed genes in an elongation-dependent but H3K36me3-independent manner. Chd1 co-localizes with the early elongating form of RNA polymerase, and affects the occupancy of RNAPII only at genes containing introns, suggesting a role in relieving splicing-related pausing of RNAPII.