DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications.

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency.

Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.

TET2 chemically modifies tRNAs and regulates tRNA fragment levels.

The ten-eleven translocation 2 (TET2) protein, which oxidizes 5-methylcytosine in DNA, can also bind RNA; however, the targets and function of TET2-RNA interactions in vivo are not fully understood. Using stringent affinity tags introduced at the Tet2 locus, we purified and sequenced TET2-crosslinked RNAs from mouse embryonic stem cells (mESCs) and found a high enrichment for tRNAs. RNA immunoprecipitation with an antibody against 5-hydroxymethylcytosine (hm5C) recovered tRNAs that overlapped with those bound to TET2 in cells. Mass spectrometry (MS) analyses revealed that TET2 is necessary and sufficient for the deposition of the hm5C modification on tRNA. Tet2 knockout in mESCs affected the levels of several small noncoding RNAs originating from TET2-bound tRNAs that were enriched by hm5C immunoprecipitation. Thus, our results suggest a new function of TET2 in promoting the conversion of 5-methylcytosine to hm5C on tRNA and regulating the processing or stability of different classes of tRNA fragments.

Mapping Native R-Loops Genome-wide Using a Targeted Nuclease Approach.

R-loops are three-stranded DNA:RNA hybrids that are implicated in many nuclear processes. While R-loops may have physiological roles, the formation of stable, aberrant R-loops has been observed in neurological disorders and cancers. Current methods to assess their genome-wide distribution rely on affinity purification, which is plagued by large input requirements, high noise, and poor sensitivity for dynamic R-loops. Here, we present MapR, a method that utilizes RNase H to guide micrococcal nuclease to R-loops, which are subsequently cleaved, released, and identified by sequencing. MapR detects R-loops formed at promoters and active enhancers that are likely to form transient R-loops due to the low transcriptional output of these regulatory elements and the short-lived nature of enhancer RNAs. MapR is as specific as existing techniques and more sensitive, allowing for genome-wide coverage with low input material in a fraction of the time.

Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2.

Kelch-like protein 6 (KLHL6) is an uncharacterized gene mutated in diffuse large B-cell lymphoma (DLBCL). Here we report that KLHL6 assembles with cullin3 to form a functional cullin-RING ubiquitin ligase. Mutations in KLHL6 inhibit its ligase activity by disrupting the interaction with cullin3. Loss of KLHL6 favours DLBCL growth and survival both in vitro and in xenograft models. We further established that the mRNA decay factor roquin2 is a substrate of KLHL6. Degradation of roquin2 is dependent on B-cell receptor activation, and requires the integrity of the Tyr691 residue in roquin2 that is essential for its interaction with KLHL6. A non-degradable roquin2(Y691F) mutant requires its RNA-binding ability to phenocopy the effect of KLHL6 loss. Stabilization of roquin2 promotes mRNA decay of the tumour suppressor and NF-κB pathway inhibitor, tumour necrosis factor-α-inducible gene 3. Collectively, our findings uncover the tumour suppressing mechanism of KLHL6.

Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH.

Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses.

The expanding epigenetic landscape of non-model organisms.

Epigenetics studies the emergence of different phenotypes from a single genotype. Although these processes are essential to cellular differentiation and transcriptional memory, they are also widely used in all branches of the tree of life by organisms that require plastic but stable adaptation to their physical and social environment. Because of the inherent flexibility of epigenetic regulation, a variety of biological phenomena can be traced back to evolutionary adaptations of few conserved molecular pathways that converge on chromatin. For these reasons chromatin biology and epigenetic research have a rich history of chasing discoveries in a variety of model organisms, including yeast, flies, plants and humans. Many more fascinating examples of epigenetic plasticity lie outside the realm of model organisms and have so far been only sporadically investigated at a molecular level; however, recent progress on sequencing technology and genome editing tools have begun to blur the lines between model and non-model organisms, opening numerous new avenues for investigation. Here, I review examples of epigenetic phenomena in non-model organisms that have emerged as potential experimental systems, including social insects, fish and flatworms, and are becoming accessible to molecular approaches.

In vivo proximity labeling for the detection of protein-protein and protein-RNA interactions.

Accurate and sensitive detection of protein-protein and protein-RNA interactions is key to understanding their biological functions. Traditional methods to identify these interactions require cell lysis and biochemical manipulations that exclude cellular compartments that cannot be solubilized under mild conditions. Here, we introduce an in vivo proximity labeling (IPL) technology that employs an affinity tag combined with a photoactivatable probe to label polypeptides and RNAs in the vicinity of a protein of interest in vivo. Using quantitative mass spectrometry and deep sequencing, we show that IPL correctly identifies known protein-protein and protein-RNA interactions in the nucleus of mammalian cells. Thus, IPL provides additional temporal and spatial information for the characterization of biological interactions in vivo.

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53.

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo.

The role of chromatin and epigenetics in the polyphenisms of ant castes.

Ants and other social insects offer a natural experimental system to investigate the molecular bases of epigenetic processes that influence the whole organism. Epigenetics is defined as the inheritance of biological variation independent of changes in the DNA sequence. As such, epigenetic research focuses on the mechanisms by which multiple phenotypes arise from a single genome. In social insects, whole individuals belong to alternative phenotypic classes (known as castes) that vary in morphology, behavior, reproductive biology and longevity. It has been proposed that the same epigenetic pathways that maintain different cell identities in vertebrates might determine the different phenotypes observed in social insects. Here, I review the current progress on investigating the role of classic epigenetic signals, such as DNA methylation and histone posttranslational modification, in the relatively unexplored paradigm of ant polyphenism.

SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors.

SFMBT1 (Scm [Sex comb on midleg] with four MBT [malignant brain tumor] domains 1) is a poorly characterized mammalian MBT domain-containing protein homologous to Drosophila SFMBT, a Polycomb group protein involved in epigenetic regulation of gene expression. Here, we show that SFMBT1 regulates transcription in somatic cells and during spermatogenesis through the formation of a stable complex with LSD1 and CoREST. When bound to its gene targets, SFMBT1 recruits its associated proteins and causes chromatin compaction and transcriptional repression. SFMBT1, LSD1, and CoREST share a large fraction of target genes, including those encoding replication-dependent histones. Simultaneous occupancy of histone genes by SFMBT1, LSD1, and CoREST is regulated during the cell cycle and correlates with the loss of RNA polymerase II at these promoters during G2, M, and G1. The interplay between the repressive SFMBT1-LSD1-CoREST complex and RNA polymerase II contributes to the timely transcriptional regulation of histone genes in human cells. SFMBT1, LSD1, and CoREST also form a stable complex in germ cells, and their chromatin binding activity is regulated during spermatogenesis.

A chromatin link to caste identity in the carpenter ant Camponotus floridanus.

In many ant species, sibling larvae follow alternative ontogenetic trajectories that generate striking variation in morphology and behavior among adults. These organism-level outcomes are often determined by environmental rather than genetic factors. Therefore, epigenetic mechanisms may mediate the expression of adult polyphenisms. We produced the first genome-wide maps of chromatin structure in a eusocial insect and found that gene-proximal changes in histone modifications, notably H3K27 acetylation, discriminate two female worker and male castes in Camponotus floridanus ants and partially explain differential gene expression between castes. Genes showing coordinated changes in H3K27ac and RNA implicate muscle development, neuronal regulation, and sensory responses in modulating caste identity. Binding sites of the acetyltransferase CBP harbor the greatest caste variation in H3K27ac, are enriched with motifs for conserved transcription factors, and show evolutionary expansion near developmental and neuronal genes. These results suggest that environmental effects on caste identity may be mediated by differential recruitment of CBP to chromatin. We propose that epigenetic mechanisms that modify chromatin structure may help orchestrate the generation and maintenance of polyphenic caste morphology and social behavior in ants.

PCGF homologs, CBX proteins, and RYBP define functionally distinct PRC1 family complexes.

The heterogeneous nature of mammalian PRC1 complexes has hindered our understanding of their biological functions. Here, we present a comprehensive proteomic and genomic analysis that uncovered six major groups of PRC1 complexes, each containing a distinct PCGF subunit, a RING1A/B ubiquitin ligase, and a unique set of associated polypeptides. These PRC1 complexes differ in their genomic localization, and only a small subset colocalize with H3K27me3. Further biochemical dissection revealed that the six PCGF-RING1A/B combinations form multiple complexes through association with RYBP or its homolog YAF2, which prevents the incorporation of other canonical PRC1 subunits, such as CBX, PHC, and SCM. Although both RYBP/YAF2- and CBX/PHC/SCM-containing complexes compact chromatin, only RYBP stimulates the activity of RING1B toward H2AK119ub1, suggesting a central role in PRC1 function. Knockdown of RYBP in embryonic stem cells compromised their ability to form embryoid bodies, likely because of defects in cell proliferation and maintenance of H2AK119ub1 levels.

Emerging topics in epigenetics: ants, brains, and noncoding RNAs.

One of the greatest wonders in biology is the high degree of molecular organization and complexity achieved by multicellular life forms, which are typically composed by hundreds of cell types, each with a unique identity and function and all sharing the same genome. Long-term maintenance of these distinct cell identities requires epigenetic signals, molecular signatures that regulate gene expression and can be inherited during cell division. Some epigenetic signals also appear to have an intimate connection with brain function, with important implications for neuroscience and medicine. To better understand these phenomena, new technologies must be developed and nonconventional model organisms should be studied. For example, the genomes of eusocial insects, such as ants and honeybees, specify drastically different morphologies (polyphenism) and behaviors (polyethism) that yield adult individuals belonging to different castes, which carry out separate functions inside the colony. These sharp epigenetic differences present unique opportunities for the experimental dissection of molecular pathways that may be conserved in other organisms, including humans.

Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator.

The organized societies of ants include short-lived worker castes displaying specialized behavior and morphology and long-lived queens dedicated to reproduction. We sequenced and compared the genomes of two socially divergent ant species: Camponotus floridanus and Harpegnathos saltator. Both genomes contained high amounts of CpG, despite the presence of DNA methylation, which in non-Hymenoptera correlates with CpG depletion. Comparison of gene expression in different castes identified up-regulation of telomerase and sirtuin deacetylases in longer-lived H. saltator reproductives, caste-specific expression of microRNAs and SMYD histone methyltransferases, and differential regulation of genes implicated in neuronal function and chemical communication. Our findings provide clues on the molecular differences between castes in these two ants and establish a new experimental model to study epigenetics in aging and behavior.

MBT domain proteins in development and disease.

The Malignant Brain Tumor (MBT) domain is a "chromatin reader", a protein module that binds to post-translational modifications on histone tails that are thought to affect a variety of chromatin processes, including transcription. More specifically, MBT domains recognize mono- and di-methylated lysines at a number of different positions on histone H3 and H4 tails. Three Drosophila proteins, SCM, L(3)MBT and SFMBT contain multiple adjacent MBT repeats and have critical roles in development, maintenance of cell identity, and tumor suppression. Although they function in different pathways, these proteins all localize to chromatin in vivo and repress transcription by a currently unknown molecular mechanism that requires the MBT domains. The human genome contains several homologues of these MBT proteins, some of which have been linked to important gene regulatory pathways, such as E2F/Rb- and Polycomb-mediated repression, and to the insurgence of certain neurological tumors. Here, we review the genetics, biochemistry, and cell biology of MBT proteins and their role in development and disease.

Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.

Bone marrow is a major reservoir and site of recruitment for central memory CD8+ T cells.

Normal bone marrow (BM) contains T cells whose function and origin are poorly understood. We observed that CD8+ T cells in BM consist chiefly of CCR7+ L-selectin+ central memory cells (TCMs). Adoptively transferred TCMs accumulated more efficiently in the BM than naive and effector T cells. Intravital microscopy (IVM) showed that TCMs roll efficiently in BM microvessels via L-, P-, and E-selectin, whereas firm arrest required the VCAM-1/alpha4beta1 pathway. alpha4beta1 integrin activation did not depend on pertussis toxin (PTX)-sensitive Galphai proteins but was reduced by anti-CXCL12. In contrast, TCM diapedesis did not require CXCL12 but was blocked by PTX. After extravasation, TCMs displayed agile movement within BM cavities, remained viable, and mounted potent antigen-specific recall responses for at least two months. Thus, the BM functions as a major reservoir for TCMs by providing specific recruitment signals that act in sequence to mediate the constitutive recruitment of TCMs from the blood.

Elimination in vivo of developing T cells by natural killer cells.

Natural killer cells gauge the absence of self class I MHC on susceptible target cells by means of inhibitory receptors such as members of the Ly49 family. To initiate killing by natural killer cells, a lack of inhibitory signals must be accompanied by the presence of activating ligands on the target cell. Although natural killer cell-mediated rejection of class I MHC-deficient bone marrow (BM) grafts is a matter of record, little is known about the targeting in vivo of specific cellular subsets by natural killer cells. We show here that development of class I MHC-negative thymocytes is delayed as a result of natural killer cell toxicity after grafting of a class I MHC-positive host with class I MHC-negative BM. Double positive thymocytes that persist in the presence of natural killer cells display an unusual T cell receptor-deficient phenotype, yet nevertheless give rise to single positive thymocytes and yield mature class I MHC-deficient lymphocytes that accumulate in the class I MHC-positive host. The resulting class I MHC-deficient CD8 T cells are functional and upon activation remain susceptible to natural killer cell toxicity in vivo. Reconstitution of class I MHC-deficient BM precursors with H2-K(b) by retroviral transduction fully restores normal thymic development.