Dual binding of chromomethylase domains to H3K9me2-containing nucleosomes directs DNA methylation in plants.

DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2, and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (BAH) and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains and reveals a distinct mechanism of interplay between DNA methylation and histone modification.

Discriminating between Interstitial and Circulating Leukocytes in Tissues of the Murine Oral Mucosa Avoiding Nasal-Associated Lymphoid Tissue Contamination.

Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis (Pg) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44hi). We report for the first time the presence of antigen-experienced Pg-specific CD4 T cells in NALT following oral feeding of mice with Pg. This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.

Pre-clinical development of gene modification of haematopoietic stem cells with chimeric antigen receptors for cancer immunotherapy.

Patients with refractory or recurrent B-lineage hematologic malignancies have less than 50% of chance of cure despite intensive therapy and innovative approaches are needed. We hypothesize that gene modification of haematopoietic stem cells (HSC) with an anti-CD19 chimeric antigen receptor (CAR) will produce a multi-lineage, persistent immunotherapy against B-lineage malignancies that can be controlled by the HSVsr39TK suicide gene. High-titer third-generation self-inactivating lentiviral constructs were developed to deliver a second-generation CD19-specific CAR and the herpes simplex virus thymidine kinase HSVsr39TK to provide a suicide gene to allow ablation of gene-modified cells if necessary. Human HSC were transduced with such lentiviral vectors and evaluated for function of both CAR and HSVsr39TK. Satisfactory transduction efficiency was achieved; the addition of the suicide gene did not impair CAR expression or antigen-specific cytotoxicity, and determined marked cytotoxicity to ganciclovir. NSG mice transplanted with gene-modified human HSC showed CAR expression not significantly different between transduced cells with or without HSVsr39TK, and expression of anti-CD19 CAR conferred anti-tumor survival advantage. Treatment with ganciclovir led to significant ablation of gene-modified cells in mouse tissues. Haematopoietic stem cell transplantation is frequently part of the standard of care for patients with relapsed and refractory B cell malignancies; following HSC collection, a portion of the cells could be modified to express the CD19-specific CAR and give rise to a persistent, multi-cell lineage, HLA-independent immunotherapy, enhancing the graft-versus-malignancy activity.

Modification of hematopoietic stem/progenitor cells with CD19-specific chimeric antigen receptors as a novel approach for cancer immunotherapy.

Chimeric antigen receptors (CARs) against CD19 have been shown to direct T-cells to specifically target B-lineage malignant cells in animal models and clinical trials, with efficient tumor cell lysis. However, in some cases, there has been insufficient persistence of effector cells, limiting clinical efficacy. We propose gene transfer to hematopoietic stem/progenitor cells (HSPC) as a novel approach to deliver the CD19-specific CAR, with potential for ensuring persistent production of effector cells of multiple lineages targeting B-lineage malignant cells. Assessments were performed using in vitro myeloid or natural killer (NK) cell differentiation of human HSPCs transduced with lentiviral vectors carrying first and second generations of CD19-specific CAR. Gene transfer did not impair hematopoietic differentiation and cell proliferation when transduced at 1-2 copies/cell. CAR-bearing myeloid and NK cells specifically lysed CD19-positive cells, with second-generation CAR including CD28 domains being more efficient in NK cells. Our results provide evidence for the feasibility and efficacy of the modification of HSPC with CAR as a strategy for generating multiple lineages of effector cells for immunotherapy against B-lineage malignancies to augment graft-versus-leukemia activity.

DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons.

The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution.