Integrated Clinical Genotype-Phenotype Characteristics of Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive neoplasm derived from plasmacytoid dendritic cells. While advances in understanding the pathophysiology of the disease have been made, integrated systematic analyses of the spectrum of immunophenotypic and molecular alterations in real-world clinical cases remain limited. We performed mutation profiling of 50 BPDCN cases and assessed our findings in the context of disease immunophenotype, cytogenetics, and clinical characteristics. Patients included 42 men and 8 women, with a median age of 68 years (range, 14–84) at diagnosis. Forty-two (84%) patients had at least one mutation, and 23 (46%) patients had ≥3 mutations. The most common mutations involved TET2 and ASXL1, detected in 28 (56%) and 23 (46%) patients, respectively. Co-existing TET2 and ASXL1 mutations were present in 17 (34%) patients. Other recurrent mutations included ZRSR2 (16%), ETV6 (13%), DNMT3A (10%), NRAS (10%), IKZF1 (9%), SRSF2 (9%), IDH2 (8%), JAK2 (6%), KRAS (4%), NOTCH1 (4%), and TP53 (4%). We also identified mutations that have not been reported previously, including ETNK1, HNRNPK, HRAS, KDM6A, RAD21, SF3A1, and SH2B3. All patients received chemotherapy, and 20 patients additionally received stem cell transplantation. With a median follow-up of 10.5 months (range, 1–71), 21 patients achieved complete remission, 4 had persistent disease, and 24 died. Patients younger than 65 years had longer overall survival compared to those who were ≥65 years (p = 0.0022). Patients who had ≥3 mutations or mutations in the DNA methylation pathway genes had shorter overall survival (p = 0.0119 and p = 0.0126, respectively). Stem cell transplantation significantly prolonged overall survival regardless of mutation status. In conclusion, the majority of patients with BPDCN have somatic mutations involving epigenetic regulators and RNA splicing factors, in addition to ETV6 and IKZF1, which are also frequently mutated. Older age, multiple mutations, and mutations in the DNA methylation pathway are poor prognostic factors.

Increase of Homocysteinemia/Hydrogen Sulfide (Hcy/H2S) Ratio Raises Cardiovascular Injuries

Increased Homocysteine Levels (HHcy) is an independent risk factor for atherosclerosis. On the other hand, hydrogen sulfide (H2S) exerts a protection against cardiovascular injuries. On the contrary, accumulating evidences showed that downregulation of defective catabolism of HHcy, with reduced H2S synthesis, is involved in the pathogenesis of a variety of cardiovascular diseases. In that occurrence, the detrimental actions on cardiovascular structures performed by HHcy are added to the negative consequences of reduced H2S (in part unlike each HHcy) on cardiovascular system. Therefore, when the reduced re-methylation pathway of Hcy towards Met (resulting in HHcy) is contemporarily added to the decreased trans-sulfuration pathway (inducing a reduction of H2S synthesis) cardiovascular impairment significantly increases.

The BHMT-betaine methylation pathway epigenetically modulates oligodendrocyte maturation

Research into the epigenome is of growing importance as a loss of epigenetic control has been implicated in the development of neurodegenerative diseases. Previous studies have implicated aberrant DNA and histone methylation in multiple sclerosis (MS) disease pathogenesis. We have previously reported that the methyl donor betaine is depleted in MS and is linked to changes in histone H3 trimethylation (H3K4me3) in neurons. We have also shown that betaine increases histone methyltransferase activity by activating chromatin bound betaine homocysteine S-methyltransferase (BHMT). Here, we investigated the role of the BHMT-betaine methylation pathway in oligodendrocytes. Immunocytochemistry in the human MO3.13 cell line, primary rat oligodendrocytes, and tissue from MS postmortem brain confirmed the presence of the BHMT enzyme in the nucleus in oligodendrocytes. BHMT expression is increased 2-fold following oxidative insult, and qRT-PCR demonstrated that betaine can promote an increase in expression of oligodendrocyte maturation genes SOX10 and NKX-2.2 under oxidative conditions. Chromatin fractionation provided evidence of a direct interaction of BHMT on chromatin and co-IP analysis indicates an interaction between BHMT and DNMT3a. Our data show that both histone and DNA methyltransferase activity are increased following betaine administration. Betaine effects were shown to be dependent on BHMT expression following siRNA knockdown of BHMT. This is the first report of BHMT expression in oligodendrocytes and suggests that betaine acts through BHMT to modulate histone and DNA methyltransferase activity on chromatin. These data suggest that methyl donor availability can impact epigenetic changes and maturation in oligodendrocytes.

The RNA-dependent DNA methylation pathway is required to restrict SPOROCYTELESS/NOZZLE expression to specify a single female germ cell precursor in Arabidopsis

ABSTRACTIn higher plants, the female germline is formed from the megaspore mother cell (MMC), a single cell in the premeiotic ovule. Previously, it was reported that mutants in the RNA-dependent DNA methylation (RdDM) pathway might be involved in restricting the female germline to a single nucellus cell. We show that the DRM methyltransferase double mutant drm1drm2 also presents ectopic enlarged cells, consistent with supernumerary MMC-like cells. In wild-type ovules, MMC differentiation requires SPOROCYTELESS/NOZZLE (SPL/NZZ), as demonstrated by the spl/nzz mutant failing to develop an MMC. We address the poorly understood upstream regulation of SPL/NZZ in ovules, showing that the RdDM pathway is important to restrict SPL/NZZ expression. In ago9, rdr6 and drm1drm2 mutants, SPL/NZZ is expressed ectopically, suggesting that the multiple MMC-like cells observed might be attributable to the ectopic expression of SPL/NZZ. We show that the ovule identity gene, SEEDSTICK, directly regulates AGO9 and RDR6 expression in the ovule and therefore indirectly regulates SPL/NZZ expression. A model is presented describing the network required to restrict SPL/NZZ expression to specify a single MMC.

Chloroplast development and genomes uncoupled signaling are independent of the RNA-directed DNA methylation pathway

The Arabidopsis genome is methylated in CG and non-CG (CHG, and CHH in which H stands for A, T, or C) sequence contexts. DNA methylation has been suggested to be critical for seed development, and CHH methylation patterns change during stratification and germination. In plants, CHH methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. To test for an involvement of the RdDM pathway in chloroplast development, we analyzed seedling greening and the maximum quantum yield of photosystem II (Fv/Fm) in Arabidopsis thaliana seedlings perturbed in components of that pathway. Neither seedling greening nor Fv/Fm in seedlings and adult plants were affected in this comprehensive set of mutants, indicating that alterations in the RdDM pathway do not affect chloroplast development. Application of inhibitors like lincomycin or norflurazon inhibits greening of seedlings and represses the expression of photosynthesis-related genes including LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN1.2 (LHCB1.2) in the nucleus. Our results indicate that the LHCB1.2 promoter is poorly methylated under both control conditions and after inhibitor treatment. Therefore no correlation between LHCB1.2 mRNA transcription and methylation changes of the LHCB1.2 promoter could be established. Moreover, we conclude that perturbations in the RdDM pathway do not interfere with gun signaling.

Transgenerational effect of mutants in the RNA-directed DNA methylation pathway on the triploid block

Hybridization of plants that differ in number of chromosome sets (ploidy) frequently causes endosperm failure and seed arrest, a phenomenon referred to as triploid block. Mutation in NRPD1, encoding the largest subunit of the plant-specific RNA Polymerase IV (Pol IV), can suppress the triploid block. Pol IV generates short RNAs required to guide de novo methylation in the RNA-directed DNA methylation (RdDM) pathway. In this study, we found that the ability of mutants in the RdDM pathway to suppress the triploid block depends on their degree of inbreeding. While nrpd1 is able to suppress in the first homozygous generation, mutants in RDR2, NRPE1, and DRM2 require at least one additional round of inbreeding to exert a suppressive effect. Inbreeding of nrpd1 was connected with a transgenerational loss of non-CG DNA methylation on sites jointly regulated by CHROMOMETHYLASES 2 and 3. Our data thus reveal that loss of RdDM function differs in its effect in early and late generations and that Pol IV acts at an early stage of triploid block establishment.One-sentence summaryInbreeding of mutants impaired in RdDM components transgenerationally enhanced their ability to suppress the triploid block.

The histone variant H2A.W promotes heterochromatin accessibility for efficient DNA methylation in Arabidopsis

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, including DNA methylation and H3K9 methylation. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. To analyze the in vivo function of H2A.W, we created a h2a.w null mutant via CRISPR-Cas9, h2a.w-2. We found that H2A.W is not essential for plant development, and that loss of H2A.W did not perturb histone methylation patterns. In contrast, we found a reduction of non-CG DNA methylation in pericentromeric heterochromatin and an increase in DNA methylation at euchromatic sites targeted by the RNA-directed DNA methylation pathway. Loss of DNA methylation in h2a.w-2 correlated with both increased H1 occupancy and decreased DNA accessibility at heterochromatin. Our results indicate that H2A.W helps stabilize the accessibility of heterochromatin and facilitates efficient DNA methylation by fine tuning the genomic distribution of H1.