lncCPSET1 acts as a scaffold for MLL2/COMPASS to regulate Bmp4 and promote the formation of chicken primordial germ cells.

Primordial germ cells (PGCs) are the ancestors of female and male germ cells. Recent studies have shown that long non-coding RNA (lncRNA) and histone methylation are key epigenetic factors affecting PGC formation; however, their joint regulatory mechanisms have rarely been studied. Here, we explored the mechanism by which lncCPSET1 and H3K4me2 synergistically regulate the formation of chicken PGCs for the first time. Combined with chromatin immunoprecipitation (CHIP) sequencing and RNA-seq of PGCs transfected with the lncCPSET1 overexpression vector, GO annotation and KEGG enrichment analysis revealed that Wnt and TGF-ß signaling pathways were significantly enriched, and Fzd2, Id1, Id4, and Bmp4 were identified as candidate genes. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ASH2L, DPY30, WDR5, and RBBP5 overexpression significantly increased the expression of Bmp4, which was up-regulated after lncCPSET1 overexpression as well. It indicated that Bmp4 is a target gene co-regulated by lncCPSET1 and MLL2/COMPASS. Interestingly, co-immunoprecipitation results showed that ASH2L, DPY30 and WDR5 combined and RBBP5 weakly combined with DPY30 and WDR5. lncCPSET1 overexpression significantly increased Dpy30 expression and co-immunoprecipitation showed that interference/overexpression of lncCPSET1 did not affect the binding between the proteins in the complexes, but interference with lncCPSET1 inhibited DPY30 expression, which was confirmed by RNA immunoprecipitation that lncCPSET1 binds to DPY30. Additionally, CHIP-qPCR results showed that DPY30 enriched in the Bmp4 promoter region promoted its transcription, thus promoting the formation of PGCs. This study demonstrated that lncCPSET1 and H3K4me2 synergistically promote PGC formation, providing a reference for the study of the regulatory mechanisms between lncRNA and histone methylation, as well as a molecular basis for elucidating the formation mechanism of PGCs in chickens.

LSD1 promotes the FSH responsive follicle formation by regulating autophagy and repressing Wt1 in the granulosa cells.

In a growing follicle, the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication. Although apoptosis and autophagy in somatic cells are involved in the process of FSH-induced follicular development, the underlying mechanisms require substantial study. According to our study, along with FSH-induced antral follicles (AFs) formation, both lysine-specific demethylase 1 (LSD1) protein levels and autophagy increased simultaneously in granulosa cells (GCs) in a time-dependent manner, we therefore evaluated the importance of LSD1 upon facilitating the formation of AFs correlated to autophagy in GCs. Conditional knockout of Lsd1 in GCs resulted in significantly decreased AF number and subfertility in females, accompanied by marked suppression of the autophagy in GCs. On the one hand, depletion of Lsd1 resulted in accumulation of Wilms tumor 1 homolog (WT1), at both the protein and mRNA levels. WT1 prevented the expression of FSH receptor (Fshr) in GCs and thus reduced the responsiveness of the secondary follicles to FSH induction. On the other hand, depletion of LSD1 resulted in suppressed level of autophagy by upregulation of ATG16L2 in GCs. We finally approved that LSD1 contributed to these sequential activities in GCs through its H3K4me2 demethylase activity. Therefore, the importance of LSD1 in GCs is attributable to its roles in both accelerating autophagy and suppressing WT1 expression to ensure the responsiveness of GCs to FSH during AFs formation.

COMPASS core subunits MpSet1 and MpSwd3 regulate Monascus pigments synthesis in Monascus purpureus.

In eukaryotes, methylation of histone H3 at lysine 4 (H3K4me) catalyzed by the complex of proteins associated with Set1 (COMPASS) is crucial for the transcriptional regulation of genes and the development of organisms. In Monascus, the functions of COMPASS in establishing H3K4me remain unclear. This study first identified the conserved COMPASS core subunits MpSet1 and MpSwd3 in Monascus purpureus and confirmed their roles in establishing H3K4me2/3. Loss of MpSet1 and MpSwd3 resulted in slower growth and development and inhibited the formation of cleistothecia, ascospores, and conidia. The loss of these core subunits also decreased the production of extracellular and intracellular Monascus pigments (MPs) by 94.2%, 93.5%, 82.7%, and 82.5%, respectively. In addition, RNA high-throughput sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) showed that the loss of MpSet1 and MpSwd3 altered the expression of 2646 and 2659 genes, respectively, and repressed the transcription of MPs synthesis-related genes. In addition, the ΔMpset1 and ΔMpswd3 strains demonstrated increased sensitivity to cell wall stress with the downregulation of chitin synthase-coding genes. These results indicated that the COMPASS core subunits MpSet1 and MpSwd3 help establish H3K4me2/3 for growth and development, spore formation, and pigment synthesis in Monascus. These core subunits also assist in maintaining cell wall integrity.

Inhibition of KDM5B participates in immune microenvironment remodeling in pancreatic cancer by inducing STING expression.

OBJECTIVE: This study aims to investigate the effect of lysine demethylase 5B (KDM5B)-mediated dimethyl-lysine 4 histone H3 (H3K4me2) demethylation on immune microenvironment remodeling in pancreatic cancer. METHODS: Pan 02 mouse pancreatic cancer cell lines were cultured and used to establish tumor model in vivo. RT-qPCR and Western blot were used to detect the expression of stimulator of interferon gene (STING) and KDM5B in pancreatic cancer tissues and Pan 02 cells. The specific demethylation domain of KDM5B was detected by isothermal titration calorimetry binding assay. The regulatory roles of KDM5B in cell apoptosis and remodeling of immune microenvironment in vitro and in vivo were explored after loss-of functions in KDM5B. RESULTS: KDM5B was highly expressed but STING was poorly expressed in pancreatic cancer tissues and Pan 02 cells. After knockdown of KDM5B, CD8+ T cells recognized and killed Pan 02 cells, which promoted the infiltration of CD8+ T cells in Pan 02 cells, thus improving the anti-tumor ability. The PHD domain in KDM5B specifically bound to H3K4me2 peptide and inhibition of KDM5B induced STING expression. Knockdown of KDM5B up-regulated STING expression to promote apoptosis, thus regulating the immune microenvironment and inhibiting the growth of tumor in mice. Meanwhile, knockdown of KDM5B and STING simultaneously counteracted the knockdown effect of KDM5B. CONCLUSION: Inhibition of KDM5B can promote the expression of STING through H3K4me2 demethylation, which promoted the recognition and killing of Pan 02 cells by CD8+ T cells, thus improving the anti-tumor ability and regulating the immune microenvironment.

Pygo2 activates BRPF1 via Pygo2-H3K4me2/3 interaction to maintain malignant progression in colon cancer.

Epigenetic alterations have essential roles during colon adenocarcinoma (COAD) progression. As the coactivator of Wnt/b-catenin signaling, Pygopus 2 (Pygo2) binds H3K4me2/3 and participate in chromatin remodeling in multiple cancers. However, It remains unclear whether the Pygo2-H3K4me2/3 association has significance in COAD. We aimed to elucidate the roles of Pygo2 in COAD. Functionally, Pygo2 inhibition attenuated cell proliferation, self-renewal capacities in vitro. Pygo2 overexpression enhanced in vivo tumor growth. Besides, Pygo2 overexpression could also enhance cell migration ability and in vivo distal metastasis. Mechanistically, Pygo2 correlates positively with BRPF1 expressions, one epigenetic reader of histone acetylation. The luciferase reporter assay and Chromatin Immunoprecipitation (ChIP)-qPCR assay were used to find that Pygo2 coordinated with H3K4me2/3 modifications to activate BRPF1 transcriptions via binding to the promoter. Both Pygo2 and BRPF1 expressed highly in tumors and Pygo2 relied on BRPF1 to accelerate COAD progression, including cell proliferation rate, migration abilities, stemness features and in vivo tumor growth. Targeting BPRF1 (GSK5959) is effective to suppress in vitro growth of Pygo2high cell lines, and has mild effect on Pygo2low cells. The subcutaneous tumor model further demonstrated that GSK5959 could effectively suppress the in vivo growth of Pygo2high COAD, but not the Pygo2low subtype. Collectively, our study represented Pygo2/BRPF1 as an epigenetic vulnerability for COAD treatment with predictive significance.

Chromatin Immunostaining of Plant Nuclei.

Recent evidence has demonstrated that specific epigenetic changes are also related to plant growth and development. Immunostaining enables the detection and characterization of chromatin modification, e.g., histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and DNA methylation (5mC) with unique and specific patterns in plant tissues. Here we describe experimental procedures to determine the histone H3 methylation (H3K4me2 and H3K9me2) patterns in 3D-chromatin in whole roots tissue and 2D-chromatin in single nuclei in rice. To analyze both iron and salinity treatments, we show how to test for changes to the epigenetic chromatin landscape using heterochromatin (H3K9me2) and euchromatin (H3K4me) markers for chromatin immunostaining, especially in the proximal meristem region. To elucidate the epigenetic impact of environmental stress and external plant growth regulators, we demonstrate how to apply a combination of salinity, auxin, and abscisic acid treatments. The results of these experiments provide insights into the epigenetic landscape during rice root growth and development.

RSI1/FLD and its epigenetic target RRTF1 are essential for the retention of infection memory in Arabidopsis thaliana.

Plants can retain a memory of previous pathogen infections to mount a more robust defense response during subsequent infections by developing systemic acquired resistance (SAR). However, the mechanism through which plants develop and retain infection memory is not known. Experiments have shown the association of epigenetic modifications of specific defense-related genes with SAR. RSI1/FLD codes for a histone demethylase and is required for the activation of SAR in Arabidopsis. Here we report the identification of RRTF1 as an epigenetic target of RSI1. RRTF1 expression is higher in pathogen-free distal tissues of the rsi1 mutant. Experiments with loss-of-function and overexpression lines suggest RRTF1 is a negative regulator of basal defense against virulent and avirulent pathogens as well as SAR. Enhanced expression of RRTF1 in a wild-type (WT) background specifically impairs SAR without impacting local resistance. RSI1 is recruited at the RRTF1 locus in a SAR-inducible manner and contributes to H3K4me2 and H3K4me3 demethylation. Introduction of the rrtf1 mutation rescues the loss-of-SAR phenotype of rsi1 plants. However, these plants fail to retain infection memory beyond 7 days post-primary inoculation, whereas WT plants retain memory for at least 11 days. Our results demonstrate that RSI1 and RRTF1 form a functional module for retaining infection memory in Arabidopsis.

H3K4me2 cooperates with Wnt/TCF7L2 to regulate TDRD1 and promote chicken spermatogonia stem cell formation.

Spermatogonia Stem Cells (SSCs) are the basis of spermatogenesis. In the poultry industry, asthenospermia and azoospermia in roosters seriously reduce economic benefits. In this study, we explored SSCs formation mechanisms in detail. TDRD1, which is a downstream target gene of TCF7L2 and is modified by histone methylation, was screened through multiomics analysis. Functionally, RT-qPCR, flow cytometry, immunohistochemistry, and indirect immunofluorescence results showed that H3K4me2 regulated TDRD1 to promote SSCs formation both in vivo and in vitro. Furthermore, ChIP-qPCR and dual luciferase assays showed that H3K4me2 was enriched in the -800 to 0 bp region of the TDRD1 promoter and positively regulated TDRD1 transcription to promote SSCs formation. Interestingly, in mechanistic terms, dual luciferase assays showed that TDRD1 transcription levels were significantly decreased after co-transfection with dCas9-LSD1-P1/P2/P3 and OETCF7L2, while TDRD1 transcript levels were not significantly altered after transfecting dCas9-LSD1-P4 and OETCF7L2. These results suggested that H3K4me2 enrichment in P1, P2, and P3 of the TDRD1 promoter promotes TDRD1 transcription by reducing enrichment of TCF7L2. This study explored the specific regulatory mechanisms involving the Wnt signaling pathway, H3K4me2, and TDRD1, enriched the regulatory network regulating the formation of SSCs, and laid a theoretical foundation for the specific application of SSCs.

Cri du chat syndrome patients have DNA methylation changes in genes linked to symptoms of the disease.

BACKGROUND: Cri du chat (also called 5p deletion, or monosomy 5p) syndrome is a genetic disease caused by deletions of various lengths in the short (p) arm of chromosome 5. Genetic analysis and phenotyping have been used to suggest dose-sensitive genes in this region that may cause symptoms when a gene copy is lost, but the heterogeneity of symptoms for patients with similar deletions complicates the picture. The epigenetics of the syndrome has only recently been looked at with DNA methylation measurements of blood from a single patient, suggesting epigenetic changes in these patients. Here, we conduct the deepest epigenetic analysis of the syndrome to date with DNA methylation analysis of eight Cri du chat patients with sibling- and age-matched controls. RESULTS: The genome-wide patterns of DNA methylation in the blood of Cri du chat patients reveal distinct changes compared to controls. In the p-arm of chromosome 5 where patients are hemizygous, we find stronger changes in methylation of CpG sites than what is seen in the rest of the genome, but this effect is less pronounced in gene regulatory sequences. Gene set enrichment analysis using patient DNA methylation changes in gene promoters revealed enrichment of genes controlling embryonic development and genes linked to symptoms which are among the most common symptoms of Cri du chat syndrome: developmental delay and microcephaly. Importantly, this relative enrichment is not driven by changes in the methylation of genes on chromosome 5. CpG sites linked to these symptoms where Cri du chat patients have strong DNA methylation changes are enriched for binding of the polycomb EZH2 complex, H3K27me3, and H3K4me2, indicating changes to bivalent promoters, known to be central to embryonic developmental processes. CONCLUSIONS: Finding DNA methylation changes in the blood of Cri du chat patients linked to the most common symptoms of the syndrome is suggestive of epigenetic changes early in embryonic development that may be contributing to the development of symptoms. However, with the present data we cannot conclude about the sequence of events between DNA methylation changes and other cellular functions-the observed differences could be directly driving epigenetic changes, a result of other epigenetic changes, or they could be a reflection of other gene regulatory changes such as changed gene expression levels. We do not know which gene(s) on the p-arm of chromosome 5 that causes epigenetic changes when hemizygous, but an important contribution from this work is making the pool of possible causative genes smaller.

Structural insight into ASH1L PHD finger recognizing methylated histone H3K4 and promoting cell growth in prostate cancer.

ASH1L is a member of the Trithorax-group protein and acts as a histone methyltransferase for gene transcription activation. It is known that ASH1L modulates H3K4me3 and H3K36me2/3 at its gene targets, but its specific mechanism of histone recognition is insufficiently understood. In this study, we found that the ASH1L plant homeodomain (PHD) finger interacts with mono-, di-, and trimethylated states of H3K4 peptides with comparable affinities, indicating that ASH1L PHD non-selectively binds to all three methylation states of H3K4. We solved nuclear magnetic resonance structures picturing the ASH1L PHD finger binding to the dimethylated H3K4 peptide and found that a narrow binding groove and residue composition in the methylated-lysine binding pocket restricts the necessary interaction with the dimethyl-ammonium moiety of K4. In addition, we found that the ASH1L protein is overexpressed in castrate-resistant prostate cancer (PCa) PC3 and DU145 cells in comparison to PCa LNCaP cells. The knockdown of ASH1L modulated gene expression and cellular pathways involved in apoptosis and cell cycle regulation and consequently induced cell cycle arrest, cell apoptosis, and reduced colony-forming abilities in PC3 and DU145 cells. The overexpression of the C-terminal core of ASH1L but not the PHD deletion mutant increased the overall H3K36me2 level but had no effect on the H3K4me2/3 level. Overall, our study identifies the ASH1L PHD finger as the first native reader that non-selectively recognizes the three methylation states of H3K4. Additionally, ASH1L is required for the deregulation of cell cycle and survival in PCas.

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers.

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

USP7 sustains an active epigenetic program via stabilizing MLL2 and WDR5 in diffuse large B-cell lymphoma.

Activated B-cell-like (ABC)-diffuse large B-cell lymphoma (ABC-DLBCL) is a common subtype of non-Hodgkin's lymphoma with poor prognosis. The survival of ABC-DLBCL relies on constitutive activation of BCR signaling, but the underlying molecular mechanism is not fully addressed. By mining The Cancer Genome Atlas database, we found that the expression of ubiquitin-specific protease 7 (USP7) is significantly elevated in three cancer types including DLBCL. Interestingly, unlike germinal center B-cell-like (GCB)-DLBCL, ABC-DLBCL shows upregulated expression of USP7. Inhibiting the enzymatic activity of USP7 (P22077) has a drastic effect on ABC-DLBCL, but not GCB-DLBCL cells. Compared to GCB-DLBCL, ABC-DLBCL cells show transcriptional upregulation of multiple components of BCR-signaling. USP7 inhibition significantly reduces the expression of upregulated components of BCR signaling. Mechanistically, USP7 inhibition greatly reduces the methylation of histone 3 on lysine 4 (H3K4me2), which is an epigenetic marker for active enhancers. USP7 inhibition greatly reduces the protein level of WDR5 and MLL2, key components of lysine-specific methyltransferase complex (complex of proteins associated with Set1 [COMPASS]). In ABC-DLBCL cells, USP7 stabilizes WDR5 and MLL2. In patients, the expression of USP7 is significantly associated with components of BCR signaling (LYN, SYK, BTK, PLCG2, PRKCB, MALT1, BCL10, and CARD11) and targets of BCR signaling (MYC and IRF4). In summary, we demonstrated an essential role of USP7 in ABC-DLBCL by organizing an oncogenic epigenetic program via stabilization of WDR5 and MLL2. Targeting USP7 might be a novel and efficient approach to treat patients with ABC-DLBCL and it might be better than targeting individual components such as BTK in BCR signaling.

Heterogeneity in the Epigenetic Landscape of Murine Testis-Specific Histone Variants TH2A and TH2B Sharing the Same Bi-Directional Promoter.

Testis-specific histone variants are crucial to promote open chromatin structure to enable nucleosome disassembly in the final stages of spermiogenesis. However, even after histone replacement, mature sperm retain a proportion of these variants, the function of which is unknown. The present study aimed to understand the functional relevance of the retained H2B and H2A variants, TH2B and TH2A. While no literature is available on the phenotype of TH2A knockouts, TH2B/TH2A double knockout male mice are reported to be infertile. In this study, ChIP-seq analysis was done for TH2B and TH2A to understand the epigenomics of the retained TH2B and TH2A, using murine caudal sperm. Distribution across genomic partitions revealed ∼35% of the TH2B peaks within ±5 kb of TSS whereas TH2A peaks distribution was sparse at TSS. Gene Ontology revealed embryo development as the most significant term associated with TH2B. Also, based on genomic regions, TH2B was observed to be associated with spindle assembly and various meiosis-specific genes, which is an important finding as TH2A/TH2B DKO mice have been reported to have defective cohesin release. A comparison of mouse and human TH2B-linked chromatin revealed 26% overlap between murine and human TH2B-associated genes. This overlap included genes crucial for embryogenesis. Most importantly, heterogeneity in the epigenetic landscape of TH2A and TH2B was seen, which is intriguing as TH2B and TH2A are well reported to be present in the same nucleosomes to promote open chromatin. Additionally, unlike TH2B, TH2A was enriched on the mitochondrial chromosome. TH2A was found to be associated with Nuclear insertion of Mitochondrial DNA sequences (NUMTs) in sperm. A comprehensive analysis of these observations indicates novel functions for the sperm-retained TH2B and TH2A.

piRNA-independent transposon silencing by the Drosophila THO complex.

piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. Here, we show that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development.

H3K4me2 Promotes the Activation of lncCPSET1 by Jun in the Chicken PGC Formation.

Primordial germ cells are the ancestors of female and male cells. Current research has shown that long non-coding RNA (lncRNA) and Histone methylation are the pivotal epigenetic factors in the PGC formation. However, there are few studies on the regulatory mechanism of lncRNA in the formation of PGC. Here, we define the lncRNA highly expressed in chicken PGC, lncCPSET1 (chicken-PGC-specifically-expressed transcript 1) This study found that compared with the interference of lncCPSET1/histone methylase Mll2 alone, the PGC formation was severely inhibited with the interference of lncCPSET1 and histone methylase Mll2 jointly in vivo and in vitro. Studies on the transcription level of lncCPSET1 found that H3K4me2 and transcription factor Jun have a positive effect on the activation of lncCPSET1; while DNA hypomethylation inhibits the expression of lncCPSET1. In terms of mechanism, compared with DNA methylation, H3K4me2 dominates lncCPSET1 activation. H3K4me2 can be enriched in the lncCPSET1 promoter, change its chromosome conformation, recruit the transcription factor Jun, and activate the expression of lncCPSET1. Taken together, we confirmed the model that H3K4me2 rather than DNA hypomethylation mediates Jun to regulate lncCPSET1 transcription, which broadens the study of lncCPSET1 pre-transcriptional mechanism.

Loss of H3K27 methylation identifies poor outcomes in adult-onset acute leukemia.

BACKGROUND: Acute leukemia is an epigenetically heterogeneous disease. The intensity of treatment is currently guided by cytogenetic and molecular genetic risk classifications; however these incompletely predict outcomes, requiring additional information for more accurate outcome predictions. We aimed to identify potential prognostic implications of epigenetic modification of histone proteins, with a focus on H3K4 and H3K27 methylation marks in relation to mutations in chromatin, splicing and transcriptional regulators in adult-onset acute lymphoblastic and myeloid leukemia. RESULTS: Histone 3 lysine 4 di- and trimethylation (H3K4me2, H3K4me3) and lysine 27 trimethylation (H3K27me3) mark expression was evaluated in 241 acute myeloid leukemia (AML), 114 B-cell acute lymphoblastic leukemia (B-ALL) and 14T-cell ALL (T-ALL) patient samples at time of diagnosis using reverse phase protein array. Expression levels of the marks were significantly lower in AML than in B and T-ALL in both bone marrow and peripheral blood, as well as compared to normal CD34+ cells. In AML, greater loss of H3K27me3 was associated with increased proliferative potential and shorter overall survival in the whole patient population, as well as in subsets with DNA methylation mutations. To study the prognostic impact of H3K27me3 in the context of cytogenetic aberrations and mutations, multivariate analysis was performed and identified lower H3K27me3 level as an independent unfavorable prognostic factor in all, as well as in TP53 mutated patients. AML with decreased H3K27me3 demonstrated an upregulated anti-apoptotic phenotype. In ALL, the relative quantity of histone methylation expression correlated with response to tyrosine kinase inhibitor in patients who carried the Philadelphia cytogenetic aberration and prior smoking behavior. CONCLUSION: This study shows that proteomic profiling of epigenetic modifications has clinical implications in acute leukemia and supports the idea that epigenetic patterns contribute to a more accurate picture of the leukemic state that complements cytogenetic and molecular genetic subgrouping. A combination of these variables may offer more accurate outcome prediction and we suggest that histone methylation mark measurement at time of diagnosis might be a suitable method to improve patient outcome prediction and subsequent treatment intensity stratification in selected subgroups.

BMP4 activates the Wnt-Lin28A-Blimp1-Wnt pathway to promote primordial germ cell formation via altering H3K4me2.

The unique developmental characteristics of chicken primordial germ cells (PGCs) enable them to be used in recovery of endangered bird species, gene editing and the generation of transgenic birds, but the limited number of PGCs greatly limits their application. Studies have shown that the formation of mammalian PGCs is induced by BMP4 signal, but the mechanism underlying chicken PGC formation has not been determined. Here, we confirmed that Wnt signaling activated via BMP4 activates transcription of Lin28A by inducing ß-catenin to compete with LSD1 for binding to TCF7L2, causing LSD1 to dissociate from the Lin28A promoter and enhancing H3K4me2 methylation in this region. Lin28A promotes PGC formation by inhibiting gga-let7a-3p maturation to initiate Blimp1 expression. Interestingly, expression of Blimp1 helped sustain Wnt5A expression by preventing LSD1 binding to the Wnt5A promoter. We thus elucidated a positive feedback pathway involving Wnt-Lin28A-Blimp1-Wnt that ensures PGC formation. In summary, our data provide new insight into the development of PGCs in chickens.

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus.

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

H3K4 Methylation Status and Lysine Specific Methyltransferase KMT2C Expression Correlate with Prognosis in Lung Adenocarcinoma.

BACKGROUND: Genetic events cannot account for the complexity of human carcinogenesis alone. Mutations of epigenetic regulators and aberrations of their expression patterns have been detected in various human malignancies. Methylation of histone H3 at lysine 4 (H3K4me), is an evolutionarily conserved histone modification that marks regions of active transcription and regulates cell growth, migration, and invasion. The MLL/KMT2 family of histone methyltransferases specifically methylate histone H3 at lysine 4. OBJECTIVE: The aim of this study was to explore the role of KMT2C/MLL3 as well as key histone modification activating markers, such as H3K4me2 and H3K4me3 in a cohort of surgically resected human lung adenocarcinomas in an effort to reveal possible biomarkers for lung adenocarcinoma diagnosis and prognosis and potential therapeutic targets. METHOD: The immunohistochemical expression of KMT2C/MLL3, H3K4me2 and H3K4me3 was analyzed in formalin fixed paraffin embedded tissue from 96 patients with lung adenocarcinoma. Results were associated with clinicopathologic parameters and patient's prognosis. RESULTS: Nuclear expression of KMT2C/MLL3 in epithelial cells was independently associated with shorter overall survival. Cytoplasmic H3K4me2 expression was associated withT stage and nuclear H3K4me2 expression was associated with female gender and patients' prognosis. The latter association persisted after multivariate analysis. No association was found between H3K4me3 expression and clinicopathological data or disease outcome in our cohort of patients. CONCLUSION: These results suggest that the pattern of histone modifications and KMT2C/MLL3 expression can be used as an independent prognostic factor in lung adenocarcinoma, revealing that chromatin remodeling is criticallyinvolved in cancer progression.

Narrow H3K4me2 is required for chicken PGC formation.

The development of primordial germ cells (PGCs) undergoes epigenetic modifications. The study of histone methylation in regulating PGCs is beneficial to understand the development and differentiation mechanism of germ stem cells. Notably, it provides a theoretical basis for directed induction and mass acquisition in vitro. However, little is known about the regulation of PGC formation by histone methylation. Here, we found the high enrichment of H3K4me2 in the blastoderm, genital ridges, and testis. Chromatin immunoprecipitation sequencing was performed and the results revealed that genomic H3K4me2 is dynamic in embryonic stem cells, PGCs, and spermatogonial stem cells. This trend was consistent with the H3K4me2 enrichment in the gene promoter region. Additionally, narrow region triggered PGC-related genes (Bmp4, Wnt5a, and Tcf7l2) and signaling pathways (Wnt and transforming growth factor-ß). After knocking down histone methylase Mll2 in vitro and vivo, the level of H3K4me2 decreased, inhibiting Cvh and Blimp1 expression, then repressing the formation of PGCs. Taken together, our study revealed the whole genome map of H3K4me2 in the formation of PGCs, contributing to improve the epigenetic study in PGC formation and providing materials for bird gene editing and rescue of endangered birds.