8a,a New Acridine Antiproliferative and Pro-Apoptotic Agent Targeting HDAC1/DNMT1

Epigenetic therapy using histone deacetylase (HDAC) inhibitors has become an attractive project in new drug development. However, DNA methylation and histone acetylation are important epigenetic ways to regulate the occurrence and development of leukemia. Given previous studies, N-(2-aminophenyl)benzamide acridine (8a), as a histone deacetylase 1 (HDAC1) inhibitor, induces apoptosis and shows significant anti-proliferative activity against histiocytic lymphoma U937 cells. HDAC1 plays a role in the nucleus, which we confirmed by finding that 8a entered the nucleus. Subsequently, we verified that 8a mainly passes through the endogenous (mitochondrial) pathway to induce cell apoptosis. From the protein interaction data, we found that 8a also affected the expression of DNA methyltransferase 1 (DNMT1). Therefore, an experiment was performed to assess the binding of 8a to DNMT1 at the molecular and cellular levels. We found that the binding strength of 8a to DNMT1 enhanced in a dose-dependent manner. Additionally, 8a inhibits the expression of DNMT1 mRNA and its protein. These findings suggested that the anti-proliferative and pro-apoptotic activities of 8a against leukemia cells were achieved by targeting HDAC1 and DNMT1.     

Estradiol-17β Regulates Expression of Luteal DNA Methyltransferases and Genes Involved in the Porcine Corpus Luteum Function In Vivo

The corpus luteum (CL) is a temporary endocrine gland vital for pregnancy establishment and maintenance. Estradiol-17β (E2) is the major embryonic signal in pigs supporting the CL’s function. The mechanisms of the luteoprotective action of E2 are still unclear. The present study aimed to determine the effect of E2 on luteal expression of factors involved in CL function. An in vivo model of intrauterine E2 infusions was applied. Gilts on day 12 of pregnancy and the estrous cycle were used as referential groups. Concentrations of E2 and progesterone were elevated in CLs of gilts receiving E2 infusions, compared to placebo-treated gilts. Estradiol-17β stimulated luteal expression of DNA-methyltransferase 1 (DNMT1), but decreased expression of DNMT3B gene and protein, as well as DNMT3A protein. Similar results for DNMT3A and 3B were observed in CLs on day 12 of pregnancy compared to day 12 of the estrous cycle. Intrauterine infusions of E2 altered luteal expression of the genes involved in CL function: PTGFR, PTGES, STAR, HSD17B1, CYP19A1, and PGRMC1. Our findings indicate a role for E2 in expression regulation of factors related to CL function and a novel potential for E2 to regulate DNA methylation as putative physiological mechanisms controlling luteal gene expression.     

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.     

The Impact of Epigenetic Modifications in Myeloid Malignancies

Myeloid malignancy is a broad term encapsulating myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Initial studies into genomic profiles of these diseases have shown 2000 somatic mutations prevalent across the spectrum of myeloid blood disorders. Epigenetic mutations are emerging as critical components of disease progression, with mutations in genes controlling chromatin regulation and methylation/acetylation status. Genes such as DNA methyltransferase 3A (DNMT3A), ten eleven translocation methylcytosine dioxygenase 2 (TET2), additional sex combs-like 1 (ASXL1), enhancer of zeste homolog 2 (EZH2) and isocitrate dehydrogenase 1/2 (IDH1/2) show functional impact in disease pathogenesis. In this review we discuss how current knowledge relating to disease progression, mutational profile and therapeutic potential is progressing and increasing understanding of myeloid malignancies.     

Aberrant Methylation of 20 miRNA Genes Specifically Involved in Various Steps of Ovarian Carcinoma Spread: From Primary Tumors to Peritoneal Macroscopic Metastases

Our work aimed to differentiate 20 aberrantly methylated miRNA genes that participate at different stages of development and metastasis of ovarian carcinoma (OvCa) using methylation-specific qPCR in a representative set of clinical samples: 102 primary tumors without and with metastases (to lymph nodes, peritoneum, or distant organs) and 30 peritoneal macroscopic metastases (PMM). Thirteen miRNA genes (MIR107, MIR124-2, MIR124-3, MIR125B-1, MIR127, MIR129-2, MIR130B, MIR132, MIR193A, MIR339, MIR34B/C, MIR9-1, and MIR9-3) were hypermethylated already at the early stages of OvCa, while hypermethylation of MIR1258, MIR137, MIR203A, and MIR375 was pronounced in metastatic tumors, and MIR148A showed high methylation levels specifically in PMM. We confirmed the significant relationship between methylation and expression levels for 11 out of 12 miRNAs analyzed by qRT-PCR. Moreover, expression levels of six miRNAs were significantly decreased in metastatic tumors in comparison with nonmetastatic ones, and downregulation of miR-203a-3p was the most significant. We revealed an inverse relationship between expression levels of miR-203a-3p and those of ZEB1 and ZEB2 genes, which are EMT drivers. We also identified three miRNA genes (MIR148A, MIR9-1, and MIR193A) that likely regulate EMT–MET reversion in the colonization of PMM. According to the Kaplan–Meier analysis, hypermethylation of several examined miRNA genes was associated with poorer overall survival of OvCa patients, and high methylation levels of MIR130B and MIR9-1 were related to the greatest relative risk of death.     

Histone Deacetylase (HDAC) Inhibitors Down-Regulate Endothelial Lineage Commitment of Umbilical Cord Blood Derived Endothelial Progenitor Cells

To test the involvement of histone deacetylases (HDACs) activity in endothelial lineage progression, we investigated the effects of HDAC inhibitors on endothelial progenitors cells (EPCs) derived from umbilical cord blood (UCB). Adherent EPCs, that expressed the endothelial marker proteins (PCAM-1, CD105, CD133, and VEGFR₂) revealed by flow cytometry were treated with three HDAC inhibitors: Butyrate (BuA), Trichostatin A (TSA), and Valproic acid (VPA). RT-PCR assay showed that HDAC inhibitors down-regulated the expression of endothelial genes such as VE-cadherin, CD133, CXCR4 and Tie-2. Furthermore, flow cytometry analysis illustrated that HDAC inhibitors selectively reduce the expression of VEGFR₂, CD117, VE-cadherin, and ICAM-1, whereas the expression of CD34 and CD45 remained unchanged, demonstrating that HDAC is involved in endothelial differentiation of progenitor cells. Real-Time PCR demonstrated that TSA down-regulated telomerase activity probably via suppression of hTERT expression, suggesting that HDAC inhibitor decreased cell proliferation. Cell motility was also decreased after treatment with HDAC inhibitors as shown by wound-healing assay. The balance of acethylation/deacethylation kept in control by the activity of HAT (histone acetyltransferases)/HDAC enzymes play an important role in differentiation of stem cells by regulating proliferation and endothelial lineage commitment.     

TET3- and OGT-Dependent Expression of Genes Involved in Epithelial-Mesenchymal Transition in Endometrial Cancer

TET3 is a member of the TET (ten-eleven translocation) proteins family that catalyzes the conversion of the 5-methylcytosine into 5-hydroxymethylcytosine. TET proteins can also affect chromatin modifications and gene expression independently of their enzymatic activity via interactions with other proteins. O-GlcNAc transferase (OGT), the enzyme responsible for modification of proteins via binding of N-acetylglucosamine residues, is one of the proteins whose action may be dependent on TET3. Here, we demonstrated that in endometrial cancer cells both TET3 and OGT affected the expression of genes involved in epithelial to mesenchymal transition (EMT), i.e., FOXC1, TWIST1, and ZEB1. OGT overexpression was caused by an increase in TWIST1 and ZEB1 levels in HEC-1A and Ishikawa cells, which was associated with increased O-GlcNAcylation of histone H2B and trimethylation of H3K4. The TET3 had the opposite effect on gene expressions and histone modifications. OGT and TET3 differently affected FOXC1 expression and the migratory potential of HEC-1A and Ishikawa cells. Analysis of gene expressions in cancer tissue samples from endometrial cancer patients confirmed the association between OGT or TET3 and EMT genes. Our results contribute to the knowledge of the role of the TET3/OGT relationship in the complex mechanism supporting endometrial cancer progression.     

Kaiso Regulates DNA Methylation Homeostasis

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.     

SDG102, a H3K36-Methyltransferase-Encoding Gene,Plays Pleiotropic Roles in Growth and Development of Maize (Zea mays L.)

Although histone lysine methylation has been studied in thale cress (Arabidopsis thaliana (L.) Heynh.) and rice (Oryza sativa L.) in recent years, its function in maize (Zea mays L.) remains poorly characterized. To better understand the function of histone lysine methylation in maize, SDG102, a H3 lysine 36 (H3K36) methylase, was chosen for functional characterization using overexpressed and knockout transgenic plants. SDG102-deficiency in maize caused multiple phenotypes including yellow leaves in seedlings, late-flowering, and increased adult plant height, while the overexpression of SDG102 led to reduced adult plant height. The key flowering genes, ZCN8/ZCN7 and MADS4/MADA67, were downregulated in SDG102-deficient plants. Chromatin immunoprecipitation (ChIP) experiments showed that H3 lysine 36 trimethylation (H3K36me3) levels were reduced at these loci. Perturbation of SDG102 expression caused the misexpression of multiple genes. Interestingly, the overexpression or knockout of SDG102 also led to genome-wide decreases and increases in the H3K36me3 levels, respectively. Together, our results suggest that SDG102 is a methyltransferase that catalyzes the trimethylation of H3K36 of many genes across the maize genome, which are involved in multiple biological processes including those controlling flowering time.     

Epigenetic Changes in Neoplastic Mast Cells and Potential Impact in Mastocytosis

Systemic mastocytosis (SM) is a hematologic neoplasm with abnormal accumulation of mast cells in various organ systems such as the bone marrow, other visceral organs and skin. So far, only little is known about epigenetic changes contributing to the pathogenesis of SM. In the current article, we provide an overview of epigenetic changes that may occur and be relevant to mastocytosis, including mutations in genes involved in epigenetic processes, such as TET2, DNMT3A and ASXL1, and global and gene-specific methylation patterns in neoplastic cells. Moreover, we discuss methylation-specific pathways and other epigenetic events that may trigger disease progression in mast cell neoplasms. Finally, we discuss epigenetic targets and the effects of epigenetic drugs, such as demethylating agents and BET-targeting drugs, on growth and viability of neoplastic mast cells. The definitive impact of these targets and the efficacy of epigenetic therapies in advanced SM need to be explored in future preclinical studies and clinical trials.     

Genome-Wide Identification of NAP1 and Function Analysis in Moso Bamboo (Phyllostachys edulis)

The nucleosome assembly protein 1 (NAP1) family is the main histone chaperone of histone H2A–H2B. To explore the function of NAP1 family genes in moso bamboo (Phyllostachys edulis), characterized by extremely rapid growth and a long flowering cycle, we originally conducted a genome-wide analysis of the PheNAP1 gene. The phylogenetic relationship, gene expression pattern, DNA methylation, and histone modification were analyzed. Eventually, 12 PheNAP1 genes were recognized from the Phyllostachys edulis genome, divided into two sorts: the NRP subfamily (four members) and the NAP subfamily (eight members). Highly conserved motifs exist in each subfamily, which are distinct between subfamilies. PheNAP1 was distributed homogeneously on 10 out of 24 chromosomes, and gene duplication contributed significantly to the enhancement of the PheNAP1 gene in the genome. Cis-acting element analysis showed that PheNAP1 family genes are involved in light, hormone, and abiotic stress responses and may play an important role in the rapid growth and flowering. PheNAP1 exhibited the highest expression level in fast-growing shoots, indicating it is closely associated with the rapid growth of moso bamboo. Besides, PheNAP1 can rescue the early-flowering phenotype of nrp1-1 nrp2-2, and it affected the expression of genes related to the flowering pathway, like BSU1, suggesting the vital role that PheNAP1 may take in the flowering process of moso bamboo. In addition, histone modification results showed that PheNAP1 could bind to phosphorylation-, acetylation-, and methylation-modified histones to further regulate gene expression. A sketch appears: that PheNAP1 can accompany histones to regulate fast-growth- and flowering-related genes in moso bamboo. The consequences of this study enrich the understanding of the epigenetic regulation mechanism of bamboo plants and lays a foundation for further studies on the role of the NAP1 gene in Phyllostachys edulis and the function of chromatin regulation in forest growth and development.     

Ectopic Overexpression of Histone H3K4 Methyltransferase CsSDG36 from Tea Plant Decreases Hyperosmotic Stress Tolerance in Arabidopsis thaliana

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.