Tanshinone I, a new EZH2 inhibitor restricts normal and malignant hematopoiesis through upregulation of MMP9 and ABCG2.

Rationale: Tanshinone, a type of diterpenes derived from salvia miltiorrhiza, is a particularly promising herbal medicine compound for the treatment of cancers including acute myeloid leukemia (AML). However, the therapeutic function and the underlying mechanism of Tanshinone in AML are not clear, and the toxic effect of Tanshinone limits its clinical application. Methods: Our work utilizes human leukemia cell lines, zebrafish transgenics and xenograft models to study the cellular and molecular mechanisms of how Tanshinone affects normal and abnormal hematopoiesis. WISH, Sudan Black and O-Dianisidine Staining were used to determine the expression of hematopoietic genes on zebrafish embryos. RNA-seq analysis showed that differential expression genes and enrichment gene signature with Tan I treatment. The surface plasmon resonance (SPR) method was used with a BIAcore T200 (GE Healthcare) to measure the binding affinities of Tan I. In vitro methyltransferase assay was performed to verify Tan I inhibits the histone enzymatic activity of the PRC2 complex. ChIP-qPCR assay was used to determine the H3K27me3 level of EZH2 target genes. Results: We found that Tanshinone I (Tan I), one of the Tanshinones, can inhibit the proliferation of human leukemia cells in vitro and in the xenograft zebrafish model, as well as the normal and malignant definitive hematopoiesis in zebrafish. Mechanistic studies illustrate that Tan I regulates normal and malignant hematopoiesis through direct binding to EZH2, a well-known histone H3K27 methyltransferase, and inhibiting PRC2 enzymatic activity. Furthermore, we identified MMP9 and ABCG2 as two possible downstream genes of Tan I's effects on EZH2. Conclusions: Together, this study confirmed that Tan I is a novel EZH2 inhibitor and suggested MMP9 and ABCG2 as two potential therapeutic targets for myeloid malignant diseases.

Trans- and cis-acting effects of Firre on epigenetic features of the inactive X chromosome.

Firre encodes a lncRNA involved in nuclear organization. Here, we show that Firre RNA expressed from the active X chromosome maintains histone H3K27me3 enrichment on the inactive X chromosome (Xi) in somatic cells. This trans-acting effect involves SUZ12, reflecting interactions between Firre RNA and components of the Polycomb repressive complexes. Without Firre RNA, H3K27me3 decreases on the Xi and the Xi-perinucleolar location is disrupted, possibly due to decreased CTCF binding on the Xi. We also observe widespread gene dysregulation, but not on the Xi. These effects are measurably rescued by ectopic expression of mouse or human Firre/FIRRE transgenes, supporting conserved trans-acting roles. We also find that the compact 3D structure of the Xi partly depends on the Firre locus and its RNA. In common lymphoid progenitors and T-cells Firre exerts a cis-acting effect on maintenance of H3K27me3 in a 26 Mb region around the locus, demonstrating cell type-specific trans- and cis-acting roles of this lncRNA.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Reproductive Long Intergenic Noncoding RNAs Exhibit Male Gamete Specificity and Polycomb Repressive Complex 2-Mediated Repression.

Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in ∼10% and ∼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.

Targeting the Notch-regulated non-coding RNA TUG1 for glioma treatment.

Targeting self-renewal is an important goal in cancer therapy and recent studies have focused on Notch signalling in the maintenance of stemness of glioma stem cells (GSCs). Understanding cancer-specific Notch regulation would improve specificity of targeting this pathway. In this study, we find that Notch1 activation in GSCs specifically induces expression of the lncRNA, TUG1. TUG1 coordinately promotes self-renewal by sponging miR-145 in the cytoplasm and recruiting polycomb to repress differentiation genes by locus-specific methylation of histone H3K27 via YY1-binding activity in the nucleus. Furthermore, intravenous treatment with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system induces GSC differentiation and efficiently represses GSC growth in vivo. Our results highlight the importance of the Notch-lncRNA axis in regulating self-renewal of glioma cells and provide a strong rationale for targeting TUG1 as a specific and potent therapeutic approach to eliminate the GSC population.

Characterization of Inhibitor Binding Through Multiple Inhibitor Analysis: A Novel Local Fitting Method.

Understanding inhibitor binding modes is a key aspect of drug development. Early in a drug discovery effort these considerations often impact hit finding strategies and hit prioritization. Multiple inhibitor experiments, where enzyme inhibition is measured in the presence of two simultaneously varied inhibitors, can provide valuable information about inhibitor binding. These experiments utilize the inhibitor concentration dependence of the observed combined inhibition to determine the relationship between two compounds. In this way, it can be determined whether two inhibitors bind exclusively, independently, synergistically, or antagonistically. Novel inhibitors can be tested against each other or reference compounds to assist hit classification and characterization of inhibitor binding. In this chapter, we discuss the utility and design of multiple inhibitor experiments and present a new local curve fitting method for analyzing these data utilizing IC50 replots. The IC50 replot method is analogous to that used for determining mechanisms of inhibition with respect to substrate, as originally proposed by Cheng and Prusoff (Cheng and Prusoff Biochem Pharmacol 22: 3099-3108, 1973). The IC50 replot generated by this method reveals distinct patterns that are diagnostic of the nature of the interaction between two inhibitors. Multiple inhibition of the histone methyltransferase EZH2 by EPZ-5687 and the reaction product S-adenosylhomocysteine is presented as an example of the method.

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

NUT Midline Carcinoma: Morphoproteomic Characterization with Genomic and Therapeutic Correlates.

NUT midline carcinoma is a rare entity arising primarily in the midline of teenagers and young adults. Genomically, it is associated with a translocation involving a nuclear protein in testis (NUT) gene with other genes, most commonly, the BRD4 gene. The resultant is a partial or near total block in differentiation of tumor cells into mature squamous elements. Such tumors are resistant to conventional therapy with a reported mean survival at less than 1 year. In this study, we investigated two cases with genomic confirmation as NUT midline carcinoma by morphoproteomic analysis using immunohistochemical antibodies. Our results showed overexpression, largely in the undifferentiated cells of the tumors of: 1) Stemness marker, SRY (sex determining region Y)-box 2 (Sox2); 2) Constitutive activation of the mTORC2 pathway with expression of total insulin-like growth factor-1 receptor (IGF-1R[Tyr1165/1166]), and nuclear p-mTOR (Ser 2448) and p-Akt (Ser 473); and 3) c-Myc, silent mating type information regulation 2 homolog 1 (Sirt1) and histone methyltransferase enhancer of Zeste, Drosophila, homolog 2 (EZH2) as molecular impediments to differentiation. These data were analyzed through the use of QIAGEN's Ingenuity(®) Pathway Analysis (IPA(®), QIAGEN Redwood City, www.qiagen.com/ingenuity). The results established the interconnection of these pathways and molecules, and identified several pharmacogenomic agents--melatonin, metformin, vorinostat, curcumin, and sulforaphane--that have the potential to remove the block in differentiation and lead to the establishment of a more benign form of NUT midline carcinoma.

Saturated lipids decrease mitofusin 2 leading to endoplasmic reticulum stress activation and insulin resistance in hypothalamic cells.

Endoplasmic reticulum (ER) and mitochondria dysfunction contribute to insulin resistance generation during obesity and diabetes. ER and mitochondria interact through Mitofusin 2 (MTF2), which anchors in the outer mitochondrial and ER membranes regulating energy metabolism. Ablation of MTF2 leads to ER stress activation and insulin resistance. Here we determine whether lipotoxic insult induced by saturated lipids decreases MTF2 expression leading to ER stress response in hypothalamus and its effects on insulin sensitivity using in vitro and in vivo models. We found that lipotoxic stimulation induced by palmitic acid, but not the monounsaturated palmitoleic acid, decreases MTF2 protein levels in hypothalamic mHypoA-CLU192 cells. Also, palmitic acid incubation activates ER stress response evidenced by increase in the protein levels of GRP78/BIP marker at later stage than MTF2 downregulation. Additionally, we found that MTF2 alterations induced by palmitic, but not palmitoleic, stimulation exacerbate insulin resistance in hypothalamic cells. Insulin resistance induced by palmitic acid is prevented by pre-incubation of the anti-inflammatory and the ER stress release reagents, sodium salicylate and 4 phenylbutirate, respectively. Finally, we demonstrated that lipotoxic insult induced by high fat feeding to mice decreases MTF2 proteins levels in arcuate nucleus of hypothalamus. Our data indicate that saturated lipids modulate MTF2 expression in hypothalamus coordinating the ER stress response and the susceptibility to insulin resistance.

Polycomb recruitment at the Class II transactivator gene.

The Class II Transactivator (CIITA) is the master regulator of Major Histocompatibility Class II (MHC II) genes. Transcription of CIITA through the IFN-γ inducible CIITA promoter IV (CIITA pIV) during activation is characterized by a decrease in trimethylation of histone H3 lysine 27 (H3K27me3), catalyzed by the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2). While EZH2 is the known catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and is present at the inactive CIITA pIV, the mechanism of PRC2 recruitment to mammalian promoters remains unknown. Here we identify two DNA-binding proteins, which interact with and regulate PRC2 recruitment to CIITA pIV. We demonstrate Yin Yang 1 (YY1) and Jumonji domain containing protein 2 (JARID2) are binding partners along with EZH2 in mammalian cells. Upon IFN-γ stimulation, YY1 dissociates from CIITA pIV while JARID2 binding to CIITA pIV increases, suggesting novel roles for these proteins in regulating expression of CIITA pIV. Knockdown of YY1 and JARID2 yields decreased binding of EZH2 and H3K27me3 at CIITA pIV, suggesting important roles for YY1 and JARID2 at CIITA pIV. JARID2 knockdown also results in significantly elevated levels of CIITA mRNA upon IFN-γ stimulation. This study is the first to identify novel roles of YY1 and JARID2 in the epigenetic regulation of the CIITA pIV by recruitment of PRC2. Our observations indicate the importance of JARID2 in CIITA pIV silencing, and also provide a novel YY1-JARID2-PRC2 regulatory complex as a possible explanation of differential PRC2 recruitment at inducible versus permanently silenced genes.

Polycomb binding precedes early-life stress responsive DNA methylation at the Avp enhancer.

Early-life stress (ELS) in mice causes sustained hypomethylation at the downstream Avp enhancer, subsequent overexpression of hypothalamic Avp and increased stress responsivity. The sequence of events leading to Avp enhancer methylation is presently unknown. Here, we used an embryonic stem cell-derived model of hypothalamic-like differentiation together with in vivo experiments to show that binding of polycomb complexes (PcG) preceded the emergence of ELS-responsive DNA methylation and correlated with gene silencing. At the same time, PcG occupancy associated with the presence of Tet proteins preventing DNA methylation. Early hypothalamic-like differentiation triggered PcG eviction, DNA-methyltransferase recruitment and enhancer methylation. Concurrently, binding of the Methyl-CpG-binding and repressor protein MeCP2 increased at the enhancer although Avp expression during later stages of differentiation and the perinatal period continued to increase. Overall, we provide evidence of a new role of PcG proteins in priming ELS-responsive DNA methylation at the Avp enhancer prior to epigenetic programming consistent with the idea that PcG proteins are part of a flexible silencing system during neuronal development.

Identification and Validation of a Putative Polycomb Responsive Element in the Human Genome.

Epigenetic cellular memory mechanisms that involve polycomb and trithorax group of proteins are well conserved across metazoans. The cis-acting elements interacting with these proteins, however, are poorly understood in mammals. In a directed search we identified a potential polycomb responsive element with 25 repeats of YY1 binding motifthatwe designate PRE-PIK3C2B as it occurs in the first intron of human PIK3C2B gene. It down regulates reporter gene expression in HEK cells and the repression is dependent on polycomb group of proteins (PcG). We demonstrate that PRE-PIK3C2B interacts directly with YY1 in vitro and recruits PRC2 complex in vivo. The localization of PcG proteins including YY1 to PRE-PIK3C2B in HEK cells is decreased on knock-down of either YY1 or SUZ12. Endogenous PRE-PIK3C2B shows bivalent marking having H3K27me3 and H3K4me3 for repressed and active state respectively. In transgenic Drosophila, PRE-PIK3C2B down regulates mini-white expression, exhibits variegation and pairing sensitive silencing (PSS), which has not been previously demonstrated for mammalian PRE. Taken together, our results strongly suggest that PRE-PIK3C2B functions as a site of interaction for polycomb proteins.

YY1 controls Igκ repertoire and B-cell development, and localizes with condensin on the Igκ locus.

Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.

X-inactivation: quantitative predictions of protein interactions in the Xist network.

The transcriptional silencing of one of the female X-chromosomes is a finely regulated process that requires accumulation in cis of the long non-coding RNA X-inactive-specific transcript (Xist) followed by a series of epigenetic modifications. Little is known about the molecular machinery regulating initiation and maintenance of chromosomal silencing. Here, we introduce a new version of our algorithm catRAPID to investigate Xist associations with a number of proteins involved in epigenetic regulation, nuclear scaffolding, transcription and splicing processes. Our method correctly identifies binding regions and affinities of protein interactions, providing a powerful theoretical framework for the study of X-chromosome inactivation and other events mediated by ribonucleoprotein associations.

Recruitment and biological consequences of histone modification of H3K27me3 and H3K9me3.

Two histone marks, H3K27me3 and H3K9me3, are well known for their repressive roles in the genic and nongenic regions of metazoan genomes. Several protein complexes are known to be responsible for generating these marks, including polycomb repression complex 2 and several H3K9 methylases. Recent studies have shown that the targeting of these histone-modifying complexes within mammalian genomes may be mediated through several DNA-binding proteins, including AEBP2, JARID2, and YY1. In this review, we discuss the potential targeting mechanisms in light of the recent results that have been derived from genome-wide chromatin immunoprecipitation sequencing data and the in vivo functions of these two histone marks in light of the results derived from mouse and human genetic studies.