Dynamic methylation of histone H3K18 in differentiating Theileria parasites.

Lysine methylation on histone tails impacts genome regulation and cell fate determination in many developmental processes. Apicomplexa intracellular parasites cause major diseases and they have developed complex life cycles with fine-tuned differentiation events. Yet, apicomplexa genomes have few transcription factors and little is known about their epigenetic control systems. Tick-borne Theileria apicomplexa species have relatively small, compact genomes and a remarkable ability to transform leucocytes in their bovine hosts. Here we report enriched H3 lysine 18 monomethylation (H3K18me1) on the gene bodies of repressed genes in Theileria macroschizonts. Differentiation to merozoites (merogony) leads to decreased H3K18me1 in parasite nuclei. Pharmacological manipulation of H3K18 acetylation or methylation impacted parasite differentiation and expression of stage-specific genes. Finally, we identify a parasite SET-domain methyltransferase (TaSETup1) that can methylate H3K18 and represses gene expression. Thus, H3K18me1 emerges as an important epigenetic mark which controls gene expression and stage differentiation in Theileria parasites.

The Transcription Factor p63 Is a Direct Effector of IL-4- and IL-13-Mediated Repression of Keratinocyte Differentiation.

Atopic Dermatitis is an inflammatory skin disease associated with broad defects in skin barrier function caused by increased levels of type-2 cytokines (IL-4 and IL-13) that repress keratinocyte (KC) differentiation. Although crucial in mediating allergic disease, the mechanisms for gene repression induced by type-2 cytokines remain unclear. In this study, we determined that gene repression requires the master regulator of the epidermal differentiation program, p63. We found that type-2 cytokine-mediated inhibition of the expression of genes involved in early KC differentiation, including keratin 1, keratin 10, and DSC-1, is reversed by p63 blockade. Type-2 cytokines, through p63, also regulate additional genes involved in KC differentiation, including CHAC-1, STC2, and CALML5. The regulation of the expression of these genes is ablated by p63 small interfering RNA as well. In addition, we found that IL-4 and IL-13 and Staphylococcus aureus lipoteichoic acid work in combination through p63 to further suppress the early KC differentiation program. Finally, we found that IL-4 and IL-13 also inhibit the activity of Notch, a transcription factor required to induce early KC differentiation. In conclusion, type-2 cytokine-mediated gene repression and blockade of KC differentiation are multifactorial, involving pathways that converge on transcription factors critical for epidermal development, p63 and Notch.

DNA Methylation-Based Epigenetic Repression of SLC22A4 Promotes Resistance to Cytarabine in Acute Myeloid Leukemia.

Reduced expression of the uptake transporter, OCTN1 (SLC22A4), has been reported as a strong predictor of poor event-free and overall survival in multiple cohorts of patients with acute myeloid leukemia (AML) receiving the cytidine nucleoside analog, cytarabine (Ara-C). To further understand the mechanistic basis of interindividual variability in the functional expression of OCTN1 in AML, we hypothesized a mechanistic connection to DNA methylation-based epigenetic repression of SLC22A4. We found increased basal SLC22A4 methylation was associated with decreased Ara-C uptake in AML cell lines. Pre-treatment with hypomethylating agents, 5-azacytidine, or decitabine, restored SLC22A4 mRNA expression, increased cellular uptake of Ara-C, and was associated with increased cellular sensitivity to Ara-C compared with vehicle-treated cells. Additionally, lower SLC22A4 methylation status was associated with distinct clinical advantages in both adult and pediatric patients with AML. These findings suggest a regulatory mechanism is involved in the interindividual variability in response to Ara-C, and provides a basis for the integration of hypomethylating agents into Ara-C-based treatment regimens.

Synthesis and Biological Evaluation of B-Cell Lymphoma 6 Inhibitors of N-Phenyl-4-pyrimidinamine Derivatives Bearing Potent Activities against Tumor Growth.

The transcriptional repressor B-cell lymphoma 6 (BCL6) is frequently misregulated in diffuse large B-cell lymphoma (DLBCL) and has emerged as an attractive drug target for the treatments of lymphoma. In this article, a series of N-phenyl-4-pyrimidinamine derivatives were designed and synthesized as potent BCL6 inhibitors by optimizing hit compound N4-(3-chloro-4-methoxyphenyl)-N2-isobutyl-5-fluoro-2,4-pyrimidinediamine on the basis of the structure-activity relationship. Among them, compound 14j displayed the most potent activities, which significantly blocked the interaction of BCL6 with its corepressors, reactivated BCL6 target genes in a dose-dependent manner, and had better effects compared with the two positive controls. Further studies indicated that a low dose of 14j could effectively inhibit germinal center formation. More importantly, 14j not only showed potent inhibition of DLBCL cell proliferation in vitro but also strongly suppressed the growth of DLBCL in vivo.

Discovery and Structure-Based Optimization of Potent and Selective WD Repeat Domain 5 (WDR5) Inhibitors Containing a Dihydroisoquinolinone Bicyclic Core.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.

Tributyrin Inhibits Ethanol-Induced Epigenetic Repression of CPT-1A and Attenuates Hepatic Steatosis and Injury.

Ethanol-mediated down-regulation of carnitine palmitoyltransferase-1 (CPT-1A) gene expression plays a major role in the development of hepatic steatosis; however, the underlying mechanisms are not completely elucidated. Tributyrin, a butyrate prodrug that can inhibit histone deacetylase (HDAC) activity, attenuates hepatic steatosis and injury. The present study examined the beneficial effect of tributyrin/butyrate in attenuating ethanol-induced pathogenic epigenetic mechanisms affecting CPT-1A promoter-histone modifications and gene expression and hepatic steatosis/injury. METHODS: Mice were fed a liquid Lieber-DeCarli diet (Research Diet Inc, New Brunswick, NJ) with or without ethanol for 4 weeks. In a subset of mice, tributyrin (2 g/kg) was administered orally by gavage. Primary rat hepatocytes were treated with 50 mmol/L ethanol and/or 2 mmol/L butyrate. Gene expression and epigenetic modifications at the CPT-1A promoter were analyzed by chromatin immunoprecipitation analysis. RESULTS: In vivo, ethanol induced hepatic CPT-1A promoter histone H3K9 deacetylation, which is indicative of a repressive chromatin state, and decreased CPT-1A gene expression. Our data identified HDAC1 as the predominant HDAC causing CPT-1A promoter histone H3K9 deacetylation and epigenetic down-regulation of gene expression. Significantly, Specificity Protein 1 (SP1) and Hepatocyte Nuclear Factor 4 Alpha (HNF4α) participated in the recruitment of HDAC1 to the proximal and distal regions of CPT-1A promoter, respectively, and mediated transcriptional repression. Importantly, butyrate, a dietary HDAC inhibitor, attenuated ethanol-induced recruitment of HDAC1 and facilitated p300-HAT binding by enabling SP1/p300 interaction at the proximal region and HNF4α/peroxisomal proliferator-activated receptor-γ coactivator-1α/p300 interactions at the distal region, leading to promoter histone acetylation and enhanced CPT-1A transcription. CONCLUSIONS: This study identifies HDAC1-mediated repressive epigenetic mechanisms that underlie an ethanol-mediated decrease in CPT-1A expression. Importantly, tributyrin/butyrate inhibits HDAC1, rescues CPT-1A expression, and attenuates ethanol-mediated hepatic steatosis and injury, suggesting its potential use in therapeutic strategies for alcoholic liver disease.

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.

Inhibiting repressive epigenetic modification promotes telomere rejuvenation in somatic cell reprogramming.

The efficiency of somatic cell nuclear transfer (SCNT) reprogramming is extremely low in terms of production of cloned animals. Here, we found that telomere rejuvenation is a critical event for SCNT reprogramming. Through small-molecule screening, we identified that melatonin significantly improved the in vitro and in vivo developmental competence of SCNT-derived embryos. Through use of embryonic biopsy, single-cell RNA sequencing, and quantitative FISH experiments, we revealed that melatonin not only attenuated the zygotic genome activation defect but also facilitated telomere elongation in the SCNT embryos. Further investigation indicated that melatonin inhibited heterochromatic epigenetic modification related to gene silencing including DNA methylation and histone H3 lysine 9 trimethylation. In addition, melatonin could increase the level of activation markers such as acetylated histone H3. This is the first study to characterize melatonin-treatment and telomere rejuvenation in SCNT-mediated reprogramming. Moreover, combinational use of melatonin-treated donor embryos and pseudopregnant recipients achieved synergistic enhancement of the production of cloned animals.-Yang, L., Liu, X., Song, L., Su, G., Di, A., Bai, C., Wei, Z., Li, G. Inhibiting repressive epigenetic modification promotes telomere rejuvenation in somatic cell reprogramming.

HOTAIR-mediated reciprocal regulation of EZH2 and DNMT1 contribute to polyphyllin I-inhibited growth of castration-resistant prostate cancer cells in vitro and in vivo.

BACKGROUND: Polyphyllin I (PPI), one of the steroidal saponins in paris polyphylla, has been reported to exhibit antitumor effects. However, the detailed molecular mechanism underlying this has not been elucidated. METHODS: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Flow cytometry assays, respectively. Cell invasion and migration were examined by Transwell invasion and wound healing assays. Western blot analysis was performed to examine the protein expressions of zeste homolog 2 (EZH2), DNA methyltransferase 1 (DNMT1). QRT-PCR was used to examine the levels of long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR). Small interfering RNAs (siRNAs) method was used to knockdown HOTAIR. Exogenously expressions of HOTAIR, DNMT1 and EZH2 were carried out by Transient transfection assays. EZH2 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro. RESULTS: We showed that PPI significantly inhibited growth, induced cell cycle arrest of castration-resistant prostate cancer (CRPC) cells. In addition, PPI also reduced the migration and invasion in CRPC cells. In mechanism, we found that PPI decreased the protein expressions of EZH2, DNMT1 and levels of HOTAIR. Interestingly, silenced HOTAIR reduced EZH2 and DNMT1 protein expressions. On the contrary, exogenously expressed HOTAIR resisted PPI-inhibited EZH2 and DNMT1 protein expressions, EZH2 promoter activity and cell growth. Moreover, excessive EZH2 antagonized PPI-suppressed DNMT1 protein expression or vice versa. Consistent with this, PPI inhibited tumor growth, HOTAIR, the protein expressions of DNMT1 and EZH2 in vivo. CONCLUSION: Our results show that PPI inhibits growth of CRPC cells through inhibition of HOTAIR expression, subsequently; this results in the repression of DNMT1 and EZH2 expressions. The interactions among HOTAIR, DNMT1 and EZH2, and reciprocal regulation of DNMT1 and EZH2 contribute to the overall responses of PPI. This study reveals a novel mechanism for HOTAIR-mediated regulating DNMT1 and EZH2 in response to PPI in inhibition of the growth of CRPC cells.

Silica nanoparticle releases SIRT6-induced epigenetic silencing of follistatin.

Follistatin (FST) plays a protective role during silica nanoparticle (SiO2 NP) exposure. SiO2 NP treatment induces FST transcription with an unknown mechanism. We herein reported that SIRT6, one of the sirtuin family members, induced epigenetic silencing of FST. The expression of FST was elevated after SIRT6 knockdown while reduced after SIRT6 overexpression. Chromatin immunoprecipitation revealed a direct interaction between SIRT6 with FST promoter. Knockdown of SIRT6 increased both Ac-H3K9 level and Ac-H3K56 level at FST promoter region. SiO2 NP treatment de-stabilized SIRT6 mRNA and reduced SIRT6 expression, leading to the activation of FST transcription. Finally, over-expression of SIRT6 increased SiO2 NP-induced apoptosis. Collectively, this study provided evidence that SIRT6 is a negative regulator of FST transcription and participates in the regulation of cell survival during silica nanoparticle exposure.

Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure.

Maintenance of phenotypic heterogeneity within cell populations is an evolutionarily conserved mechanism that underlies population survival upon stressful exposures. We show that the genomes of a cancer cell subpopulation that survives treatment with otherwise lethal drugs, the drug-tolerant persisters (DTPs), exhibit a repressed chromatin state characterized by increased methylation of histone H3 lysines 9 and 27 (H3K9 and H3K27). We also show that survival of DTPs is, in part, maintained by regulators of H3K9me3-mediated heterochromatin formation and that the observed increase in H3K9me3 in DTPs is most prominent over long interspersed repeat element 1 (LINE-1). Disruption of the repressive chromatin over LINE-1 elements in DTPs results in DTP ablation, which is partially rescued by reducing LINE-1 expression or function.

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells.

Epigenetic regulation of RGS2 (Regulator of G-protein signaling 2) in chemoresistant ovarian cancer cells.

Regulator of G-protein signaling 2 (RGS2) is a GTPase-activating protein functioning as an inhibitor of G-protein coupled receptors (GPCRs). RGS2 dysregulation was implicated in solid tumour development and RGS2 downregulation has been reported in prostate and ovarian cancer progression. However, the molecular mechanism by which RGS2 expression is suppressed in ovarian cancer remains unknown. The expression and epigenetic regulation of RGS2 in chemosensitive and chemoresistant ovarian cancer cells were determined by qRT-PCR and chromatin immunoprecipitation assays, respectively. In the present study, the molecular mechanisms contributing to the loss of RGS2 expression were determined in ovarian cancer. The data indicated that suppression of RGS2 gene in chemoresistant ovarian cancer cells, in part, due to accumulation of histone deacetylases (HDACs) and DNA methyltransferase I (DNMT1) at the promoter region of RGS2. Inhibition of HDACs or DNMTs significantly increases RGS2 expression. These results suggest that epigenetic changes in histone modifications and DNA methylation may contribute to the loss of RGS2 expression in chemoresistant ovarian cancer cells. The results further suggest that class I HDACs and DNMT1 contribute to the suppression of RGS2 during acquired chemoresistance and support growing evidence that inhibition of HDACs/DNMTs represents novel therapeutic approaches to overcome ovarian cancer chemoresistance.

Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene.

Sensitization of androgen refractory prostate cancer cells to anti-androgens through re-expression of epigenetically repressed androgen receptor - Synergistic action of quercetin and curcumin.

Epigenetic repression of Androgen Receptor (AR) gene by hypermethylation of its promoter causes resistance in prostate cancer (CaP) to androgen deprivation therapy with anti-androgens. Some dietary phytocompounds like quercetin (Q) and curcumin (C) with reported DNMT-inhibitory activity were tested for their ability to re-express the AR in AR-negative CaP cell lines PC3 and DU145. Combined treatment with Q+C was much more effective than either Q or C in inhibiting DNMT, causing global hypomethylation, restoring AR mRNA and protein levels and causing apoptosis via mitochondrial depolarization of PC3 and DU145. The functional AR protein expressed in Q+C treated cells sensitized them to dihydrotestosterone (DHT)-induced proliferation, bicalutamide-induced apoptosis, bound to androgen response element to increase luciferase activity in gene reporter assay and was susceptible to downregulation by AR siRNA. Bisulfite sequencing revealed high methylation of AR promoter CpG sites in AR-negative DU145 and PC3 cell lines that was significantly demethylated by Q+C treatment, which restored AR expression. Notable synergistic effects of Q+C combination in re-sensitizing androgen refractory CaP cells to AR-mediated apoptosis, their known safety in clinical use, and epidemiological evidences relating their dietary consumption with lower cancer incidences indicate their potential for use in chemoprevention of androgen resistance in prostate cancer.

MEIS3 is repressed in A549 lung epithelial cells by deoxynivalenol and the repression contributes to the deleterious effect.

Deoxynivalenol (DON) is an important Fusarium toxin of concern for food safety. The inhalation of powder contaminated with DON is possible and may cause lung toxicity. In this study, we analyzed the gene expression profile of A549 cells treated for 24 hr with 0.2 µg/mL DON by microarray analysis. In total, 16 genes and 5 noncoding RNAs were significantly affected by DON treatment. The repression of B3GALT4, MEIS3, AK7, SEMA3A, KCNMB4, and SCARA5 was confirmed by quantitative PCR. We investigated the DON toxicity on A549 cells that exogenously expressed these 6 genes. The result indicated that A549 cells that transiently expressed MEIS3 were tolerant to the deleterious effects of DON. Our data show that DON affected the expression of genes with various functions, and suggest that the repression of MEIS3 plays roles in the deleterious effect of DON in A549 lung epithelial cells.

Pb2+ induces gastrin gene expression by extracellular signal-regulated kinases 1/2 and transcription factor activator protein 1 in human gastric carcinoma cells.

Divalent lead ions (Pb(2+) ) are toxic environmental pollutants known to cause serious health problems in humans and animals. Absorption of Pb(2+) from air, water, and food takes place in the respiratory and digestive tracts. The ways in which absorbed Pb(2+) affects cell physiology are just beginning to be understood at the molecular level. Here, we used reverse transcription PCR and Western blotting to analyze cultures of human gastric carcinoma cells exposed to 10 µM lead nitrate. We found that Pb(2+) induces gastrin hormone gene transcription and translation in a time-dependent manner. Promoter deletion analysis revealed that activator protein 1 (AP1) was necessary for gastrin gene transcription in cells exposed to Pb(2+) . MitogIen-activated protein kinase (MAPK)/ERK kinase inhibitor PD98059 suppressed the Pb(2+) -induced increase in messenger RNA. Epidermal growth factor receptor (EGFR) inhibitors AG1478 and PD153035 reduced both transcription and phosphorylation by extracellular signal-regulated kinase (ERK1/2). Cells exposed to Pb(2+) also increased production of c-Jun protein, a component of AP1, and over-expression of c-Jun enhanced activation of the gastrin promoter. In sum, the findings suggest the EGFR-ERK1/2-AP1 pathway mediates the effects of Pb(2+) on gastrin gene activity in cell culture.

EZH2-induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in ovarian cancer.

Epithelial ovarian cancer (EOC) is the second leading cause of death from gynecological malignancies worldwide. Enhancer of zeste homology 2 (EZH2), participating in gene expression silencing by trimethylating histone 3 lysine 27 (H3K27me3), is often up-regulated in EOC. ARHI, an imprinted tumor-suppressor gene, is markedly down-regulated or even undetectable in the majority of EOC. To explore the correlation between EZH2 and ARHI expression in EOC as well as the possible mechanism of EZH2-ARH1 interaction. We used immunohistochemical staining to evaluate the expression of EZH2 and ARHI in EOC and normal ovarian tissue specimens; western blotting, shRNA, and chromatin immunoprecipitation were used to study the expression correlation of EZH2 and ARHI in EOC and normal ovarian epithelial cells and to further explore the mechanism of EZH2 regulation of ARHI expression. Cell viability assay was used to evaluate the influence of these two genes on cell survival. (1) The expression of EZH2 inversely correlated with ARHI expression levels and predicted shorter overall survival in EOC patients; (2) EZH2 promoted repression of ARHI by catalyzing trimethylation on H3K27; (3) ARHI was synergistically silenced by DNA methylation and histone modification; and (4) DZNep, an inhibitor of EZH2, significantly reduced survival rate of EOC cells by restoring ARHI expression. EZH2-″induced H3K27me3 is associated with epigenetic repression of the ARHI tumor-suppressor gene in EOC. Suppression of EZH2 by DZNep, as a way of restoring the expression of ARHI, could be a potential treatment modality to EOC.

Hormone-responsive enhancer-activity maps reveal predictive motifs, indirect repression, and targeting of closed chromatin.

Steroid hormones act as important developmental switches, and their nuclear receptors regulate many genes. However, few hormone-dependent enhancers have been characterized, and important aspects of their sequence architecture, cell-type-specific activating and repressing functions, or the regulatory roles of their chromatin structure have remained unclear. We used STARR-seq, a recently developed enhancer-screening assay, and ecdysone signaling in two different Drosophila cell types to derive genome-wide hormone-dependent enhancer-activity maps. We demonstrate that enhancer activation depends on cis-regulatory motif combinations that differ between cell types and can predict cell-type-specific ecdysone targeting. Activated enhancers are often not accessible prior to induction. Enhancer repression following hormone treatment seems independent of receptor motifs and receptor binding to the enhancer, as we show using ChIP-seq, but appears to rely on motifs for other factors, including Eip74. Our strategy is applicable to study signal-dependent enhancers for different pathways and across organisms.

Autism's cancer connection: the anti-proliferation hypothesis and why it may matter.

Autism remains an idiopathic disorder in 90% of cases. Recent prevalence, heritability, and genetic studies are suggestive that epigenetic and, therefore, environmental factors are important in autism pathogenesis. Among the environmental factors, only some uncommon viral infections and certain drugs have been conclusively linked to autism causation. Thalidomide, valproate, terbutaline and, most recently, antidepressants are the main drugs reported to elevate autism risk. This article discusses a phenomenal relationship between the drugs reported to elevate autism risk and the antiproliferative effects of the same drugs and/or analogs of the drugs in cancer cells. Cancer treatment has entered a new era-epigenetic therapy. In cancer cell lines, thalidomide is antiangiogenic and antiproliferative via suppression of tumor necrosis factor-alpha (TNF-α) and downstream effects on the nuclear factor (NFκB) cascade. Valproate shares similar mechanisms with thalidomide, but is best known in cancer therapy for its epigenetic effects as a histone deacetylase inhibitor. Terbutaline, a beta-adrenergic agonist, acts via adenylyl cyclase and cAMP-PKA signal transduction. Current cancer therapy aims to exploit this epigenetic pathway by developing site-selective cAMP analogs. Last, it has long been noted in preclinical studies that some antidepressants are antiproliferative in cancer cells but the mechanisms remain unclear. Based on a systematic review of these drugs, it is hypothesized that all central nervous system-acting drugs, which show antiproliferative effects in cancer cell lines, share the potential to elevate autism risk when administered prenatally. It is further posited that, in autism, the drugs act as "triggers" that disturb the pro-proliferative fetal milieu using the same, mainly epigenetic, mechanisms that they demonstrate in rapidly proliferating cancer cells. In addition to their direct antiproliferative effects, evidence is suggestive that the drugs may lock in the pro-inflammatory bias of the prenatal immune system by preventing normal perinatal dendritic cell maturation. This unifying hypothesis for how structurally different drugs elevate autism risk could help focus research on other drugs, or other xenobiotics, that may elevate autism risk. For example, there is evidence that an old class of drugs, the phenothiazines, is antiproliferative in cancer cell lines via inhibition of calmodulin and/or histaminic pathways. Promethazine, one of the first heterocyclic phenothiazines synthesized, is commonly prescribed during pregnancy; however, its role in elevating the risk of autism has not been investigated. Based on the anti-proliferation hypothesis, more studies of promethazine and other similar drugs are suggested to evaluate their potential to elevate autism risk following prenatal exposures.