Transcriptional Derepression Research Articles

The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Interplay between Active Chromatin Marks and RNA-Directed DNA Methylation in Arabidopsis thaliana

DNA methylation is an epigenetic mark that is associated with transcriptional repression of transposable elements and protein-coding genes. Conversely, transcriptionally active regulatory regions are strongly correlated with histone 3 lysine 4 di- and trimethylation (H3K4m2/m3). We previously showed that Arabidopsis thaliana plants with mutations in the H3K4m2/m3 demethylase JUMONJI 14 (JMJ14) exhibit a mild reduction in RNA-directed DNA methylation (RdDM) that is associated with an increase in H3K4m2/m3 levels. To determine whether this incomplete RdDM reduction was the result of redundancy with other demethylases, we examined the genetic interaction of JMJ14 with another class of H3K4 demethylases: LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 1 and LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 2 (LDL1 and LDL2). Genome-wide DNA methylation analyses reveal that both families cooperate to maintain RdDM patterns. ChIP-seq experiments show that regions that exhibit an observable DNA methylation decrease are co-incidental with increases in H3K4m2/m3. Interestingly, the impact on DNA methylation was stronger at DNA-methylated regions adjacent to H3K4m2/m3-marked protein-coding genes, suggesting that the activity of H3K4 demethylases may be particularly crucial to prevent spreading of active epigenetic marks. Finally, RNA sequencing analyses indicate that at RdDM targets, the increase of H3K4m2/m3 is not generally associated with transcriptional de-repression. This suggests that the histone mark itself—not transcription—impacts the extent of RdDM.

Abstract CN07-04: Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling.

Abstract Thyroid cancers are associated with mutually exclusive point mutations of BRAF (57%) and RAS (NRAS >HRAS; 12%), and fusion events activating BRAF, or the RTKs RET, NTRK1 or NTRK3. The molecular taxonomy of the disease is different in more advanced forms of thyroid cancer. Although BRAF mutations also predominate, poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC) are relatively enriched for RAS mutations (23-44%). As predicted from the genetics of the disease, thyroid cancers, like melanomas, are ERK-driven tumors. However, they exhibit lineage-specific properties that offer distinct therapeutic opportunities and challenges. The 10-year survival of patients with metastatic thyroid cancers that retain radioiodine (RAI) avidity is ∼60%, whereas it is only 10% if the metastases are refractory to RAI therapy (1). There are several lines of evidence indicating that constitutive MAPK activity causes decreased radioiodine responsiveness. Conditional expression of oncogenic RET/PTC, RAS or BRAF in thyroid PCCL3 cells downregulates expression of the sodium iodide symporter (NIS) and other key genes required to organify and retain iodide (2, 3), which is restored by treatment with RAF or MEK inhibitors (2, 4). Mice with dox-inducible expression of BRAFV600E in thyroid cells develop invasive papillary thyroid cancers with profoundly impaired expression of genes required for iodine uptake and metabolism and of 124-iodine uptake in vivo, which are reversed when dox is discontinued, but only partially restored by RAF or MEK inhibitors (5). Based on these findings we performed a phase 2 trial of the MEK inhibitor selumetinib (AZD6244) in patients with RAI-refractory thyroid cancer. Selumetinib increased iodide incorporation at metastatic sites in 12/20 patients as determined by 124I PET scanning, and in ∼40% (8/20) the increase in uptake was sufficient to enable treatment with 131I, with remarkable clinical responses (6). Responses were greater in patients whose tumors harbored RAS as compared to BRAF mutations. An international multicenter phase 3 registration study of selumetinib vs placebo to increase remission rates of high risk thyroid cancer patients receiving post-surgical adjuvant radioiodine therapy is now in progress (the ASTRA trial). There is concern that responses to monotherapy with MEK or RAF inhibitors may not be optimal, particularly in BRAF-mutant disease. We find that BRAF-mutant human thyroid cancer cell lines have decreased responsiveness to RAF and MEK inhibitors because of relief of negative feedback resulting in derepression of HER3 transcription, followed by neuregulin-1- induced HER3/HER2 signaling and reactivation of the MEK-ERK and PI3K-AKT pathways, which is overcome by co-treatment with HER kinase inhibitors (7). This mechanism of adaptive resistance differs in significant ways from the responses seen in BRAF-mutant melanoma and colorectal cell lines. Our preclinical data predicts that optimal restoration of RAI responsiveness would be best achieved by compounds that inhibit MAPK output in a profound and sustained way. Indeed, recent experiments show that such strategies have excellent potential to further augment the specialized functions of thyroid cells, and lead to even more effective therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN07-04. Citation Format: James A. Fagin. Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN07-04.

Abstract IA17: Targeting the methylome for epigenetic resensitization of ovarian cancer

Abstract Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. Although most patients respond to platinum-based chemotherapy, relapses are common, leading to platinum-resistant OC, which is uniformly fatal. OC progression is associated with accumulation of epigenetic alterations. In particular, deoxycytosine methylation of CpG islands in promoter regions of tumor suppressor genes (TSGs) plays a prominent role in the development and progression of drug-resistant epithelial OC. We recently demonstrated for the first time in a clinical trial that therapeutic interventions targeting the OC methylome reverse drug resistance and induce meaningful clinical responses. We showed that repetitive low-dose decitabine reactivated silenced genes and restored sensitivity to carboplatin, providing strong clinical and biological support for further study of hypomethylating agents in heavily pre-treated, platinum-resistant ovarian cancer patients. While the FDA-approved demethylating agent decitabine is prone to deamination by cytidine deaminase, SGI-110 (Astex Pharmaceuticals, Inc.), a dinucleotide analogue of decitabine, is more stable, less toxic, and a promising alternative to restoring silenced TSG expression in cancer cells by reversal of DNA methylation. Our preclinical evaluation demonstrated that SGI-110 resensitized platinum-resistant OC cell lines to cisplatin (CDDP) (3-fold reduction in IC50) and reduced the CDDP IC50. SGI-110 treatment induced significant demethylation and subsequent transcriptional derepression of tumor suppressors and differentiation-associated genes in OC cells. SGI-110 alone or in combination with CDDP was well tolerated in non-tumor bearing mice. Significant antitumor activity was observed in mice harboring subcutaneous OC tumors and treated with single SGI-110 and SGI-110 + CDDP treatment in both a biweekly and daily (QD5) regimen. In addition to reducing tumor growth in xenografts, SGI therapy was effective in causing global as well as TSG demethylation and gene reexpression in vivo. Furthermore, the antitumor activity of SGI-110 was associated with reduced chromatin compaction, allowing greater CDDP intercalation into DNA, as assessed by increased DNA platinum adducts in SGI-treated OC cells. The results of our preclinical study support our recently activated clinical trial NCT01696032 using SGI-110 in combination with carboplatin in patients with recurrent, platinum-resistant OC. Clinical specimens (tumor and plasma samples) will be analyzed for epigenetic biomarker changes. We seek to bring forward the new concept of epigenetic targeting in platinum resistant OC by priming the tumors with SGI-110 and set the stage for interventions targeting the OC epigenome, as well as guide and impact the design of future clinical investigations in OC. Citation Format: Kenneth P. Nephew, Daniela Matei, Pietro Taverna, Fang Fang, Jessica Tang, Gavin Choy, John Lyons, Mohammad Azab, Jay Pilrose, John Turchi. Targeting the methylome for epigenetic resensitization of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA17.

Dissociation of the H3K36 demethylase Rph1 from chromatin mediates derepression of environmental stress-response genes under genotoxic stress inSaccharomyces cerevisiae

Cells respond to environmental signals by altering gene expression through transcription factors. Rph1 is a histone demethylase containing a Jumonji C (JmjC) domain and belongs to the C(2)H(2) zinc-finger protein family. Here we investigate the regulatory network of Rph1 in yeast by expression microarray analysis. More than 75% of Rph1-regulated genes showed increased expression in the rph1-deletion mutant, suggesting that Rph1 is mainly a transcriptional repressor. The binding motif 5'-CCCCTWA-3', which resembles the stress response element, is overrepresented in the promoters of Rph1-repressed genes. A significant proportion of Rph1-regulated genes respond to DNA damage and environmental stress. Rph1 is a labile protein, and Rad53 negatively modulates Rph1 protein level. We find that the JmjN domain is important in maintaining protein stability and the repressive effect of Rph1. Rph1 is directly associated with the promoter region of targeted genes and dissociated from chromatin before transcriptional derepression on DNA damage and oxidative stress. Of interest, the master stress-activated regulator Msn2 also regulates a subset of Rph1-repressed genes under oxidative stress. Our findings confirm the regulatory role of Rph1 as a transcriptional repressor and reveal that Rph1 might be a regulatory node connecting different signaling pathways responding to environmental stresses.

New regulatory circuit controlling spatial and temporal gene expression in the sea urchin embryo oral ectoderm GRN

The sea urchin oral ectoderm gene regulatory network (GRN) model has increased in complexity as additional genes are added to it, revealing its multiple spatial regulatory state domains. The formation of the oral ectoderm begins with an oral–aboral redox gradient, which is interpreted by the cis-regulatory system of the nodal gene to cause its expression on the oral side of the embryo. Nodal signaling drives cohorts of regulatory genes within the oral ectoderm and its derived subdomains. Activation of these genes occurs sequentially, spanning the entire blastula stage. During this process the stomodeal subdomain emerges inside of the oral ectoderm, and bilateral subdomains defining the lateral portions of the future ciliary band emerge adjacent to the central oral ectoderm. Here we examine two regulatory genes encoding repressors, sip1 and ets4, which selectively prevent transcription of oral ectoderm genes until their expression is cleared from the oral ectoderm as an indirect consequence of Nodal signaling. We show that the timing of transcriptional de-repression of sip1 and ets4 targets which occurs upon their clearance explains the dynamics of oral ectoderm gene expression. In addition two other repressors, the direct Nodal target not, and the feed forward Nodal target goosecoid, repress expression of regulatory genes in the central animal oral ectoderm thereby confining their expression to the lateral domains of the animal ectoderm. These results have permitted construction of an enhanced animal ectoderm GRN model highlighting the repressive interactions providing precise temporal and spatial control of regulatory gene expression.

NDRG2 positively regulates E-cadherin expression and prolongs overall survival in colon cancer patients

To discover the molecular mechanism of N-Myc downstream-regulated gene 2 (NDRG2), a newly found differentiation-related tumor suppressor, the relationships between NDRG2 and E-cadherin were investigated in tumor cells and tissues. Positive correlations between the expression of E-cadherin and NDRG2 were shown in several colon cancer cell lines as well as in colon cancer tissues. According to the transcription assays using a reporter plasmid containing E-cadherin promoter region (-368~+51), NDRG2 introduction into colon cancer cell lines induced upregulation of E-cadherin promoter activity and its transcription. On the contrary, inhibition of NDRG2 expression by siRNA treatment caused the decrease of E-cadherin transcription. Snail, a zinc-finger transcriptional repressor, was shown to be a mediator of NDRG2-regulated E-cadherin expression. The enhancement of glycogen synthase kinase 3β (GSK-3β) activity by NDRG2 overexpression caused proteasomal degradation of Snail transcription factor followed by transcriptional de-repression of E-cadherin. We also found that NDRG2 could mediate cell density-regulated E-cadherin expression. The increase of NDRG2 expression with cell density preceded E-cadherin expression, and the regulation of Snail activity by GSK-3β was also related to this process.

Structural Basis of Multisite Single-Stranded DNA Recognition and ACTA2 Repression by Purine-Rich Element Binding Protein B (Purβ)

A hallmark of dysfunctional fibroblast to myofibroblast differentiation associated with fibrotic disorders is persistent expression of ACTA2, the gene encoding the cyto-contractile protein smooth muscle α-actin. In this study, a PURB-specific gene knockdown approach was used in conjunction with biochemical analyses of protein subdomain structure and function to reveal the mechanism by which purine-rich element binding protein B (Purβ) restricts ACTA2 expression in mouse embryo fibroblasts (MEFs). Consistent with the hypothesized role of Purβ as a suppressor of myofibroblast differentiation, stable short hairpin RNA-mediated knockdown of Purβ in cultured MEFs promoted changes in cell morphology, actin isoform expression, and cell migration indicative of conversion to a myofibroblast-like phenotype. Promoter-reporter assays in transfected Purβ knockdown MEFs confirmed that these changes were attributable, in part, to derepression of ACTA2 transcription. To map the domains in Purβ responsible for ACTA2 repression, several recombinant truncation mutants were generated and analyzed based on hypothetical, computationally derived models of the tertiary and quaternary structure of Purβ. Discrete subdomains mediating sequence- and strand-specific cis-element binding, protein-protein interaction, and inhibition of a composite ACTA2 enhancer were identified using a combination of biochemical, biophysical, and cell-based assays. Our results indicate that the Purβ homodimer possesses three separate but unequal single-stranded DNA-binding modules formed by subdomain-specific inter- and intramolecular interactions. This structural arrangement suggests that the cooperative assembly of the dimeric Purβ repressor on the sense strand of the ACTA2 enhancer is dictated by the association of each subdomain with distinct purine-rich binding sites within the enhancer.

Specific Requirement of Gli Transcription Factors in Hedgehog-mediated Intestinal Development

Hedgehog (Hh) signaling is involved in multiple aspects of embryonic gut development, including mesenchymal growth and smooth muscle differentiation. The Gli family transcription factors is thought to collectively mediate Hh signaling in mammals. However, the function of different Gli proteins in gut development remains uncharacterized. Here, we genetically dissect the contribution of Gli transcriptional activation and de-repression in intestinal growth and patterning. We find that removal of the Gli3 repressor is dispensable for intestinal development and does not play a major role in Hh-controlled gut development. However, Gli2 activation is able to fully rescue the Smoothened (Smo)-null intestinal phenotype, suggesting that the Gli2 transcription factor is the main effector for Hh signaling in the intestine. To understand further the molecular mechanism underlying Hh/Gli function in the developing gut, we identify a subset of small leucine-rich glycoproteins (SLRPs) that may function downstream of Hh signaling in the mesenchyme. We show that osteoglycin, a SLRP, inhibits Hh-induced differentiation toward the smooth muscle lineage in C3H10T1/2 pluripotent mesenchymal cells. Taken together, our study reveals, for the first time, the distinct roles of Gli proteins in intestine development and suggests SLRPs as novel regulators of smooth muscle cell differentiation.

CK2-NCoR signaling cascade promotes prostate tumorigenesis

The aberrant expressions of casein kinase 2 (CK2) was found in prostate cancer patient and cell lines, but little is known of the detailed mechanisms implicated in prostate tumorigenesis. In this study, we report that both CK2 activity and CK2-mediated NCoR phosphorylation are significantly elevated in the androgen-independent prostate cancer cell line DU145 and PC-3 compared with RWPE1 and LNCaP cells. Increased phosphorylation inversely correlates with the mRNA level of the NCoR-regulated gene, interferon-γ-inducible protein 10 (IP-10). CK2 inhibition abrogated NCoR phosphorylation, IP-10 transcriptional repression, and the invasion activity of PC-3 cells. Inhibition of the CK2-NCoR network significantly reduced in vivo PC-3 cell tumorigenicity, likely due to transcriptional derepression of IP-10. Clinicopathological analyses revealed that increased CK2-mediated NCoR phosphorylation significantly correlates with poor survival among prostate cancer patients. These findings elucidate a CK2-modulated oncogenic cascade in prostate tumorigenesis.

Abstract 4623: The novel, small molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer.

Abstract Background: Ovarian cancer (OC) progression is associated with accumulation of epigenetic changes leading to transcriptional silencing of tumor suppressor and chemo-responsiveness associated genes. We recently demonstrated for the first time in a clinical trial that therapeutic interventions targeting the OC methylome reverse drug resistance and induce meaningful clinical responses. While the FDA-approved demethylating agent decitabine is prone to deamination by cytidine deaminase, SGI-110 (Astex Pharmaceuticals, Inc.), a dinucleotide analogue of decitabine, is more stable, less toxic, and a promising alternative to restoring silenced TSG expression in cancer cells by reversal of DNA methylation. Our previous preclinical evaluation demonstrated that SGI-110 resensitized platinum-resistant OC cell lines to cisplatin (CDDP) (3-fold reduction in IC50) and reduced the CDDP IC50 by >2-fold in the A2780 platinum sensitive OC cell line. SGI-110 treatment induced significant demethylation and subsequent transcriptional derepression of tumor suppressors and differentiation-associated genes in A2780 ovarian cancer cells. In addition, non-tumor bearing mice tolerated SGI-110 alone or in combination with CDDP. Methods: In the current study, we assessed the activity of SGI-110 in combination with CDDP to retard the growth of platinum sensitive or drug resistant human ovarian cancer xenografts. Mice were injected with SGI-110 (2 mg/kg or 5 mg/kg, SQ.) or CDDP (2 mg/kg or 4 mg/kg, IP.) treatment or in combination in a biweekly or QD5 regimen. Results: Significant antitumor activity was observed in the single SGI-110 and SGI-110 + CDDP treatment in both the biweekly and QD5 regimen mice bearing a subcutaneous A2780 OC xenograft. The effect of SGI-110 treatment alone was greater than CDDP treatment alone. Pyrosequencing analysis was use to reaffirm global demethylation by SGI-110 treatment, based on significant demethylation of LINE1 in peripheral blood mononuclear cells. As expected, SGI-110 treatment resulted in derepression of TSGs (MLH1 and RASSF1A), HOXA10 and HOXA11 (differentiation-associated genes), CSNK1D (DNA repair), IL2RG (receptor subunit in Jak/STAT pathway), and STAT5B (transcription factor) in tumors (as observed in the in vitro study). To test the hypothesis that SGI-110 treatment influenced DNA adduct formation and repair of cisplatin-DNA damage, we are currently analyzing the amount of platinum-DNA damage induced by CDDP treatment using mass spectrometry. Conclusions: The results of our preclinical study support our recently activated clinical trial using SGI-110 in combination with carboplatin in patients with recurrent, platinum-resistant OC. Citation Format: Jessica Tang, Fang Fang, Yinu Wang, Pietro Taverna, David F.B. Miller, Gavin Choy, Mohammad Azab, Daniela Matei, Katherine S. Pawelczak, Pamela VanderVere-Carozza, Michael Wagner, John J. Turchi, Kenneth P. Nephew. The novel, small molecule DNA methylation inhibitor SGI-110 as an ovarian cancer chemosensitizer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4623. doi:10.1158/1538-7445.AM2013-4623

Characterization of the ospZ Promoter in Shigella flexneri and Its Regulation by VirB and H-NS

OspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between -731 and -412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirB-dependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression.

Collapsin response mediator protein 3 deacetylates histone H4 to mediate nuclear condensation and neuronal death

CRMP proteins play critical regulatory roles during semaphorin-mediated neurite outgrowth, neuronal differentiation and death. Albeit having a high degree of structure and sequence resemblance to that of liver dihydropyrimidinase, purified rodent brain CRMPs do not hydrolyze dihydropyrimidinase substrates. Here we found that mouse CRMP3 has robust histone H4 deacetylase activity. During excitotoxicity-induced mouse neuronal death, calpain-cleaved, N-terminally truncated CRMP3 undergoes nuclear translocation to cause nuclear condensation through deacetylation of histone H4. CRMP3-mediated deacetylation of H4 leads to de-repression of the E2F1 gene transcription and E2F1-dependent neuronal death. These studies revealed a novel mechanism of CRMP3 in neuronal death. Together with previous well established bodies of literature that inhibition of histone deacetylase activity provides neuroprotection, we envisage that inhibition of CRMP3 may represent a novel therapeutic approach towards excitotoxicity-induced neuronal death.

Long Non-Coding RNA Targeting and Transcriptional De-Repression

Our current understanding of the molecular events that functionally characterize cellular biology continues to be revised. Recent observations find that the vast majority of the human genome is transcribed and may be functionally relevant. Many of these previously unrecognized transcripts, both short and long non-coding RNAs, have been found to be active modulators of protein coding gene function. While such observations were in the past relegated to imprinted genes, it is now becoming apparent that several different genes in differentiated cells may be under some form of non-coding RNA based regulatory control. Emerging evidence suggests that some of these long non-coding RNAs are functional in controlling gene transcription by the targeted recruitment of epigenetic silencing complexes to homology-containing loci in the genome. Most notably when these repressor non-coding RNAs are targeted using small RNA-based inhibitors (such as with RNA interference), a de-repression of the targeted gene can occur resulting in activation of gene expression. Knowledge of this emerging RNA based epigenetic regulatory network has implications not only in cellular evolution but also for the development of an entirely new area of pharmacology.

Allosteric Inhibition of a Zinc-Sensing Transcriptional Repressor: Insights into the Arsenic Repressor (ArsR) Family

The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal-sensing proteins and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97′ NHε2•••O=C His67 quaternary structural hydrogen bond is an energetically significant contributor to the magnitude of the allosteric coupling free energy, ∆Gc. A “cavity” introduced just beneath this hydrogen bond in V66A/L68V CzrA results in a significant reduction in regulation by Zn(II) despite adopting a wild-type global structure and Zn(II) binding and DNA binding affinities only minimally affected from wild type. The energetics of Zn(II) binding and heterotropic coupling free energies (∆Hc, −T∆Sc) of the double mutant are also radically altered and suggest that increased internal dynamics leads to poorer allosteric negative regulation in V66A/L68V CzrA. A statistical coupling analysis of 3000 ArsR proteins reveals a sector that links the DNA-binding determinants and the α5 Zn(II)-sensing sites through V66/L68 in CzrA. We propose that distinct regulatory sites uniquely characteristic of individual ArsR proteins result from evolution of distinct connectivities to this sector, each capable of driving the same biological outcome, transcriptional derepression.

Ring1b bookmarks genes in pancreatic embryonic progenitors for repression in adult β cells

Polycomb-mediated gene repression is essential for embryonic development, yet its precise role in lineage-specific programming is poorly understood. Here we inactivated Ring1b, encoding a polycomb-repressive complex 1 subunit, in pancreatic multipotent progenitors (Ring1b(progKO)). This caused transcriptional derepression of a subset of direct Ring1b target genes in differentiated pancreatic islet cells. Unexpectedly, Ring1b inactivation in differentiated islet β cells (Ring1b(βKO)) did not cause derepression, even after multiple rounds of cell division, suggesting a role for Ring1b in the establishment but not the maintenance of repression. Consistent with this notion, derepression in Ring1b(progKO) islets occurred preferentially in genes that were targeted de novo by Ring1b during pancreas development. The results support a model in which Ring1b bookmarks its target genes during embryonic development, and these genes are maintained in a repressed state through Ring1b-independent mechanisms in terminally differentiated cells. This work provides novel insights into how epigenetic mechanisms contribute to shaping the transcriptional identity of differentiated lineages.

SMRT-GPS2 corepressor pathway dysregulation coincides with obesity-linked adipocyte inflammation

Low-grade chronic inflammation is a major characteristic of obesity and results from deregulated white adipose tissue function. Consequently, there is interest in identifying the underlying regulatory mechanisms and components that drive adipocyte inflammation. Here, we report that expression of the transcriptional corepressor complex subunits GPS2 and SMRT was significantly reduced in obese adipose tissue, inversely correlated to inflammatory status, and was restored upon gastric bypass surgery-induced weight loss in morbid obesity. These alterations correlated with reduced occupancy of the corepressor complex at inflammatory promoters, providing a mechanistic explanation for elevated inflammatory transcription. In support of these correlations, RNAi-mediated depletion of GPS2 and SMRT from cultured human adipocytes promoted derepression of inflammatory transcription and elevation of obesity-associated inflammatory markers, such as IL-6 and MCP-1. Furthermore, we identified a regulatory cascade containing PPARγ and TWIST1 that controlled the expression of GPS2 and SMRT in human adipocytes. These findings were clinically relevant, because treatment of diabetic obese patients with pioglitazone, an antidiabetic and antiinflammatory PPARγ agonist, restored expression of TWIST1, GPS2, and SMRT in adipose tissue. Collectively, our findings identify alterations in a regulatory transcriptional network in adipocytes involving the dysregulation of a specific corepressor complex as among the initiating events promoting adipose tissue inflammation in human obesity.

XPD mutations in trichothiodystrophy hamper collagen VI expression and reveal a role of TFIIH in transcription derepression

Mutations in the XPD subunit of the transcription/DNA repair factor (TFIIH) give rise to trichothiodystrophy (TTD), a rare hereditary multisystem disorder with skin abnormalities. Here, we show that TTD primary dermal fibroblasts contain low amounts of collagen type VI alpha1 subunit (COL6A1), a fundamental component of soft connective tissues. We demonstrate that COL6A1 expression is downregulated by the sterol regulatory element-binding protein-1 (SREBP-1) whose removal from the promoter is a key step in COL6A1 transcription upregulation in response to cell confluence. We provide evidence for TFIIH being involved in transcription derepression, thus highlighting a new function of TFIIH in gene expression regulation. The lack of COL6A1 upregulation in TTD is caused by the inability of the mutated TFIIH complexes to remove SREBP-1 from COL6A1 promoter and to sustain the subsequent high rate of COL6A1 transcription. This defect might account for the pathologic features that TTD shares with hereditary disorders because of mutations in COL6A genes.

A Novel Evolutionarily Conserved Element Is a General Transcriptional Repressor of p21WAF1/CIP1

The effective induction of p21(WAF1/CIP1/Cdkn1a) (p21) expression in p53-negative cancer cells is an important avenue in cancer management. We investigated the ability of various common chemotherapeutic drugs to induce p21 expression in p53-negative cancer cells and showed that the induction of p21 expression by oxaliplatin is caused by the derepression of a previously unrecognized negative regulatory element with a Sp1/Sp3 palindrome sequence core at -216 to -236 of the p21 proximal promoter. Electrophoretic mobility shift and antibody super-shift assays confirmed the specific binding of Sp1/Sp3, and showed that the oxaliplatin-mediated derepression of p21 transcription was associated with an increased Sp1/Sp3 phosphorylation and binding affinity to the oxaliplatin-responsive element. A search of the ENCODE database for vertebrate-conserved genomic elements identified the Sp1/Sp3 palindrome element as the only vertebrate-conserved element within the 500-bp proximal p21 promoter region, indicating its fundamental importance. In in vivo competition assays, transfected synthetic Sp1/Sp3 palindrome elements derepressed the cotransfected or endogenous p21 promoter in a dosage-dependent manner. This derepression was not seen in oxaliplatin-treated cells, suggesting that the exogenous Sp1/Sp3 palindrome and oxaliplatin had the same downstream signaling target. Taken together, our results revealed, for the first time, this evolutionarily conserved Sp1/Sp3 palindrome element in the proximal p21 promoter that serves as a regulatory repressor to maintain p21 basal level expression.

Structural genes of wheat and barley 5-methylcytosine DNA glycosylases and their potential applications for human health

Wheat supplies about 20% of the total food calories consumed worldwide and is a national staple in many countries. Besides being a key source of plant proteins, it is also a major cause of many diet-induced health issues, especially celiac disease. The only effective treatment for this disease is a total gluten-free diet. The present report describes an effort to develop a natural dietary therapy for this disorder by transcriptional suppression of wheat DEMETER (DME) homeologs using RNA interference. DME encodes a 5-methylcytosine DNA glycosylase responsible for transcriptional derepression of gliadins and low-molecular-weight glutenins (LMWgs) by active demethylation of their promoters in the wheat endosperm. Previous research has demonstrated these proteins to be the major source of immunogenic epitopes. In this research, barley and wheat DME genes were cloned and localized on the syntenous chromosomes. Nucleotide diversity among DME homeologs was studied and used for their virtual transcript profiling. Functional conservation of DME enzyme was confirmed by comparing the motif and domain structure within and across the plant kingdom. Presence and absence of CpG islands in prolamin gene sequences was studied as a hallmark of hypo- and hypermethylation, respectively. Finally the epigenetic influence of DME silencing on accumulation of LMWgs and gliadins was studied using 20 transformants expressing hairpin RNA in their endosperm. These transformants showed up to 85.6% suppression in DME transcript abundance and up to 76.4% reduction in the amount of immunogenic prolamins, demonstrating the possibility of developing wheat varieties compatible for the celiac patients.