Repertoire Of Target Genes Research Articles

Orphan nuclear receptor NR2F6 acts as an essential gatekeeper of Th17 CD4+ T cell effector functions.

Members of the evolutionarily conserved family of the chicken ovalbumin upstream promoter transcription factor NR2F/COUP-TF orphan receptors have been implicated in lymphocyte biology, ranging from activation to differentiation and elicitation of immune effector functions. In particular, a CD4+ T cell intrinsic and non-redundant function of NR2F6 as a potent and selective repressor of the transcription of the pro-inflammatory cytokines interleukin (Il) 2, interferon y (ifng) and consequently of T helper (Th)17 CD4+ T cell-mediated autoimmune disorders has been discovered. NR2F6 serves as an antigen receptor signaling threshold-regulated barrier against autoimmunity where NR2F6 is part of a negative feedback loop that limits inflammatory tissue damage induced by weakly immunogenic antigens such as self-antigens. Under such low affinity antigen receptor stimulation, NR2F6 appears as a prototypical repressor that functions to “lock out” harmful Th17 lineage effector transcription. Mechanistically, only sustained high affinity antigen receptor-induced protein kinase C (PKC)-mediated phosphorylation has been shown to inactivate NR2F6, thereby displacing pre-bound NR2F6 from the DNA and, subsequently, allowing for robust NFAT/AP-1- and RORγt-mediated cytokine transcription. The NR2F6 target gene repertoire thus identifies a general anti-inflammatory gatekeeper role for this orphan receptor. Investigating these signaling pathway(s) will enable a greater knowledge of the genetic, immune, and environmental mechanisms that lead to chronic inflammation and of certain autoimmune disorders in a given individual.

Abstract P5-07-02: Mutant GATA3 causes precocious lobuloalveolar development in the mouse mammary gland and is associated with more aggressive human breast cancer

Abstract GATA3 is an essential mediator of mammary gland development and is expressed at high levels in Estrogen Receptor positive tumors. This gene has recently emerged as one of the most frequently mutated genes in Estrogen Receptor positive breast cancer. GATA3 mutant tumors tend to retain and express the wild-type GATA3 allele which, together with the established role for GATA3 in maintaining luminal epithelial differentiation, has led to the suggestion that GATA3 is a haploinsufficient tumor suppressor gene in the mammary gland. We describe a striking contrast between the GATA3 mutation spectrum in breast cancer with that found in a true GATA3 haploinsufficiency syndrome and propose that this is indicative of a strong selective pressure to mutate and retain expression of a substantial portion of the GATA3 coding sequence. We demonstrate that the proteins encoded by GATA3 mutant alleles lack the capacity to bind to canonical GATA sequence motifs and acquire a new recognition specificity governed by the N-terminal zinc finger domain. Transgenic expression of the mutant protein results in a pronounced precocious lobuloalveolar development in 9 week old female mice, suggesting that the mutant gene is not a null allele and can regulate cell proliferation. In addition, we demonstrate that Estrogen Receptor positive GATA3 mutant human breast tumors arise at earlier ages than Estrogen Receptor GATA3 wild-type tumors, which further emphasizes the clinical relevance of these mutations. Based on the estimated mutation prevalence from the TCGA study, approximately 22,000 new cases of GATA3-mutant invasive breast cancer are diagnosed in the US each year. Collectively, our data indicate that GATA3 mutant tumors may comprise a breast cancer subset with distinct biological and clinical behavior and that a detailed understanding of the novel repertoire of target genes may yield useful clinical targets for this population. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-02.

Fishful thinking: the rise and fall of MITF in melanoma

Fishful thinking: the rise and fall of <scp>MITF</scp> in melanoma

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BMP Induces Cochlin Expression to Facilitate Self-renewal and Suppress Neural Differentiation of Mouse Embryonic Stem Cells

BMP4 maintains self-renewal of mouse embryonic stem cells (ESCs) in collaboration with LIF. Here, we report the identification of a novel key BMP target gene, cochlin (Coch) in mouse ESCs. Coch can be significantly up-regulated by BMP4 specifically in ESCs but not in somatic differentiated cells, and this up-regulation is dependent on the BMP signaling mediators Smad1/5 and Smad4. Overexpression of Coch can partially substitute BMP4 to promote self-renewal of mouse ESCs together with LIF, whereas knockdown of Coch impairs self-renewal marker gene expression even in the presence of both BMP4 and LIF. Further studies showed that COCH could mimic BMP4 in repressing neural differentiation of mouse ESCs upon LIF withdrawal and the inhibitory effect of BMP4 on neural differentiation is compromised by Coch knockdown. Taken together, our data suggest that COCH is a part of the downstream target network of BMP signaling and serves as another important effector to fine-tune mouse ESC fates.

Genes involved in cell adhesion and signaling: A new repertoire of Retinoic Acid Receptors target genes in mouse embryonic fibroblasts

Nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

Abstract 1429: Forkhead box M1 (FoxM1) is a direct transcriptional target of EWS/FLI1 and a regulator of Ewing's sarcoma cell morphology, adhesiveness and tumorigenicity

Abstract Ewing's sarcomas (EWS) are tumors typically formed by undifferentiated small round cells that predominantly affect children and young adults. The aggressiveness of EWS is illustrated by both the fact that about 25-30% patients present with metastases at diagnosis and the elevated rate of lethal recurrence among them. In spite of therapeutic combinations including chemotherapy, radiation and surgery, the 5-year disease-free survival rate for EWS patients has remained low for many years. Poor prognosis, lethal recurrence and long-term side effects point to the urgent requirement of novel EWS targeted therapies. EWS is cytogenetically characterized by the presence of chromosomal translocations resulting in the fusion of the EWS gene with several ETS genes, most frequently with FLI1, leading to the expression of chimeric proteins that contain the transcriptional activation domain of EWS and the FLI1 DNA binding domain. The EWS/FLI1 aberrant transcription factors are responsible for the malignant phenotype of EWS and regulate a large repertoire of target genes, many of which are critical determinants for EWS development, and thus have the potential of providing novel therapeutic options for EWS. In order to understand mechanisms of cell cycle regulation by EWS/FLI1, we performed comparative array analyses of EWS cells before and after knocking down EWS/FLI1 expression. Results showed that EWS/FLI1 knockdown consistently down-regulated FoxM1, and chromatin immunoprecipitation (ChIP) assays demonstrated that FoxM1 is a novel direct transcriptional target of EWS/FLI1. Western hybridization and immunohistochemical analyses demonstrated that FoxM1 is indeed abundantly expressed in EWS cell lines, patient tumors and EWS cell derived tumors in nude mouse xenografts. Down-regulation of FoxM1 expression in by shRNA-mediated gene knockdown caused morphological alterations, slowed the growth of EWS cells, and increased their adhesion to the substrate in culture-plates. Accordingly, FoxM1 down-regulation in EWS cells diminished their migration and invasiveness as well as their tumorigenicity in nude mice. Alterations in the pattern of integrin expression observed in FoxM1 knocked-down EWS cells may likely contribute to their altered morphology, increased adhesion and the overall downgrading of their malignant phenotype. These data identify FoxM1 as a direct EWS/FLI1 target and as an important regulator of the proliferation, morphology and tumorigenicity of EWS cells, and imply that targeting FoxM1 may allow the development of new molecular therapeutic strategies for EWS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1429. doi:1538-7445.AM2012-1429

Genome-Wide Analysis of N1ICD/RBPJ Targets In Vivo Reveals Direct Transcriptional Regulation of Wnt, SHH, and Hippo Pathway Effectors by Notch1

The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these datasets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH, and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.

A Novel Nonconsensus Xenobiotic Response Element Capable of Mediating Aryl Hydrocarbon Receptor-Dependent Gene Expression

The aryl hydrocarbon receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. The AhR functions as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with the heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE highlighting the growing realization that AhR-mediated gene expression seems to involve additional mechanisms distinct from the well characterized process involving the XRE. The present study characterized a novel nonconsensus XRE (NC-XRE) in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene that recruits a novel protein-DNA complex responsible for TCDD-inducible expression. DNA binding studies and reporter assays identified key residues in the NC-XRE necessary for protein-DNA binding and function, respectively. Functional studies with AhR expression constructs confirm that TCDD-inducibility is AhR-dependent and requires direct AhR-DNA binding to the NC-XRE. Chromatin immunoprecipitation and RNA interference studies reveal that the Arnt protein is not a component of the NC-XRE-bound AhR complex, suggesting that in contrast to the XRE, AhR-dependent gene expression mediated through the NC-XRE may involve a new DNA binding partner.

Molecular pathogenesis of Ewing sarcoma: new therapeutic and transcriptional targets.

Approximately one-third of sarcomas contain specific translocations. Ewing sarcoma is the prototypical member of this group of sarcomas; it was the first to be recognized pathologically as a singular entity and to have its signature translocation defined cytogenetically, which led to the identification of its key driver alteration, the EWS-FLI1 gene fusion that encodes this aberrant, chimeric transcription factor. We review recent progress in selected areas of Ewing sarcoma research, including the application of genome-wide chromatin immunoprecipitation analyses, to provide a comprehensive view of the EWS-FLI1 target gene repertoire, the identification of EWS-FLI1 target genes that may also point to therapeutically targetable pathways, and data from model systems as they relate to the elusive cell of origin of Ewing sarcoma and its possible similarities to mesenchymal stem cells.

Let-7a Is a Direct EWS-FLI-1 Target Implicated in Ewing's Sarcoma Development

Ewing's sarcoma family tumors (ESFT) are the second most common bone malignancy in children and young adults, characterized by unique chromosomal translocations that in 85% of cases lead to expression of the EWS-FLI-1 fusion protein. EWS-FLI-1 functions as an aberrant transcription factor that can both induce and suppress members of its target gene repertoire. We have recently demonstrated that EWS-FLI-1 can alter microRNA (miRNA) expression and that miRNA145 is a direct EWS-FLI-1 target whose suppression is implicated in ESFT development. Here, we use miRNA arrays to compare the global miRNA expression profile of human mesenchymal stem cells (MSC) and ESFT cell lines, and show that ESFT display a distinct miRNA signature that includes induction of the oncogenic miRNA 17–92 cluster and repression of the tumor suppressor let-7 family. We demonstrate that direct repression of let-7a by EWS-FLI-1 participates in the tumorigenic potential of ESFT cells in vivo. The mechanism whereby let-7a expression regulates ESFT growth is shown to be mediated by its target gene HMGA2, as let-7a overexpression and HMGA2 repression both block ESFT cell tumorigenicity. Consistent with these observations, systemic delivery of synthetic let-7a into ESFT-bearing mice restored its expression in tumor cells, decreased HMGA2 expression levels and resulted in ESFT growth inhibition in vivo. Our observations provide evidence that deregulation of let-7a target gene expression participates in ESFT development and identify let-7a as promising new therapeutic target for one of the most aggressive pediatric malignancies.

Target Gene Repression Mediated by miRNAs miR-181c and miR-9 Both of Which Are Down-regulated by Amyloid-β

MicroRNAs (miRNAs) are small non-coding RNA regulators of protein synthesis that are essential for normal brain development and function. Their profiles are significantly altered in neurodegenerative diseases such as Alzheimer's disease (AD) that is characterized by amyloid-β (Aβ) and tau deposition in brain. How deregulated miRNAs contribute to AD is not understood, as their dysfunction could be both a cause and a consequence of disease. To address this question we had previously profiled miRNAs in models of AD. This identified miR-9 and -181c as being down-regulated by Aβ in hippocampal cultures. Interestingly, there was a remarkable overlap with those miRNAs that are deregulated in Aβ-depositing APP23 transgenic mice and in human AD tissue. While the Aβ precursor protein APP itself is a target of miRNA regulation, the challenge resides in identifying further targets. Here, we expand the repertoire of miRNA target genes by identifying the 3' untranslated regions (3' UTRs) of TGFBI, TRIM2, SIRT1 and BTBD3 as being repressed by miR-9 and -181c, either alone or in combination. Taken together, our study identifies putative target genes of miRNAs miR-9 and 181c, which may function in brain homeostasis and disease pathogenesis.

Epigenetic regulation of signaling pathways in cancer: Role of the histone methyltransferase EZH2

EZH2 is the histone H3 lysine 27 methyltransferase of polycomb-repressive complex 2. It transcriptionally silences cohorts of developmental regulators in stem/progenitors and cancer cells. EZH2 is essential in maintaining stem cell identity by globally repressing differentiation programs. Analogously, it plays a key role in oncogenesis by targeting signaling molecules that control cell differentiation. Emerging data indicate that EZH2 promotes cancer formation and progression through epigenetic activation of oncogenic signaling cascades and inhibition of pro-differentiation pathways. Genome-wide mapping analysis has been expanding the repertoire of target genes and the associated signaling pathways regulated by EZH2. Better understanding of the molecular basis of such regulations in various cancer types will help establish EZH2-mediated epigenetic silencing as a therapeutic target.

Ronin/Hcf-1 binds to a hyperconserved enhancer element and regulates genes involved in the growth of embryonic stem cells

Self-renewing embryonic stem (ES) cells have an exceptional need for timely biomass production, yet the transcriptional control mechanisms responsible for meeting this requirement are largely unknown. We report here that Ronin (Thap11), which is essential for the self-renewal of ES cells, binds with its transcriptional coregulator, Hcf-1, to a highly conserved enhancer element that previously lacked a recognized binding factor. The subset of genes bound by Ronin/Hcf-1 function primarily in transcription initiation, mRNA splicing, and cell metabolism; genes involved in cell signaling and cell development are conspicuously underrepresented in this target gene repertoire. Although Ronin/Hcf-1 represses the expression of some target genes, its activity at promoter sites more often leads to the up-regulation of genes essential to protein biosynthesis and energy production. We propose that Ronin/Hcf-1 controls a genetic program that contributes to the unimpeded growth of ES cells.

Abstract 3413: Tumor-specific transcriptional control networks regulating DNA replication genes in Ewing's sarcoma

Abstract Surveillance of DNA replication licensing is often perturbed in cancer cells allowing for genomic aberrations. Although this process contributes to cellular transformation, it is not well understood to what extent and how oncogenes might deregulate genes involved in DNA replication and in the control of replication licensing. Previous findings on the EWS-FLI1 fusion oncogene (which is the genetic hallmark Ewing's sarcoma), and our recent findings on MYC targets in this aggressive childhood tumor suggested that both these oncogenes constitute a regulatory feed-back loop. Our gene expression data from Ewing's cells in which MYC had been knocked down, we propose that MYC mediates at least part of the oncogenic EWS-FLI1 functions since it controls most essential biological functions required for cell proliferation. Gene expression correlation and gene set enrichment analysis in tumors and cell lines suggests the MYC/EWS-FLI1 loop as a central regulator in a regulatory network that controls DNA replication. To further narrow down the target gene repertoire and to identify direct transcriptional targets, we performed a genome wide chromatin-immunoprecipitation study using an anti MYC antibody in combindation with ChIP/CHIP analysis. We further extended our model whereby the core EWS-FLI1/MYC feedback loop could be linked to other candidate transcriptional regulators of DNA replication. Several replication licensing genes including MCM, ORC or licensing control genes such as CDT1 and CDC6 were found to be under direct and/or indirect control of the MYC/EWS-FLI1 loop and their associated transcription factor network. A subset of DNA replication genes is particularly deregulated in Ewing's cells when compared to mesenchymal progenitor cells that are considered the tissue of origin of Ewing's sarcoma. When comparing Ewing's sarcoma with other childhood tumors such as e.g. neuroblastoma, we found Ewing's-specific versus more general regulatory connections between the MYC/EWS-FLI1 loop and DNA replication genes. Together, our data provide first insight into a newly defined MYC/EWS-FLI1-driven regulatory network underlying transcriptional DNA replication control. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3413.

Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells

All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-beta)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RAR alpha and RAR gamma receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-beta pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA.

REST and CoREST Modulate Neuronal Subtype Specification, Maturation and Maintenance

BackgroundThe repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is a master regulator of neuronal gene expression. REST functions as a modular scaffold for dynamic recruitment of epigenetic regulatory factors including its primary cofactor, the corepressor for element-1-silencing transcription factor (CoREST), to genomic loci that contain the repressor element-1 (RE1) binding motif. While REST was initially believed to silence RE1 containing neuronal genes in neural stem cells (NSCs) and non-neuronal cells, emerging evidence shows an increasingly complex cell type- and developmental stage-specific repertoire of REST target genes and functions that include regulation of neuronal lineage maturation and plasticity.Methodology/Principal FindingsIn this study, we utilized chromatin immunoprecipitation on chip (ChIP-chip) analysis to examine REST and CoREST functions during NSC-mediated specification of cholinergic neurons (CHOLNs), GABAergic neurons (GABANs), glutamatergic neurons (GLUTNs), and medium spiny projection neurons (MSNs). We identified largely distinct but overlapping profiles of REST and CoREST target genes during neuronal subtype specification including a disproportionately high percentage that are exclusive to each neuronal subtype.Conclusions/SignificanceOur findings demonstrate that the differential deployment of REST and CoREST is an important regulatory mechanism that mediates neuronal subtype specification by modulating specific gene networks responsible for inducing and maintaining neuronal subtype identity. Our observations also implicate a broad array of factors in the generation of neuronal diversity including but not limited to those that mediate homeostasis, cell cycle dynamics, cell viability, stress responses and epigenetic regulation.

Integrated Approach for the Identification of Human Hepatocyte Nuclear Factor 4α Target Genes Using Protein Binding Microarrays

Hepatocyte nuclear factor 4 alpha (HNF4alpha), a member of the nuclear receptor superfamily, is essential for liver function and is linked to several diseases including diabetes, hemophilia, atherosclerosis, and hepatitis. Although many DNA response elements and target genes have been identified for HNF4alpha, the complete repertoire of binding sites and target genes in the human genome is unknown. Here, we adapt protein binding microarrays (PBMs) to examine the DNA-binding characteristics of two HNF4alpha species (rat and human) and isoforms (HNF4alpha2 and HNF4alpha8) in a high-throughput fashion. We identified approximately 1400 new binding sequences and used this dataset to successfully train a Support Vector Machine (SVM) model that predicts an additional approximately 10,000 unique HNF4alpha-binding sequences; we also identify new rules for HNF4alpha DNA binding. We performed expression profiling of an HNF4alpha RNA interference knockdown in HepG2 cells and compared the results to a search of the promoters of all human genes with the PBM and SVM models, as well as published genome-wide location analysis. Using this integrated approach, we identified approximately 240 new direct HNF4alpha human target genes, including new functional categories of genes not typically associated with HNF4alpha, such as cell cycle, immune function, apoptosis, stress response, and other cancer-related genes. We report the first use of PBMs with a full-length liver-enriched transcription factor and greatly expand the repertoire of HNF4alpha-binding sequences and target genes, thereby identifying new functions for HNF4alpha. We also establish a web-based tool, HNF4 Motif Finder, that can be used to identify potential HNF4alpha-binding sites in any sequence.

Thyroid hormone receptor mutants implicated in human hepatocellular carcinoma display an altered target gene repertoire

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of normal physiology and development. Mutations in TRs have been identified at high frequency in certain cancers, including human hepatocellular carcinomas (HCCs). The majority of HCC TR mutants bear lesions within their DNA recognition domains, and we have hypothesized that these lesions change the mutant receptors' target gene repertoire in a way crucial to their function as oncoproteins. Using stable cell transformants and expression array analysis, we determined that mutant TRs isolated from two different HCCs do, as hypothesized, display a target gene repertoire distinct from that of their normal TR progenitors. Only a subset of genes regulated by wild-type TRs were regulated by the corresponding HCC-TR mutants. More surprisingly, the HCC-TR mutants also gained the ability to regulate additional target genes not recognized by the wild-type receptors, and were not simply restricted to repression, but could also activate a subset of their target genes. We conclude that the TR mutants isolated from HCC have sustained multiple alterations from their normal progenitors that include not only changes in their transcriptional outputs, but also changes in the genes they target; both are likely to contribute to neoplasia.

Isoform-Specific Transcriptional Activity of Overlapping Target Genes that Respond to Thyroid Hormone Receptors α1 and β1

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of physiology and development. TRs are expressed in vertebrates as a series of distinct isoforms that exert distinct biological roles. We wished to determine whether the two most widely expressed isoforms, TR alpha 1 and TR beta 1, exert their different biological effects by regulating different sets of target genes. Using stably transformed HepG2 cells and a microarray analysis, we were able to demonstrate that TR alpha 1 and TR beta 1 regulate a largely overlapping repertoire of target genes in response to T(3) hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T(3)-mediated regulation by TR alpha 1 than by TR beta 1, to near equal regulation by both isoforms. We also identified TR alpha 1 and TR beta 1 target genes that were regulated by these receptors in a hormone-independent fashion. We suggest that it is this gene-specific, isoform-specific amplitude of transcriptional regulation that is the likely basis for the appearance and maintenance of TR alpha 1 and TR beta 1 over evolutionary time. In essence, TR alpha 1 and TR beta 1 adjust the magnitude of the transcriptional response at different target genes to different levels; by altering the ratio of these isoforms in different tissues or at different developmental times, the intensity of T(3) response can be individually tailored to different physiological and developmental requirements.