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Glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1), the protein that shuttles LPL to the capillary lumen, is essential for plasma triglyceride metabolism. When GPIHBP1 is absent, LPL remains stranded within the interstitial spaces and plasma triglyceride hydrolysis is impaired, resulting in severe hypertriglyceridemia. While the functions of GPIHBP1 in intravascular lipolysis are reasonably well understood, no one has yet identified DNA sequences regulating GPIHBP1 expression. In the current studies, we identified an enhancer element located ∼3.6 kb upstream from exon 1 of mouse Gpihbp1. To examine the importance of the enhancer, we used CRISPR/Cas9 genome editing to create mice lacking the enhancer (Gpihbp1(Enh/Enh)). Removing the enhancer reduced Gpihbp1 expression by >90% in the liver and by ∼50% in heart and brown adipose tissue. The reduced expression of GPIHBP1 was insufficient to prevent LPL from reaching the capillary lumen, and it did not lead to hypertriglyceridemia—even when mice were fed a high-fat diet. Compound heterozygotes (Gpihbp1(Enh/−) mice) displayed further reductions in Gpihbp1 expression and exhibited partial mislocalization of LPL (increased amounts of LPL within the interstitial spaces of the heart), but the plasma triglyceride levels were not perturbed. The enhancer element that we identified represents the first insight into DNA sequences controlling Gpihbp1 expression.

FSH is critical for fertility. Transcription of FSHB, the gene encoding the beta subunit, is rate-limiting in FSH production and is regulated by both GnRH and activin. Activin signals through SMAD transcription factors. Although the mechanisms and importance of activin signaling in mouse Fshb transcription are well-established, activin regulation of human FSHB is less well understood. We previously reported a novel enhancer of FSHB that contains a fertility-associated single nucleotide polymorphism (rs10031006) and requires a region resembling a full (8 base-pair) SMAD binding element (SBE). Here, we investigated the role of the putative SBE within the enhancer in activin and GnRH regulation of FSHB. In mouse gonadotrope-derived LβT2 cells, the upstream enhancer potentiated activin induction of both the human and mouse FSHB proximal promoters and conferred activin responsiveness to a minimal promoter. Activin induction of the enhancer required the SBE and was blocked by the inhibitory SMAD7, confirming involvement of the classical SMAD signaling pathway. GnRH induction of FSHB was also potentiated by the enhancer and dependent on the SBE, consistent with known activin/GnRH synergy regulating FSHB transcription. In DNA pull-down, the enhancer SBE bound SMAD4, and chromatin immunoprecipitation demonstrated SMAD4 enrichment at the enhancer in native chromatin. Combined activin/GnRH treatment elevated levels of the active transcriptional histone marker, histone 3 lysine 27 acetylation, at the enhancer. Overall, this study indicates that the enhancer is directly targeted by activin signaling and identifies a novel, evolutionarily conserved mechanism by which activin and GnRH can regulate FSHB transcription.

Chromatin remodeling impacts the structural neighborhoods and regulates gene expression. However, the role of enhancer-guided chromatin remodeling in the gene regulation remains unclear. Here, using RNA-seq and ChIP-seq, we identified for the first time that neurotensin (NTS) serves as a key oncogene in uveal melanoma and that CTCF interacts with the upstream enhancer of NTS and orchestrates an 800 kb chromosomal loop between the promoter and enhancer. Intriguingly, this novel CTCF-guided chromatin loop was ubiquitous in a cohort of tumor patients. In addition, a disruption in this chromosomal interaction prevented the histone acetyltransferase EP300 from embedding in the promoter of NTS and resulted in NTS silencing. Most importantly, in vitro and in vivo experiments showed that the ability of tumor formation was significantly suppressed via deletion of the enhancer by CRISPR-Cas9. These studies delineate a novel onco-enhancer guided epigenetic mechanism and provide a promising therapeutic concept for disease therapy.

The classical definition of genetic cis-regulatory modules such as enhancers identifies these elements as insensitive to changes in position and orientation relative to the transcription start site (TSS). While this theory is well supported, some recent studies have uncovered examples of constraint in the position or orientation of particular enhancers. This study examines the position and orientation sensitivity of two upstream enhancer elements found in the gene Pax6, which regulates development of the eyes and central nervous system in vertebrates. Late embryonic stages of the organism used in this study, Ciona intestinalis, show expression of Pax6 in the nerve cord and sensory vesicle. For this investigation Pax6 constructs were created in which the enhancers were repositioned or inverted relative to the TSS. These constructs were electroporated into embryos of C. intestinalis, and expression of the Pax6-GFP fusion gene was measured both by total fluorescence and by number of positive embryos. Alteration of either position or orientation of these enhancers was found to cause a strong decrease in measured Pax6 expression. These results support the conclusion that the upstream enhancers must be in the proper location and arrangement to be fully functional, a finding that is consistent with the results of many other studies of cis-regulatory elements.

We observed increased expression of ADAMDEC1 RNA in monocytes from patients with systemic lupus erythematosus. The precise role of ADAMDEC1 is uncertain and uniquely among metalloproteinases it utilizes a zinc-coordinating aspartic acid residue which allows it to escape inhibition by tissue inhibitor of metalloprotease-3 (TIMP-3). A closely related gene encodes the protein ADAM28, which is not up-regulated in lupus. We leveraged the ability to look at both gene’s promoters and enhancers simultaneously. ADAMDEC1 was up-regulated by LPS while ADAM28 was not upregulated in the short term. We identified MAP kinases and NFκB as critical cell pathways regulating the expression of ADAMDEC1. These same pathways were implicated in driving the expression of the ADAMDEC1 upstream enhancer RNAs. We demonstrated that binding of the enhancer RNAs produced from the upstream enhancer were critically important and that p300 bound to both the RNA from the enhancer and the DNA at the enhancer. P300 binding to the enhancer was dependent on NFκB. These data define the critical pathways regulating the expression of ADAMDEC1 and extend our knowledge of the roles of enhancer RNAs and mechanistically links p300 and enhancer RNAs.

The SERPINB2 gene is strongly upregulated in inflammatory states. In monocytes, it can constitute up to 1% of total cellular protein. It functions in protection from proteotoxic stress and plays a role in angioedema. The purpose of this study was to define the roles of enhancer RNAs embedded in the SERPIN gene complex. We found that the upstream enhancer RNAs upregulated SERPINB2 and the enhancer RNAs were expressed prior to those of SERPINB2 mRNA. Studies of the SERPINB2 promoter demonstrated the presence of an RNA polymerase II pause-inducing protein, NELF. Stimulation with LPS led to recruitment of the pause-releasing kinase P-TEFb and departure of the pause-inducing protein NELF. RNA immunoprecipitation revealed that NELF and the CDK9 component of P-TEFb bound to the enhancer RNAs after stimulation with distinct kinetics. Knock-down of the enhancer RNAs compromised stimulus induction of promoter and enhancer chromatin changes. Conversely, over-expression was associated with enhanced recruitment of c-JUN and increased expression of SERPINB2 mRNA expression. This study is the first to associate enhancer RNAs with SERPINB2 and is the first demonstration of acquisition of NELF binding by enhancer RNAs on chromatin.

EKLF/KLF1 is an essential transcription factor that plays a global role in erythroid transcriptional activation. It’s own regulation is of interest, as it displays a highly restricted expression pattern, limited to erythroid cells and its progenitors. Here we use biochemical affinity purification to identify the Ddx5/p68 protein as an activator of KLF1 by virtue of its interaction with the erythroid-specific DNAse hypersensitive site upstream enhancer element (EHS1). We postulate that its range of interactions with other proteins known to interact with this element render it part of the enhanseosome complex critical for optimal expression of KLF1. These individual interactions provide quantitative contributions that, in sum, establish high level activity of the KLF1 promoter and suggest they can be selectively manipulated for clinical benefit.

Long non-coding RNAs (lncRNAs) function in a wide range of processes by diverse mechanisms, though their roles in regulation of oncogenes and/or tumor suppressors remain rather elusive. We performed a global search for lncRNAs affecting MYC activity using a systems biology-based approach with a K supercomputer and the GIMLET algorism based on local distance correlations. Consequently, MYMLR was identified and experimentally shown to maintain MYC transcriptional activity and cell cycle progression despite the low levels of expression. A proteomic search for MYMLR-binding proteins identified PCBP2, while it was also found that MYMLR places a 557-kb upstream enhancer region in the proximity of the MYC promoter in cooperation with PCBP2. These findings implicate a crucial role for MYMLR in regulation of the archetypical oncogene MYC and warrant future studies regarding the involvement of low copy number lncRNAs in regulation of other crucial oncogenes and tumor suppressor genes.

SOX2 is a prominent member of the SOX family of transcription factors that has many different functional roles. This pleiotropy is made possible by multiple regulatory mechanisms that direct appropriate spatial and temporal patterns of expression, and therefore action. The current study concerns the mechanisms that determine Sox2 gene expression in the adult mammalian brain, where SOX2 protein is absent in general, but is selectively and abundantly expressed in a majority of neurons within a ventral diencephalic brain structure, the suprachiasmatic nucleus (SCN). In this study, a comparative bioinformatic and biochemical analysis of different adult rat brain regions was conducted in order to identify SCN-selective (immaturity-related) regulatory mechanisms. The approach incorporated an integrated analysis of Sox2 enhancers, CTCF binding sites, and also expression of the Sox2-overlapping, long non-coding (lnc)RNA, Sox2ot. Initial experiments revealed brain region-specific Sox2ot expression (including region-specific novel transcripts), indicating a significant diversity of Sox2ot expression across the adult brain. However, the pattern and abundance of Sox2ot expression in the SCN, relative to selected control areas of the brain, did not indicate an overt relationship to Sox2 gene expression. Furthermore, although multiple individual Sox2ot exon sequences were shown to overlap annotated Sox2 gene enhancers at different sites across the Sox2 locus, again there was no indication of a SCN-specific functional correlation. Further integration with an analysis of selectively-active CTCF sites within the Sox2 locus directed attention to one site with both a prominent peak of activity in immature brain, and proximity to a functionally-characterized, ventral diencephalic, Sox2 enhancer termed U6 (upstream enhancer 6). Ex vivo analysis of the U6-associated CTCF site revealed SCN-selective CTCF binding, and these sequences were both localized within a known (brain region-selective) super-enhancer. Bioinformatic analysis of the U6 enhancer sequence revealed an abundance of consensus sites for the SCN-selective transcription factor LHX1, and over-expression of this factor enhanced the activity of cloned U6 sequence in transfected cells. However, despite this compelling evidence for a molecular mechanism that underlies adult brain expression of SOX2, further analysis of LHX1-SOX2 co-expression in the SCN confounded this view, indicating the presence of other concurrent mechanisms in the different cell populations of the SCN.

Polycystic ovary syndrome (PCOS) is the most common cause of female infertility and is associated with an increased risk of type 2 diabetes and cardiovascular disease. Understanding the etiology of PCOS is crucial to relieve this tremendous health burden. Twin studies estimate that 70% of PCOS etiology can be explained by genetic factors, motivating studies to identify these factors. Recent genome wide association studies identified a PCOS risk locus that encompasses the gene encoding the follicle-stimulating hormone beta (FSHβ) subunit. The most significant single nucleotide polymorphism (SNP) within this locus, rs11031006 (rs06), resides within a region of DNA (~450 base pairs) that is highly conserved among mammals. As FSH supplementation can restore ovulation in some PCOS patients, deficient FSH signaling may be a causative factor of anovulation and potentially other facets of PCOS. FSH transcription and release is stimulated by gonadotropin-releasing hormone (GnRH) produced in the hypothalamus, as well as activin produced by the gonads and pituitary. We previously hypothesized that the conserved region containing rs06 acts as an enhancer of FSHβ transcription, and we determined through luciferase reporter assays that this highly conserved region enhances transcription ~2 fold compared to the minimal FSHβ promoter alone for human and 2.5 fold for mouse. The rs06 SNP minor allele further increased both mouse and human FSHβ luciferase expression. In the present study, we sought to identify interactions between the enhancer and the hormones, GnRH and activin. With DNA constructs from both human and mouse, we identified synergistic interactions between the enhancer and GnRH- or activin-induction of FSHβ. These interactions were maintained after mutation of the rs06 site to the minor allele, suggesting that this SNP alters basal and not hormone-induced FSHβ transcription. We are currently investigating the effect of the enhancer and rs06 on activin/GnRH synergy. Overall, our findings provide evidence that the enhancer functions as a novel mediator of activin and GnRH regulation of FSHβ transcription in both human and mouse.

The oncogenic microRNA-155 (miR-155) is the most frequently upregulated miRNA in Epstein-Barr virus (EBV)-positive B cell malignancies and is upregulated in other non-viral lymphomas. Both the EBV nuclear antigen 2 (EBNA2), and B cell transcription factor, interferon regulatory factor 4 (IRF4) are known to activate transcription of the host cell gene from which miR-155 is processed (miR-155HG,BIC). EBNA2 also activatesIRF4transcription indicating that EBV may upregulate miR-155 through direct and indirect mechanisms. The mechanism of transcriptional regulation ofIRF4andmiR-155HGby EBNA2 however has not been defined. We demonstrate that EBNA2 can activateIRF4andmiR-155HGexpression through specific upstream enhancers that are dependent on the Notch signaling transcription factor RBPJ, a known binding partner of EBNA2. We demonstrate that in addition to activation of themiR-155HGpromoter, IRF4 can also activatemiR-155HGvia the upstream enhancer also targeted by EBNA2. Gene editing to remove the EBNA2- and IRF4-responsivemiR-155HGenhancer located 60 kb upstream ofmiR-155HGled to reducedmiR155HGexpression in EBV-infected cells. Our data therefore demonstrate that specific RBPJ-dependent enhancers regulate the IRF4-miR-155 expression network and play a key role in the maintenance of miR-155 expression in EBV-infected B cells. These findings provide important insights that will improve our understanding of miR-155 control in B cell malignancies.IMPORTANCEMicroRNA-155 (miR-155) is expressed at high level in many human cancers particularly lymphomas. Epstein-Barr virus (EBV) infects human B cells and drives the development of numerous lymphomas. Two EBV-encoded genes (LMP1 and EBNA2) upregulate miR-155 expression and miR-155 expression is required for the growth of EBV-infected B cells. We show that the EBV transcription factor EBNA2 upregulates miR-155 expression by activating an enhancer upstream from the miR-155 host gene (miR-155HG) from which miR-155 is derived. We show that EBNA2 also indirectly activatesmiR-155expression through enhancer-mediated activation ofIRF4.IRF4 then activates both themiR-155HGpromoter and the upstream enhancer, independently of EBNA2. Gene editing to remove themiR-155HGenhancer leads to a reduction inmiR-155HGexpression. We therefore identify enhancer-mediated activation ofmiR-155HGas a critical step in promoting B cell growth and a likely driver of lymphoma development.

The repression of telomerase activity during cellular differentiation promotes replicative aging and functions as a physiological barrier for tumorigenesis in long-lived mammals, including humans. However, the underlying mechanisms remain largely unclear. Here we describe how miR-615-3p represses hTERT expression. mir-615-3p is located in an intron of the HOXC5 gene, a member of the highly conserved homeobox family of transcription factors controlling embryogenesis and development. Unexpectedly, we found that HoxC5 also represses hTERT expression by disrupting the long-range interaction between hTERT promoter and its distal enhancer. The 3′UTR of hTERT and its upstream enhancer region are well conserved in long-lived primates. Both mir-615-3p and HOXC5 are activated upon differentiation, which constitute a feed-forward loop that coordinates transcriptional and post-transcriptional repression of hTERT during cellular differentiation. Deregulation of HOXC5 and mir-615-3p expression may contribute to the activation of hTERT in human cancers., The expression of telomerase catalytic subunit hTERT is frequently upregulated in many cancers. Here, the authors show HoxC5 and miR-615-3p can negatively regulate hTERT to impede tumorigenesis by targeting the newly evolved cis-regulatory genomic elements of hTERT.

Exogenous gene expression assays during development, including reporters under the control of 5′ upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5′ upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

We report here the isolation and functional characterization of the as yet undescribed rat Thyroglobulin gene upstream enhancer element.

Gene promoters are frequently insufficient to drive the spatiotemporal patterns of gene expression during cell differentiation and organism development. Enhancers convey these properties through diverse mechanisms, including long-distance interactions with target promoters via their association with specific transcription factors. Despite unprecedented progress in the knowledge of enhancer mechanisms of action, there are still many unanswered questions. In particular, the contribution of an enhancer's local chromatin configuration to its mechanism of action is not completely understood. Here we describe a novel regulatory element, the Upstream Enhancer Element (UEE), which modulates the activity of the chicken α-globin 3′ enhancer by regulating its chromatin structure, specifically by positioning a nucleosome upstream of the core enhancer. This element binds nuclear factors and confers a more restricted activation on the α-globin 3′ enhancer, suggesting a progressive/rheostatic model for enhancer activity. Our results suggest that the UEE activity contributes to the positioning of a nucleosome that is necessary for the α-globin 3′ enhancer activation.

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.

Recent genome editing techniques, including CRISPR mutagenesis screens, offer unparalleled opportunities to study the regulatory non-coding genomic regions, enhancers, promoters, and functional non-coding RNAs. Heterozygous point mutations in FOXF1 and genomic deletion copy-number variants at chromosomal region 16q24.1 involving FOXF1 or its regulatory region mapping ~300 kb upstream of FOXF1 and leaving it intact have been identified in the vast majority of patients with a lethal neonatal lung disease, alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Homozygous Foxf1 −/− mice have been shown to die by embryonic day 8.5 because of defects in the development of extraembryonic and lateral mesoderm-derived tissues, whereas heterozygous Foxf1 +/− mice exhibit features resembling ACDMPV. We have previously defined a human lung-specific enhancer region encoding two long non-coding RNAs, LINC01081 and LINC01082, expressed in the lungs. To investigate the biological significance of lncRNAs in the Foxf1 enhancer region, we have generated a CRISPR/Cas9-mediated ~2.4 kb deletion involving the entire lncRNA-encoding gene Gm26878, located in the mouse region syntenic with the human Foxf1 upstream enhancer. Very recently, this mouse genomic region has been shown to function as a Foxf1 enhancer. Our results indicate that homozygous loss of Gm26878 is neonatal lethal with low penetrance. No changes in Foxf1 expression were observed, suggesting that the regulation of Foxf1 expression differs between mouse and human.

The human and mouse genes that code for the alpha2 chain of collagen I (COL1A2 and Col1a2, respectively) share a common chromatin structure and nearly identical proximal promoter and far upstream enhancer sequences. Despite these homologies, species-specific differences have been reported regarding the function of individual cis-acting elements, such as the first intron sequence. In the present study, we have investigated the transcriptional contribution of the unique open chromatin site in the first intron of COL1A2 using a transgenic mouse model. DNase I footprinting identified a cluster of three distinct areas of nuclease protection (FI1-3) that span from nucleotides +647 to +760, relative to the transcription start site, and which contain consensus sequences for GATA and interferon regulatory factor (IRF) transcription factors. Gel mobility shift and chromatin immunoprecipitation assays corroborated this last finding by documenting binding of GATA-4 and IRF-1 and IRF-2 to the first intron sequence. Moreover, a short sequence encompassing the three footprints was found to inhibit expression of transgenic constructs containing the COL1A2 proximal promoter and far upstream enhancer in a position-independent manner. Mutations inserted into each of the footprints restored transgenic expression to different extents. These results therefore indicated that the unique open chromatin site of COL1A2 corresponds to a repressor, the activity of which seems to be mediated by the concerted action of GATA and IRF proteins. More generally, the study reiterated the existence of species-specific difference in the regulatory networks of the mammalian alpha2(I) collagen coding genes.

We have examined the chromatin structure around and upstream of the transcriptional start site of the human alpha2(I) collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites (HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences and at locations comparable to the mouse gene. HS3, HS4, and HS5 were likewise mapped approximately 20 kilobases upstream of COL1A2 at about the same position as the mouse far-upstream enhancer and within a remarkably homologous genomic segment. DNase I footprinting identified twelve areas of nuclease protection in the far-upstream region (FU1-12) and within stretches nearly identical to the mouse sequence. The region containing HS3-5 was found to confer high and tissue-specific expression in transgenic mice to the otherwise minimally active COL1A2 promoter. Characterization of the human element documented functional differences with the mouse counterpart. Enhancer activity substantially decreased without the segment containing FU1-7 and HS5, and inclusion of AluI repeats located 3' of HS3 augmented position-independent expression of the transgene. Hence, subtle differences may characterize the regulation of mammalian alpha2(I) collagen genes by evolutionarily conserved sequences.

The brown fat specific UnCoupling Protein 1 (UCP1) is involved in thermogenesis, a process by which energy is dissipated as heat in response to cold stress and excess of caloric intake. Thermogenesis has potential implications for body mass control and cellular fat metabolism. In fact, in humans, the variability of the UCP1 gene is associated with obesity, fat gain and metabolism. Since regulation of metabolism is one of the key-pathways in lifespan extension, we tested the possible effects of UCP1 variability on survival. Two polymorphisms (A-3826G and C-3740A), falling in the upstream promoter region of UCP1 , were analyzed in a sample of 910 subjects from southern Italy (475 women and 435 men; age range 40–109). By analyzing haplotype specific survival functions we found that the A-C haplotype favors survival in the elderly. Consistently, transfection experiments showed that the luciferase activity of the construct containing the A-C haplotype was significantly higher than that containing the G-A haplotype. Interestingly, the different UCP1 haplotypes responded differently to hormonal stimuli. The results we present suggest a correlation between the activity of UCP1 and human survival, indicating once again the intricacy of mechanisms involved in energy production, storage and consumption as the key to understanding human aging and longevity.

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1–Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.

Previous studies have demonstrated that STAT5 is critical for expression of granulysin and antimicrobial activity. Because the signaling pathway and the resultant microbicidal activity are defective in HIV-infected patients, the mechanism by which STAT5 leads to granulysin expression is of great interest. In the current study, IL-2–stimulated CRL-2105 CD4+ T cells expressed granulysin and killed Cryptococcus neoformans similar to primary CD4+ T cells. The enhancer activity of the upstream element of the granulysin promoter was analyzed in primary CD4+ T cells and CRL-2105 T cells with a luciferase reporter assay, and a STAT5 binding site, 18,302 to 18,177 bp upstream of the transcription start site, was identified as an enhancer. Additionally, the enhancer functioned in the context of heterologous SV40 promoter irrespective of its transcriptional orientation. Chromatin immunoprecipitation and EMSAs demonstrated that the enhancer element bound STAT5 both in vivo and in vitro, and mutation of the STAT5 binding site abrogated its enhancer activity. Furthermore, overexpression of a dominant negative STAT5a abolished the enhancer activity of the STAT5 binding site and abrogated the anticryptococcal activity of IL-2–stimulated primary CD4+ T cells. Taken together, these data provide details about the complex regulation leading to granulysin expression and anticryptococcal activity in primary CD4+ T cells.

CHRNA5, encoding the nicotinic α5 subunit, is implicated in multiple disorders, including nicotine addiction and lung cancer. Previous studies demonstrate significant associations between promoter polymorphisms and CHRNA5 mRNA expression, but the responsible sequence variants remain uncertain. To search for cis-regulatory variants, we measured allele-specific mRNA expression of CHRNA5 in human prefrontal cortex autopsy tissues and scanned the CHRNA5 locus for regulatory variants. A cluster of six frequent single-nucleotide polymorphisms (rs1979905, rs1979906, rs1979907, rs880395, rs905740, and rs7164030), in complete linkage disequilibrium (LD), fully account for a >2.5-fold allelic expression difference and a fourfold increase in overall CHRNA5 mRNA expression. This proposed enhancer region resides more than 13 kilobases upstream of the CHRNA5 transcription start site. The same upstream variants failed to affect CHRNA5 mRNA expression in peripheral blood lymphocytes, indicating tissue-specific gene regulation. Other promoter polymorphisms were also correlated with overall CHRNA5 mRNA expression in the brain, but were inconsistent with allelic mRNA expression ratios, a robust and proximate measure of cis-regulatory variants. The enhancer region and the nonsynonymous polymorphism rs16969968 generate three main haplotypes that alter the risk of developing nicotine dependence. Ethnic differences in LD across the CHRNA5 locus require consideration of upstream enhancer variants when testing clinical associations.

Mammalian kidney development requires the functions of the Wilms tumor gene WT1 and the WNT/beta-catenin signaling pathway. Recent studies have shown that WT1 negatively regulates WNT/beta-catenin signaling, but the molecular mechanisms by which WT1 inhibits WNT/beta-catenin signaling are not completely understood. In this study, we identified a gene, CXXC5, which we have renamed WID (WT1-induced Inhibitor of Dishevelled), as a novel WT1 transcriptional target that negatively regulates WNT/beta-catenin signaling. WT1 activates WID transcription through the upstream enhancer region. In the developing kidney, Wid and Wt1 are coexpressed in podocytes of maturing nephrons. Structure-function analysis demonstrated that WID interacts with Dishevelled via its C-terminal CXXC zinc finger and Dishevelled binding domains and potently inhibits WNT/beta-catenin signaling in vitro and in vivo. WID is evolutionarily conserved, and ablation of wid in zebrafish embryos with antisense morpholino oligonucleotides perturbs embryonic kidney development. Taken together, our results demonstrate that the WT1 negatively regulates WNT/beta-catenin pathway via its target gene WID and further suggest a role for WID in nephrogenesis.

In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.

Myeloperoxidase (MPO), an enzyme active against bacterial and fungal infections, is expressed specifically in myeloblasts and promyelocytes and minimal in other cell types. We recently identified and partially characterized an upstream enhancer located between -4100 and -3844 bp of the MPO gene. We showed that an AML1 site contributes to enhancer activity and specificity. We now demonstrate three additional footprints within the MPO enhancer and provide evidence that C/EBP and c-Myb sites contribute to its functional, tissue-specific activity. This distal enhancer appears to play an important role in the control of MPO transcription during differentiation of myeloid cells.

Nitric oxide synthase (NOS)2, an inducible enzyme that produces NO during inflammation, is transcriptionally regulated. Our goal was to determine whether high mobility group (HMG)A1 contributes to human (h)NOS2 gene regulation. Using a small molecule inhibitor of HMGA1 binding to DNA, or a dominant-negative form of HMGA1, we blunted the induction of hNOS2 by pro-inflammatory stimuli. Binding of HMGA1 in the region −3506 to −3375 of the hNOS2 promoter, a region not previously known to be involved in hNOS2 regulation, contributed to the induction of hNOS2 promoter in conjunction with upstream enhancer regions. We demonstrate a previously unknown role for HMGA1 in the regulation of hNOS2.

Identification of genes with cancer-specific overexpression offers the potential to efficiently discover cancer-specific activities in an unbiased manner. We apply this paradigm to study mesothelin (MSLN) overexpression, a nearly ubiquitous, diagnostically and therapeutically useful characteristic of pancreatic cancer. We identified an 18-bp upstream enhancer, termed CanScript, strongly activating transcription from an otherwise weak tissue-nonspecific promoter and operating selectively in cells having aberrantly elevated cancer-specific MSLN transcription. Introducing mutations into CanScript showed two functionally distinct sites: an Sp1-like site and an MCAT element. Gel retardation and chromatin immunoprecipitation assays showed the MCAT element to be bound by transcription enhancer factor (TEF)-1 (TEAD1) in vitro and in vivo. The presence of TEF-1 was required for MSLN protein overexpression as determined by TEF-1 knockdown experiments. The cancer specificity seemed to be provided by a putative limiting cofactor of TEF-1 that could be outcompeted by exogenous TEF-1 only in a MSLN-overexpressing cell line. A CanScript concatemer offered enhanced activity. These results identify a TEF family member as a major regulator of MSLN overexpression, a fundamental characteristic of pancreatic and other cancers, perhaps due to an upstream and highly frequent aberrant cellular activity. The CanScript sequence represents a modular element for cancer-specific targeting, potentially suitable for nearly a third of human malignancies. [Cancer Res 2007;67(19):9055–65]

Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.

Both transcriptional and post-transcriptional CYP2E1 regulatory mechanisms are known, resulting in 20-fold or greater variation in CYP2E1 expression. To evaluate functional regulatory elements controlling transcription, CYP2E1 promoter constructs were used to make adenovirus vectors containing CYP2E1 promoter-driven luciferase reporters for analyses in both primary human hepatocytes and HepG2 cells. A 1.2-kilobase pair portion of the CYP2E1 promoter was associated with 5- to 10-fold greater luciferase activity. This upstream region contained five direct repeats of 59 base pairs (bp) that increased thymidine kinase-driven luciferase reporter activity in HepG2 cells more than 5-fold, regardless of orientation. Electrophoretic mobility shift assays (EMSAs) identified sequence-specific nuclear protein binding to the 59-bp repeats that was dependent on a 17-bp sequence containing a canonical GATA binding site (WGATAR). Competitive and supershift EMSA identified the participation of GATA4, another GATA family member or GATA-like factor, and a third factor unrelated to the GATA family. Involvement of the tricho-rhino-phalangeal syndrome-1 factor, which also binds a GATA sequence, was eliminated. Rather, competitive EMSA using known binding sequences for the orphan nuclear receptors, steroidogenic factor-1 (or NR5A1), and fetoprotein transcription factor (or NR5A2) implicated an NR5A member in binding a sequence overlapping the canonical GATA. Chromatin immunoprecipitation assay demonstrated in vivo binding of NR5A2 to the enhancer sequence in human hepatocytes. The enhancer sequence is conserved within the human population but seems species-specific. The identification of this novel enhancer and its putative mechanism adds to the complexities of human CYP2E1 regulation.

In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4alpha (HNF4alpha) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4alpha-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4alpha-specific antiserum. Chromatin-immunoprecipitation assays of HNF4alpha-deficient (H4LivKO) and control (H4Flox) livers with HNF4alpha antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor beta1 (TRbeta1) were exclusively expressed in pericentral hepatocytes. In H4LivKO liver, this pericentral expression remained unaffected, but the genes were additionally expressed in the periportal hepatocytes, albeit at a lower level. The expression of the periportal enzyme phosphoenolpyruvate carboxykinase had declined in HNF4alpha-deficient hepatocytes. GS-negative cells, which were present as single, large hepatocytes or as groups of small cells near portal veins, did express HNF4alpha. Clusters of very small GS- and HNF4alpha-negative, and PCNA- and OV6-positive cells near portal veins were contiguous with streaks of brightly HNF4alpha-positive, OV6-, PCNA-, and PEPCK-dim cells. Conclusion: Our findings show that HNF4alpha suppresses the expression of pericentral proteins in periportal hepatocytes, possibly via a HDAC1-mediated mechanism. Furthermore, we show that HNF4alpha deficiency induces foci of regenerating hepatocytes. (HEPATOLOGY 2007;45:433-444.). AD - AMC Liver Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Carbamoylphosphate synthetase-I is the flux-determining enzyme of the ornithine cycle, and neutralizes toxic ammonia by converting it to urea. An 80 bp glucocorticoid response unit located 6.3 kb upstream of the transcription start site mediates hormone responsiveness and liver-specific expression of carbamoylphosphate synthetase-I. The glucocorticoid response unit consists of response elements for the glucocorticoid receptor, forkhead box A, CCAAT/enhancer-binding protein, and an unidentified protein. With only four transcription factor response elements, the carbamoylphosphate synthetase-I glucocorticoid response unit is a relatively simple unit. The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined by comparing a carbamoylphosphate synthetase-I-expressing hepatoma cell line with a carbamoylphosphate synthetase-I-negative fibroblast cell line. DNaseI hypersensitivity assays revealed an open chromatin configuration of the carbamoylphosphate synthetase-I enhancer in hepatoma cells only. In vivo footprinting assays showed that the accessory transcription factors of the glucocorticoid response unit bound to their response elements in carbamoylphosphate synthetase-I-positive cells, irrespective of whether carbamoylphosphate synthetase-I expression was induced with hormones. In contrast, the binding of glucocorticoid receptor to the carbamoylphosphate synthetase-I glucocorticoid response unit was dependent on treatment of the cells with glucocorticoids. Only forkhead box A was exclusively present in hepatoma cells, and therefore appears to be an important determinant of the observed tissue specificity of carbamoylphosphate synthetase-I expression. As the glucocorticoid receptor is the only DNA-binding protein specifically recruited to the glucocorticoid response unit upon stimulation by glucocorticoids, it is likely to be directly responsible for the transcriptional activation mediated by the glucocorticoid response unit.

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is ~10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamine. AD - AMC Liver Center and Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 69-71, 1105 BK Amsterdam, The Netherlands.

Sigma54-RNA polymerase (Esigma54) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of Esigma54 that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma54-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of Esigma54, DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound Esigma54 in the closed complex. Therefore, these results provide evidence that the activator must approach the Esigma54 closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-Esigma54 complex.

During Strongylocentrotus purpuratus embryogenesis, aboral ectoderm-specific expression of spec2a relies on an upstream enhancer that confers its spatial specificity largely through repression. The purpose of this study was to determine how spec2a expression is repressed in endoderm and oral ectoderm territories. A 78-base pair DNA sequence within the enhancer contains five tightly spaced cis-regulatory elements including proximal (TAATCT) and distal (TAATCC) elements that bind to both SpOtx, a broadly distributed transcriptional activator, and SpGoosecoid (SpGsc), an oral ectoderm-restricted transcriptional repressor. We show here that these two seemingly redundant Otx/Gsc elements have distinct functions. The proximal element bound to SpGATA-E, an endomesoderm-specific transcription factor. Treatment with SpGATA-E and SpGsc morpholino antisense oligonucleotides (MASOs) resulted in enhanced transcriptional activity from the proximal element, suggesting that both factors functioned as repressors at this site. SpGATA-E MASO-treated embryos failed to express ectoderm markers, indicating a role for SpGATA-E in ectoderm differentiation. The spec2a proximal element was distinct from the corresponding element in the related spec1 enhancer, and swaps between spec1 and spec2a cis-regulatory elements indicated, that for optimal repression, the proximal element had to interact with a nearby CCAAT-binding factor element. Our results show that the recently evolved proximal element contributes to the repression of spec2a in endomesoderm and oral ectoderm territories.

Reproductive function is controlled by the hypothalamic neuropeptide, GnRH, which serves as the central regulator of the hypothalamic-pituitary-gonadal axis. GnRH expression is limited to a small population of neurons in the hypothalamus. Targeting this minute population of neurons (as few as 800 in the mouse) requires regulatory elements upstream of the GnRH gene that remain to be fully characterized. Previously, we have identified an evolutionarily conserved promoter region (−173 to −1) and an enhancer (−1863 to −1571) in the rat gene that targets a subset of the GnRH neurons in vivo. In the present study, we used phylogenetic sequence comparison between human and rodents and analysis of the transcription factor clusters within conserved regions in an attempt to identify additional upstream regulatory elements. This approach led to the characterization of a new upstream enhancer that regulates expression of GnRH in a cell-specific manner. Within this upstream enhancer are nine binding sites for Octamer-binding transcription factor 1 (OCT1), known to be an important transcriptional regulator of GnRH gene expression. In addition, we have identified nuclear factor I (NF1) binding to multiple elements in the GnRH-regulatory regions, each in close proximity to OCT1. We show that OCT1 and NF1 physically and functionally interact. Moreover, the OCT1 and NF1 binding sites in the regulatory regions appear to be essential for appropriate GnRH gene expression. These findings indicate a role for this upstream enhancer and novel OCT1/NF1 complexes in neuron-restricted expression of the GnRH gene.

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. We report here that the bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 ( ngn1 ) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together our data demonstrate a role of Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homologue Hairy.

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken alpha-globin domain, 11-16 kb upstream of the embryonic alpha-type pi gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two alpha-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific alpha-globin gene domain located in a permanently open chromatin area.

Expression of a Hoxa-7/lacZ reporter construct in transgenic mouse embryos is shifted anteriorly when the upstream enhancer is multimerized. The shift occurs in spinal ganglia, neurectoderm and in both paraxial and lateral plate mesoderms. Much of the multimer effect is inhibited by destruction of a single caudal (cdx) binding motif in the additional copies of the enhancer. These observations are in agreement with earlier enhancer multimerization analyses made for Hoxb-8 (Charite et al., 1998). Our findings therefore provide further evidence that the anterior limit of a Hox gene's expression domain is normally dependent upon and is determined by, the dosage of transcription factor(s) which bind to its enhancer element(s) and that these factors may be, or must include, the cdx proteins. We consider these findings in terms of both instructional (morphogen-like) gradient and timing models for the establishment of Hox gene expression domains. Enhancer multimerization results in an earlier onset of Hoxa-7/lacZ activity in the embryo. In neurectoderm at 8.7 days and in mesoderm at 10.5 days, the anterior boundaries of expression are located posterior to those seen at some earlier stages of development. We discuss how these findings are in keeping with a model where Hox expression boundaries become set along instructional cdx gradients, formed by cdx decay in cells moving away from the primitive streak region.

Graves' thyroid tissue has been shown to express elevated levels of human sodium/iodide symporter (hNIS) mRNA and protein. In the present work, we demonstrate for the first time that hNIS gene expression in Graves' disease (GD) is up-regulated by Graves' IgG. Here, in transient transfection experiments using FRTL-5 cells, hNIS promoter and enhancer/luciferase construct showed an up to six-fold increase in transcriptional activity after incubation with purified Graves' IgG. Mutation of a CRE site in hNIS enhancer inhibited Graves' IgG response. In addition, mutation of a novel putative regulatory region in hNIS promoter reduced the stimulation three-fold. This discovered putative regulatory sequence might play a role in hNIS up-regulation by Graves' IgG and TSH. The data presented here complement our current knowledge of the pathogenesis of GD and will contribute to a better understanding of mechanisms regulating the thyroid iodide concentrating activity.

This research was originally published in the Journal of Biological Chemistry. Tomoaki Niimi, Yoshitaka Hayashi and Kiyotoshi Sekiguchi. Identification of an Upstream Enhancer in the Mouse Lamininα1 Gene Defining Its High Level of Expression in Parietal Endoderm Cells. J. Biol. Chem. 2003; 278: 9332–9338 © the American Society for Biochemistry and Molecular Biology, Laminin-1 is the major component of the embryonic basement membrane and consists of α1, β1, and γ1 chains. The expression of laminin-1 is induced in mouse F9 embryonal carcinoma cells upon differentiation into parietal endoderm through transcriptional up-regulation of the genes encoding these subunits. Here, we identified a 435-bp enhancer in the 5′-flanking region of the mouse laminin α1 (LAMA1) gene that activated its transcription in a differentiation-dependent manner. This enhancer was also active in PYS-2 parietal yolk sac-derived cells but not in NIH/3T3 fibroblasts, indicating that it was a parietal endoderm-specific enhancer. This enhancer was also active in Engelbreth-Holm-Swarm (EHS) tumor-derived cells characterized by excessive production of laminin-1 and other basement membrane components, suggesting that EHS tumors have a transcriptional control mechanism similar to that of parietal endoderm cells. Electrophoretic mobility shift analyses revealed four protein binding sites (PBS1-PBS4) in the 435-bp region. However, these DNA-binding proteins were detected not only in parietal endoderm cells (i.e. differentiated F9 cells, PYS-2 cells, and EHS tumor-derived cells) but also in undifferentiated F9 cells and NIH/3T3 cells. Mutational analyses revealed that three of these binding sites (PBS2, PBS3, and PBS4) function synergistically to confer the parietal endoderm-specific enhancer activity. The proteins binding to PBS2 and PBS4 were identified as the Sp1/Sp3 family of transcription factors and YY1, respectively.

Recent studies reveal important and distinct roles for cardiac α1a adrenergic receptors (α1aARs). Surprisingly, given their importance in myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as frequent use of rat cardiomyocyte model systems, the rat α1aAR gene promoter has never been characterized. Therefore, we isolated 3.9 kb of rat α1aAR 5′-untranslated region and 5′-regulatory sequences and identified multiple transcription initiation sites. One proximal (P1) and several clustered upstream distal promoters (P2, P3, and P4) were delineated. Sequences surrounding both proximal and distal promoters lack typical TATA or CCAAT boxes but contain cis-elements for multiple myocardium-relevant nuclear regulators including Sp1, GATA, and CREB, findings consistent with enhanced cardiac basal α1aAR expression seen in Northern blots and reporter constructs. Promoter analysis using deletion reporter constructs reveals, in addition to a powerful upstream enhancer, a key region (–558/−542) important in regulating all α1aAR promoters with hypoxic stress. Gel shift analysis of this 14-bp region confirms a hypoxia-induced shift independent of direct hypoxia-inducible factor binding. Mutational analysis of this sequence identifies a novel 9-bp hypoxia response element, the loss of which severely attenuates hypoxia-mediated repression of α1aAR transcription. These findings for the α1a gene should facilitate elucidation of α1AR-mediated mechanisms involved in distinct myocardial pathologies.

Prostate-specific antigen (PSA) is considered as an important marker for prostate cancer. Regulation of PSA gene expression is mediated by androgens bound to androgen receptors via androgen response elements (AREs) in its promoter and far upstream enhancer regions. In addition, GATA proteins contribute to PSA gene transcription by interacting with GATA motifs present in the PSA enhancer sequence. The TRPS1 gene contains a single GATA zinc finger domain and not only binds to forward consensus GATA motifs but also to an inverse GATA motif overlapping the ARE III in the far upstream enhancer of the PSA gene. Overexpression of TRPS1 in androgen-dependent human LNCaP prostate cancer cells inhibited the transcription of a transiently transfected PSA enhancer/promoter-driven luciferase reporter construct. Furthermore, overexpression of TRPS1 reduced the androgen-induced endogenous PSA levels secreted in culture medium of LNCaP cells. Our results suggest a role of TRPS1 in androgen regulation of PSA gene expression.

We analyzed the order of recruitment of factors to the HNF-4α regulatory regions upon the initial activation of the gene during enterocyte differentiation. An initially independent assembly of regulatory complexes at the proximal promoter and the upstream enhancer regions was followed by the tracking of the entire DNA-protein complex formed on the enhancer along the intervening DNA until it encountered the proximal promoter. This movement correlated with a unidirectional spreading of histone hyperacetylation. Transcription initiation coincided with the formation of a stable enhancer-promoter complex and remodeling of the nucleosome situated at the transcription start site. The results provide experimental evidence for the involvement of a dynamic process culminating in enhancer-promoter communication during long-distance gene activation.

The human 8q24 gene desert contains multiple enhancers that form tissue-specific long-range chromatin loops with the MYC oncogene, but how chromatin looping at the MYC locus is regulated remains poorly understood. Here we demonstrate that a long noncoding RNA (lncRNA), CCAT1-L, is transcribed specifically in human colorectal cancers from a locus 515 kb upstream of MYC. This lncRNA plays a role in MYC transcriptional regulation and promotes long-range chromatin looping. Importantly, the CCAT1-L locus is located within a strong super-enhancer and is spatially close to MYC. Knockdown of CCAT1-L reduced long-range interactions between the MYC promoter and its enhancers. In addition, CCAT1-L interacts with CTCF and modulates chromatin conformation at these loop regions. These results reveal an important role of a previously unannotated lncRNA in gene regulation at the MYC locus.

The expression of the pre-T cell receptor α (pTa) gene occurs exclusively in immature T lymphocytes and is regulated by poorly defined mechanisms. We have analyzed the role of the upstream enhancer in pTa expression using conventional and bacterial artificial chromosome (BAC) reporter transgenes. The deletion of the enhancer completely abolished the expression of pTa BAC reporter in transgenic mice. Conversely, the combination of pTa enhancer and promoter targeted transgenes specifically to immature thymocytes, recapitulating the expression pattern of pTa. The core enhancer is conserved between mice and humans and contains a critical binding site for the transcription factor c-Myb. We also show that pTa promoter contains a conserved tandem E box site activated by E protein, HEB. These data establish the enhancer as a critical element regulating pTa gene expression and identify additional targets for c-Myb and E proteins in T cell development.