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Human CYP19 spans a region of chromosome 15 of approximately 130 kb and encodes aromatase, an enzyme required for estrogen synthesis. In the human granulosa cell-line KGN, there are seven open chromatin regions within the CYP19 locus. In this study, we demonstrate that two of these regions ~40 kb upstream and ~15 kb downstream of the CYP19 promoter are cohesin-loading sites, physically interacting with the promoter to negatively and positively regulate transcription, respectively. These observations suggest that CYP19 expression is controlled by a balance between the upstream silencer and downstream enhancer. When cohesin is depleted, CYP19 expression is elevated since the silencer is 2.5-fold further from the promoter than the enhancer and most likely depends on cohesin-mediated tethering to influence expression.

To extend the applications of engineered nanomaterials, such as graphene oxide (GO), it is necessary to minimize cytotoxicity. However, the mechanisms underlying this cytotoxicity are unclear. Dynamic chromosomal interactions have been used to illustrate the molecular bases of gene expression, which offers a more sensitive and cutting-edge technology to elucidate complex biological processes associated with epigenetic regulations. In this study, the role of GO-triggered chromatin interactions in the activation of cox2, a hallmark of inflammation, was investigated in normal human cells. Using chromosome conformation capture technology, we showed that GO triggers physical interactions between the downstream enhancer and the cox2 promoter in human embryonic kidney 293T (293T) via p65 and p300 complex-mediated dynamic chromatin looping, which was required for high cox2 expression. Moreover, tumor necrosis factor-α (TNF-α), located upstream of the p65 signaling pathway, contributed to the regulation of cox2 activation through dynamic chromatin architecture. Compared with pristine GO and aminated GO (GO-NH2), poly (acrylic acid)-functionalized GO (GO-PAA) induced a weaker inflammatory response and a weaker effect on chromatin architecture. Our results mechanistically link GO-mediated chromatin interactions with the regulation of cox2 and suggest that GO derivatives may minimize toxicity in practical applications.

We recently described a new class of long noncoding RNAs (lncRNAs) that are distinguished by especially tight chromatin association and whose presence is strongly correlated to expression of nearby genes. Here, we examine the cis-enhancer mechanism of this class of chromatin-enriched RNA (cheRNA) across multiple human cell lines. cheRNAs are largely cell type specific and provide the most reliable chromatin signature to predict cis-gene transcription in every human cell type examined. Targeted depletion of three cheRNAs decreases expression of their neighboring genes, indicating potential co-activator function, and single-molecule fluorescence in situ hybridization (smFISH) of one cheRNA-distal target gene pair suggests a spatial overlap consistent with a role in chromosome looping. Additionally, the cheRNA HIDALGO stimulates the fetal hemoglobin subunit gamma 1 (HBG1) gene during erythroid differentiation by promoting contacts to a downstream enhancer. Our results suggest that multiple cheRNAs activate proximal lineage-specific gene transcription.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

Heteroblasty refers to a phenomenon that a plant produces morphologically or functionally different lateral organs in an age‐dependent manner. In the model plant Arabidopsis thaliana, the production of trichomes (epidermal leaf hairs) on the abaxial (lower) side of leaves is a heteroblastic mark for the juvenile‐to‐adult transition. Here, we show that the heteroblastic development of abaxial trichomes is regulated by a spatiotemporally regulated complex comprising the leaf abaxial fate determinant (KAN1) and the developmental timer (miR172‐targeted AP2‐like proteins). We provide evidence that a short‐distance chromatin loop brings the downstream enhancer element into close association with the promoter elements of GL1, which encodes a MYB transcription factor essential for trichome initiation. During juvenile phase, the KAN1‐AP2 repressive complex binds to the downstream sequence of GL1 and represses its expression through chromatin looping. As plants age, the gradual reduction in AP2‐like protein levels leads to decreased amount of the KAN1‐AP2 complex, thereby licensing GL1 expression and the abaxial trichome initiation. Our results thus reveal a novel molecular mechanism by which a heteroblastic trait is governed by integrating age and leaf polarity cue in plants.

In previous work it was shown that mutation of site 1 in the downstream enhancer sequence (DE) led to ablation of enhancement. Mutation of the Wilms tumour factor element (Wt), situated in the Amh promoter between the tata box and the start of translation (TSS), also led to ablation of enhancement. This suggested that these sites may be the anchor points for a specific duplex factor bridging remote DNA elements to the promoter. Mutation analysis of the DNA sequence between sections 1 and 2 of DE was carried out by site directed mutagenesis. It is reported here that site 4 lying between DE1 and DE2, plays a key role in controlling the level of enhancement.

In vertebrates, the periodic formation of somites from the presomitic mesoderm (PSM) is driven by the molecular oscillator known as the segmentation clock. The hairy-related gene, hes6/her13.2, functions as a hub by dimerizing with other oscillators of the segmentation clock in zebrafish embryos. Although hes6 exhibits a posterior–anterior expression gradient in the posterior PSM with a peak at the tailbud, the detailed mechanisms underlying this unique expression pattern have not yet been clarified. By establishing several transgenic lines, we found that the transcriptional regulatory region downstream of hes6 in combination with the hes6 3′UTR recapitulates the endogenous gradient of hes6 mRNA expression. This downstream region, which we termed the PT enhancer, possessed several putative binding sites for the T-box and Ets transcription factors that were required for the regulatory activity. Indeed, the T-box transcription factor (Tbx16) and Ets transcription factor (Pea3) bound specifically to the putative binding sites and regulated the enhancer activity in zebrafish embryos. In addition, the 3′UTR of hes6 is required for recapitulation of the endogenous mRNA expression. Furthermore, the PT enhancer with the 3′UTR of hes6 responded to the inhibition of retinoic acid synthesis and fibroblast growth factor signaling in a manner similar to endogenous hes6. The results showed that transcriptional regulation by the T-box and Ets transcription factors, combined with the mRNA stability given by the 3′UTR, is responsible for the unique expression gradient of hes6 mRNA in the posterior PSM of zebrafish embryos.

To elucidate the transcriptional regulation that underlies specification of the otic placode, we investigated the Sox3 downstream enhancer Otic1 of the chicken, the activity of which is restricted to and distributed across the entire otic placode. The 181-bp Otic1 enhancer sequence was dissected into a 68-bp minimal activating sequence, which exhibited dimer enhancer activity in the otic placode and cephalic neural crest, and this was further reduced to a 25-bp Otic1 core sequence, which also showed octamer enhancer activity in the same regions. The Otic1 core octamer was activated by the combined action of Sall4 and the SoxE transcription factors (TFs) Sox8 or Sox9. Binding of Sall4, Sox8 and Sox9 to the Otic1 sequence in embryonic tissues was confirmed by ChIP-qPCR analysis. The core-adjoining 3' side sequences of Otic1 augmented its enhancer activity, while inclusion of the CAGGTG sequence in the immediate 3' end of the 68-bp sequence repressed its enhancer activity outside the otic placode. The CAGGTG sequence likely serves as the binding sites of the repressor TFs δEF1 (Zeb1), Sip1 (Zeb2), and Snail2, all of which are expressed in the cephalic neural crest but not in the otic placode. Therefore, the combination of Sall4-Sox8-dependent activation and CAGGTG sequence-dependent repression determines otic placode development. Although the Otic1 sequence is not conserved in mammals or fishes, the activation mechanism is, as Otic1 was also activated in otic placode tissues developed from mouse embryonic stem cells and transient transgenic zebrafish embryos.

We report an unusual coincidence of two Mendelian disorders in a proband presenting with short stature and cafe-au-lait spots. Genetic testing yielded both a deletion in the downstream enhancer region of SHOX gene related to Leri–Weill dyschondrosteosis, and Neurofibromin gene truncating mutation resulting in Neurofibromatosis 1 phenotype. Pedigree analysis indicated a crossover in the pseudoautosomal region 1 (PAR1) in one of the family members gametes. To the best of our knowledge, this specific combination has not been previously reported.

The vertebrate T-box transcription factor gene Tbx18 performs a vital role in development of multiple organ systems. Tbx18 insufficiency manifests as recessive phenotypes in the upper urinary system, cardiac venous pole, inner ear, and axial skeleton; homozygous null mutant animals die perinatally. Here, we report a new regulatory mutation of Tbx18, a reciprocal translocation breaking 78kbp downstream of the gene. 12Gso homozygotes present urinary and vertebral defects very similar to those associated with Tbx18-null mutations, but 12Gso is clearly not a global null allele since homozygotes survive into adulthood. We show that 12Gso down-regulates Tbx18 expression in a manner that is both spatially- and temporally-specific; combined with other data, the mutation points particularly to the presence of an essential urogenital enhancer located near the translocation breakpoint site. In support of this hypothesis, we identify a distal enhancer element, ECR1, which is active in developing urogenital and other tissues; we propose that disruption of this element leads to premature loss of Tbx18 function in 12Gso mutant mice. These data reveal a long-range regulatory architecture extending far downstream of Tbx18, identify a novel and likely essential urogenital enhancer, and introduce a new tool for dissecting postnatal phenotypes associated with dysregulation of Tbx18.

Human blood monocytes comprise at least 3 subpopulations that differ in phenotype and function. Here, we present the first in-depth regulome analysis of human classical (CD14(++)CD16(-)), intermediate (CD14(+)CD16(+)), and nonclassical (CD14(dim)CD16(+)) monocytes. Cap analysis of gene expression adapted to Helicos single-molecule sequencing was used to map transcription start sites throughout the genome in all 3 subsets. In addition, global maps of H3K4me1 and H3K27ac deposition were generated for classical and nonclassical monocytes defining enhanceosomes of the 2 major subsets. We identified differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validated novel downstream enhancer elements at the CD14 locus. In addition to known subset-specific features, pathway analysis revealed marked differences in metabolic gene signatures. Whereas classical monocytes expressed higher levels of genes involved in carbohydrate metabolism, priming them for anaerobic energy production, nonclassical monocytes expressed higher levels of oxidative pathway components and showed a higher mitochondrial routine activity. Our findings describe promoter/enhancer landscapes and provide novel insights into the specific biology of human monocyte subsets.

The LIM class of homeodomain protein 3 (LHX3) transcription factor is essential for pituitary gland and nervous system development in mammals. In humans, mutations in the LHX3 gene underlie complex pediatric syndromes featuring deficits in anterior pituitary hormones and defects in the nervous system. The mechanisms that control temporal and spatial expression of the LHX3 gene are poorly understood. The proximal promoters of the human LHX3 gene are insufficient to guide expression in vivo and downstream elements including a conserved enhancer region appear to play a role in tissue-specific expression in the pituitary and nervous system. Here we characterized the activity of this downstream enhancer region in regulating gene expression at the cellular level during development. Human LHX3 enhancer-driven Cre reporter transgenic mice were generated to facilitate studies of enhancer actions. The downstream LHX3 enhancer primarily guides gene transcription in α-glycoprotein subunit -expressing cells secreting the TSHβ, LHβ, or FSHβ hormones and expressing the GATA2 and steroidogenic factor 1 transcription factors. In the developing nervous system, the enhancer serves as a targeting module active in V2a interneurons. These results demonstrate that the downstream LHX3 enhancer is important in specific endocrine and neural cell types but also indicate that additional regulatory elements are likely involved in LHX3 gene expression. Furthermore, these studies revealed significant gonadotrope cell heterogeneity during pituitary development, providing insights into the cellular physiology of this key reproductive regulatory cell. The human LHX3 enhancer-driven Cre reporter transgenic mice also provide a valuable tool for further developmental studies of cell determination and differentiation in the pituitary and nervous system.

Bcl11b is a T-cell specific gene in hematopoiesis that begins expression during T-lineage commitment and is required for this process. Aberrant expression of BCL11B or proto-oncogene translocation to the vicinity of BCL11B can be a contributing factor in human T-ALL. To identify the mechanism that controls its distinctive T-lineage expression, we corrected the identified Bcl11b transcription start site and mapped a cell-type-specific differentially methylated region bracketing the Bcl11b promoter. We identified a 1.9-kb region 850 kb downstream of Bcl11b, "Major Peak," distinguished by its dynamic histone marking pattern in development that mirrors the pattern at the Bcl11b promoter. Looping interactions between promoter-proximal elements including the differentially methylated region and downstream elements in the Major Peak are required to recapitulate the T-cell specific expression of Bcl11b in stable reporter assays. Functional dissection of the Major Peak sequence showed distinct subregions, in which TCF-1 sites and a conserved element were required for T-lineage-specific activation and silencing in non-T cells. A bacterial artificial chromosome encompassing the full Bcl11b gene still required the addition of the Major Peak to exhibit T-cell specific expression. Thus, promoter-proximal and Major Peak sequences are cis-regulatory elements that interact over 850 kb to control expression of Bcl11b in hematopoietic cells.

Amh (anti-Mullerian hormone) is a single copy gene which is expressed strongly in Sertoli cells in the foetal testis and participates in the onset of sexual differentiation. Its promoter driving the expression of a reporter gene (d2EGFP) has been used to analyse the role of certain defined putative elements and a downstream enhancer element in gene expression. These experiments were carried out in vitro using a line of pre-pubertal mouse Sertoli cells, transienly transfected with circular DNA constructs with variously mutated promoter elements. A downstream enhancer element, situated immediately 3’ of the polyadenylation (PA) signal for Amh, has been inserted in an equivalent position in the d2EGFP construct. When the Amh promoter is unmodified, the downstream enhancer (DE) is positively associated with a large increase in EGFP expression. This is at least partly the consequence of an increased rate of expression by individual cells. Experiments using variously truncated Amh promoters indicate that an upstream region (-214 to -336) may play a minor role in facilitating enhancement. However mutation of the Wilms tumour factor-1 element, situated between the tata box and the start of translation, results in an almost complete suppression of enhancement.

Environmental factors interact with the genome throughout life to determine gene expression and, consequently, tissue function and disease risk. One such factor that is known to play an important role in determining long-term metabolic health is diet during critical periods of development. Epigenetic regulation of gene expression has been implicated in mediating these programming effects of early diet. The precise epigenetic mechanisms that underlie these effects remain largely unknown. Here, we show that the transcription factor Hnf4a , which has been implicated in the etiology of type 2 diabetes (T2D), is epigenetically regulated by maternal diet and aging in rat islets. Transcriptional activity of Hnf4a in islets is restricted to the distal P2 promoter through its open chromatin configuration and an islet-specific interaction between the P2 promoter and a downstream enhancer. Exposure to suboptimal nutrition during early development leads to epigenetic silencing at the enhancer region, which weakens the P2 promoter–enhancer interaction and results in a permanent reduction in Hnf4a expression. Aging leads to progressive epigenetic silencing of the entire Hnf4a locus in islets, an effect that is more pronounced in rats exposed to a poor maternal diet. Our findings provide evidence for environmentally induced epigenetic changes at the Hnf4a enhancer that alter its interaction with the P2 promoter, and consequently determine T2D risk. We therefore propose that environmentally induced changes in promoter-enhancer interactions represent a fundamental epigenetic mechanism by which nutrition and aging can influence long-term health.

Léri-Weill dyschondrosteosis (LWD, MIM 127300), is a dominantly inherited skeletal dysplasia with disproportionate short stature, mesomelic limb shortening, and the characteristic Madelung deformity. Two regions of the pseudoautosomal region 1 (PAR1) have been shown to be involved in LWD, SHOX (short-stature homeobox-containing gene) and the downstream enhancer region. We report our genetic findings of a young girl clinically diagnosed with LWD. We analyzed the proband and her family using MLPA and microsatellite analysis. We identified a deletion, 726-866 kb in size, of the downstream SHOX enhancer region in the proband. Neither parent carried the deletion. Microsatellite analysis showed that the deleted allele was of paternal origin. The mutation is more likely to have arisen from a de novo event but paternal gonadal mosaicism cannot be excluded. In conclusion, we report the clinical and molecular details of the first case of a de novo deletion of the downstream PAR1 region in an LWD individual. De novo deletions of SHOX and the downstream enhancer region must be therefore considered in cases of isolated LWD.

Summary Transcription of the lysozyme gene is rapidly induced by proinflammatory stimuli such as treatment with bacterial lipopolysaccharide (LPS). Here we show that this induction involves both the relief of repression mediated by the enhancer-blocking protein CTCF that binds to a negative regulatory element at −2.4 kb, and the activation of two flanking enhancer elements. The downstream enhancer has promoter activity, and LPS stimulation initiates the transient synthesis of a noncoding RNA (LINoCR) transcribed through the −2.4 kb element. Expression of LINoCR is correlated with IKKα recruitment, histone H3 phosphoacetylation in the transcribed region, the repositioning of a nucleosome over the CTCF binding site, and, eventually, CTCF eviction. Each of these events requires transcription elongation. Our data reveal a transcription-dependent mechanism of chromatin remodeling that switches a cis-regulatory region from a repressive to an active conformation.

The spatial configuration of the chicken alpha-globin gene domain in erythroid and lymphoid cells was studied by using the Chromosome Conformation Capture (3C) approach. Real-time PCR with TaqMan probes was employed to estimate the frequencies of cross-linking of different restriction fragments within the domain. In differentiated cultured erythroblasts and in 10-day chick embryo erythrocytes expressing 'adult' alpha(A) and alpha(D) globin genes the following elements of the domain were found to form an 'active' chromatin hub: upstream Major Regulatory Element (MRE), -9 kb upstream DNase I hypersensitive site (DHS), -4 kb upstream CpG island, alpha(D) gene promoter and the downstream enhancer. The alpha(A) gene promoter was not present in the 'active' chromatin hub although the level of alpha(A) gene transcription exceeded that of the alpha(D) gene. Formation of the 'active' chromatin hub was preceded by the assembly of multiple incomplete hubs containing MRE in combination with either -9 kb DHS or other regulatory elements of the domain. These incomplete chromatin hubs were present in proliferating cultured erythroblasts which did not express globin genes. In lymphoid cells only the interaction between the alpha(D) promoter and the CpG island was detected.

The mouse Igκ locus has three known transcriptional enhancers: the matrix association region/intronic enhancer, the 3′ enhancer (E3′), and the further downstream enhancer (Ed). Previous studies have shown that both matrix association region/intronic and E3′ enhancers are required for maximal gene rearrangement of the locus, and that E3′ is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germline deletion of Ed. Ed deleted homozygous mice (Ed−/−) have moderately reduced numbers of Igκ expressing B cells and correspondingly increased numbers of Igλ expressing B cells in spleen. Ed−/− mice also have decreased Igκ mRNA expression in resting and T cell-dependent activated splenic B cells and reduced Igκ chains in sera. However, our analysis indicates that Igκ gene rearrangement is normal in Ed−/− mice. In addition, our results show that Ed−/− mice exhibit reduced SHM in the Igκ gene J-C intronic region in germinal center B cells from Peyer’s patches. We conclude that Ed positively regulates Igκ gene expression and SHM, but not gene rearrangement.

We tested the hypothesis that a transcriptional chorionic enhancer (CE), previously identified to increase human renin expression in choriodecidual cells is required to mediate tissue-specific, cell-specific, and regulated expression of human renin in transgenic mice. Recombineering was used to delete the CE upstream of the renin gene alone or in combination with the kidney enhancer (KE) in a large artificial chromosome construct containing the entire human renin gene and extensive flanking sequences. Deletion of the CE had no qualitative or quantitative effect on the tissue-specific expression of human renin, nor on the cellular localization of human renin in the kidney or placenta. Combined deletion of both the CE and KE caused a decrease in the level of renal renin expression consistent with the established role of the KE. We also considered the possibility that the CE is a downstream enhancer of the KiSS1 gene, which lies directly upstream of renin and is also expressed in the placenta. Deletion of the CE alone, or the CE and KE together, had no effect on the level of KiSS1 expression in the placenta. These data provide convincing evidence that the CE is silent in vivo, at least in the mouse. The absence of a phenotype caused by deletion of the CE is consistent with the observation that the sequence is not evolutionarily conserved.

Previous studies have suggested that juxtaposition of a downstream enhancer to the fetal gamma-globin gene results in reactivation of the gamma-gene in adult erythrocytes of individuals with hereditary persistence of fetal hemoglobin (HPFH). To test the hypothesis in a much stricter basis, we produced beta locus YAC transgenic mice carrying an exact beta locus replicate of a deletional HPFH mutation, HPFH 2. Although the gamma-globin gene was expressed in the HPFH 2/beta locus YAC (HPFH2/YAC) transgenic mice in the early stage of development, it was completely silenced in the adult mice. The failure of gamma-gene reactivation by the juxtaposed HPFH2 enhancer contradicts the results of previous studies. We speculate that the discrepant results reflect differences in the distance between the locus of region (LCR) and the gamma-globin gene characteristic of the plasmid, cosmid or YAC constructs used for production of transgenic mice. The difference in the phenotype of the HPFH2/YAC transgenic mice and the humans with HPFH2 mutation suggests that in addition to juxtaposition of HPFH enhancers, the upstream region that is absent in the beta-YAC construct might be involved in gamma-gene reactivation in HPFH individuals. The DNase I hypersensitive sites of the LCR were well formed and the chromatin histones were acetylated. A moderate level of pol II binding was detected in the LCR, despite the fact that no transcription occurred in the globin-genes of the adult HPFH2/YAC transgenic mice. The results suggest that formation of the LCR chromatin structure in erythroid cells is independent of globin-gene transcription in the locus.

Fragile X syndrome, the most common form of mental impairment, is caused by expansion of a (CGG)n trinucleotide repeat element located in the 5' untranslated region of the fragile X mental retardation 1 (FMR1) gene. Repeat expansion is known to influence both transcription and translation; however, the mechanisms by which the CGG element exerts its effects are not known. In the current work, we have utilized 5'-RLM-RACE to examine the influence of CGG repeat number on the utilization of transcription start sites in normal (n

The human nonmuscle myosin heavy chain B gene contains a 30-nucleotide alternative exon, N30, that is included in the mRNA from neural cells but is skipped in all other cells. We have previously identified an intronic distal downstream enhancer (IDDE) region that is required for neural cell-specific inclusion of N30. In this study, we investigated the mechanism by which the IDDE promotes N30 exon usage. In vitro splicing analysis using neural cell nuclear extracts and two-exon pre-mRNA substrates, which consist of the N30 exon and either the upstream (E5) or downstream (E6) exon, demonstrates that the IDDE activates upstream E5-N30 splicing by facilitating early prespliceosome complex formation. The IDDE has no effect on N30-E6 splicing where the IDDE resides. Inspection of splice site consensus sequences shows that a polypyrimidine (Py) tract preceding N30 is suboptimal for U2AF binding. Optimizing the Py tract completely relieves the requirement for the IDDE in E5-N30 splicing in vitro. In transfected cells, the wild-type minigene transcripts, which consist of three exons, E5, N30, and E6, undergo neural cell-specific and IDDE-dependent alternative splicing of N30. Optimizing the Py tract in minigenes also completely relieves the requirement for the IDDE in N30 inclusion. Furthermore, overexpression of the truncated U2AF65, which contains the arginine and serine dipeptide-rich domain and linker domain, but lacks the RNA binding domain, selectively inhibits the IDDE-mediated N30 inclusion in mRNA from the wild-type minigene in a dominant negative fashion. These results support the hypothesis that the IDDE facilitates the recognition of the 3' splice site preceding N30 by a network of protein-protein interactions implicated in the recruitment of U2AF to a suboptimal Py tract.

Embryonal carcinoma (EC) cells are used widely as a model system for studying the expression of developmentally regulated genes, in particular genes that are regulated at the transcriptional level when EC cells differentiate. This review focuses on the molecular mechanisms that govern the transcription of the fibroblast growth factor-4 (FGF-4) gene, which appears to be the first FGF expressed during mammalian development. Interest in this gene has increased considerably with the finding that FGF-4 is essential for mammalian embryogenesis. The FGF-4 gene has also generated considerable interest because it is inhibited at the transcriptional level when EC cells undergo differentiation and because this gene is regulated by a powerful distal enhancer located 3 kb downstream of the transcription start site in the last exon of the gene. Hence, study of the FGF-4 gene is likely to shed light on the molecular mechanisms by which distal enhancers regulate gene expression. In addition to being regulated by the downstream enhancer, the expression of this gene is influenced by a regulatory region located just upstream of the transcription start site, which contains two Sp1 motifs and a CCAAT box motif. Examination of the downstream enhancer has identified three functional cis-regulatory elements: a high mobility group (HMG) protein binding motif, an octamer binding motif, and an Sp1 motif, which are likely to bind Sox-2, Oct-3, and Sp1/Sp3, respectively, in vivo. Interestingly, Sox-2 and Oct-3 expression, like FGF-4 expression, decreases when EC cells differentiate, which suggests that the loss of these transcription factors is responsible, at least in part, for the transcriptional turn-off of the FGF-4 gene. In view of these and other findings, we present a model for the differential expression of the FGF-4 gene that includes not only the contributions of specific transcription factors, but also the contribution of chromatin structure before and after differentiation.

It has been shown previously that the FGF-4 gene is regulated by a powerful downstream enhancer in embryonal carcinoma (EC) cells. This enhancer contains an essential HMG motif; however, the transcription factor that binds to the HMG motif in EC cells has not been determined definitively. In earlier studies, this HMG motif was shown to bind a heat-stable, redox-insensitive factor expressed by F9 EC cells. Others have proposed that the transcription factor Sox-2 binds to the FGF-4 enhancer HMG motif. In this study, we demonstrate that the N-terminal half of Sox-2, which contains the DNA binding domain, binds to the FGF-4 enhancer HMG motif and we show that this binding is unaffected by heat and oxidation. In addition, we employed two experimental approaches to demonstrate that Sox-2 regulates the transcription of the FGF-4 gene in EC cells. As part of these studies, an expression plasmid that codes for a dominant-negative form of Sox-2 was used in transient expression assays. In other experiments, a Sox-2 antisense expression plasmid was used. When co-transfected into F9 EC cells along with an FGF-4 promoter/reporter gene construct, each expression plasmid caused a significant reduction in reporter activity. Our studies also demonstrate that Sox-2 affects the expression of the FGF-4 gene in the multipotent EC cell line, P19. Taken together, these studies argue strongly that Sox-2 plays an important role in the expression of the FGF-4 gene in vivo.

Programmed ribosomal frameshifting in viral messenger RNA occurs in response to neighboring sequence elements consisting of: a frameshift site, a spacer, and a downstream enhancer sequence. In human immunodeficiency virus type 1 (HIV-1) mRNA, this sequence element has a potential to form either a stem-loop or a pseudoknot structure. Based on many mutational studies, the stem-loop structure has been proposed for the downstream enhancer region of the HIV-1 mRNA. This stimulatory stem-loop structure is separated from the shift site by a spacer of seven nucleotides. In contrast, a recent report has proposed an alternative model in which the bases in the spacer sequence form a pseudoknot structure as the downstream enhancer sequence [Du et al., Biochemistry 35 (1996) 4187–4198.]. Using UV melting and enzymatic mapping analyses, we have investigated the conformation of the sequence region involved in ribosomal frameshifting in HIV-1. Our S1, V1, and T1 endonuclease mappings, together with UV melting analysis, clearly indicate that this sequence element of the HIV-1 mRNA frameshift site forms a stem-loop structure, not a pseudoknot structure. This finding further supports the stem-loop structure proposed by many mutational studies for the downstream enhancer sequence of the HIV-1 mRNA.

The mouse M-lysozyme downstream enhancer has been previously characterized on several levels of gene regulation. The enhancer was co-localized with a DNase I hypersensitive site in the chromatin of mature macrophages, the in vivo interaction of transcription factor GABP with the enhancer core (MLDE) demonstrated binding being restricted to mature macrophage cells, and analysis of the MLDE methylation state revealed a correlation between demethylation of CpG dinucleotides and the in vivo GABP binding. Here, we analyzed in detail the full-length enhancer in addition to the core element. We identified a total of nine binding sites for nuclear factors. Most of these factors are found ubiquitously in all cell types tested. These factors include several unknown proteins as well as the transcription factor NF-Y. In addition, three binding sites for a new single-stranded DNA binding protein were found. The presence of this factor in mature macrophages correlates with the in vivo DNA melting of one of the binding sites and with the enhancer strength.

The mouse lysozyme downstream enhancer was previously colocalized with the DNase I-hypersensitive site in the chromatin of mature macrophages. This hypersensitive site was shown to be macrophage differentiation-dependent. Demethylation of CpG sequences within the enhancer is correlated with lysozyme expression in mature macrophages. Binding of the GABP heterotetrameric transcription factor to the enhancer core element (MLDE), only seen in vivo on the demethylated MLDE element in macrophages, is inhibited by DNA methylation. Here, we analyzed the DNA sequences required for demethylation. In electrophoretic mobility shift experiments we found that in addition to the complete methylated MLDE the hemimethylated form of the lower strand inhibits GABP binding as well. Therefore, GABP is unlikely to be the mediator of demethylation. In addition, we show by stable DNA transfections of methylated mouse lysozyme enhancer sequences that MLDE-flanking sequences are required for demethylation. We narrowed down these DNA elements to two short regions of 163 and 79 base pairs on either side of the MLDE, each of which is sufficient to mediate demethylation of the GABP site.

Haploinsufficiency of SHOX on the short arm pseudoautosomal region (PAR1) leads to Leri–Weill dyschondrosteosis (LWD), and nullizygosity of SHOX results in Langer mesomelic dysplasia (LMD). Molecular defects of LWD/LMD include various microdeletions in PAR1 that involve exons and/or the putative upstream or downstream enhancer regions of SHOX, as well as several intragenic mutations. Here, we report on a Japanese male infant with mild manifestations of LMD and hitherto unreported microdeletions in PAR1. Clinical analysis revealed mesomelic short stature with various radiological findings indicative of LMD. Molecular analyses identified compound heterozygous deletions, that is, a maternally inherited ∼46 kb deletion involving the upstream region and exons 1–5 of SHOX, and a paternally inherited ∼500 kb deletion started from a position ∼300 kb downstream from SHOX. In silico analysis revealed that the downstream deletion did not affect the known putative enhancer regions of SHOX, although it encompassed several non-coding elements which were well conserved among various species with SHOX orthologs. These results provide the possibility of the presence of a novel enhancer for SHOX in the genomic region ∼300 to ∼800 kb downstream of the start codon. © 2013 Wiley Periodicals, Inc.

The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-kappaB to an enhancer located just downstream of its 3' UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-kappaB to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-kappaB.

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

Embryonal carcinoma (EC) cells and their embryo-derived counterparts, embryonic stem (ES) cells, have been used extensively to study the transcriptional regulation of the fibroblast growth factor-4 (FGF-4) gene. The FGF-4 gene is expressed in EC cells and ES cells, but it is repressed in their retinoic acid (RA)-induced differentiated counterparts. Previous studies have shown that the transcription of the FGF-4 gene is controlled by cis-regulatory elements located in the 5' flanking region of the gene, and by a powerful enhancer located approximately 3 kb downstream from the transcription start site. In the current study, gel mobility shift analysis was used to examine the binding of nuclear proteins to cis-regulatory elements involved in the transcription of the FGF-4 gene. We demonstrate that the transcription factors Sp1 and Sp3 in nuclear extracts prepared from EC cells bind to three Sp1 motifs, one located in the downstream enhancer, and two located in the 5' flanking region of the gene. We also show that Sp1 and Sp3 bind to each of the Sp1 motifs when nuclear extracts prepared from EC-derived differentiated cells are used. In contrast, differentiation of EC cells and ES cells drastically reduces the ability of nuclear factors to bind to an octamer motif and an adjacent High Mobility Group (HMG) motif, which have been shown previously to play essential roles in the functioning of the FGF-4 enhancer. Together, these findings provide a mechanistic explanation of how the distant FGF-4 enhancer promotes transcription of this gene in EC cells and ES cells, and how differentiation of these cells represses transcription of the FGF-4 gene.

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.

In vitro DNAase I footprinting and gel mobility shift assays have shown that the activities of several nuclear factors (GATA-1, Sp1) that bind to the promoter and downstream enhancer regions of the chicken histone H5 gene are reduced in mature erythrocytes relative to those in immature erythrocytes. In this study we investigated site occupancy in the promoter and downstream enhancer regions of the H5 gene in mature and immature erythrocytes. The ligation-mediated polymerase chain reaction was used to detect DNAase I footprints generated in situ. Most of the sites that bound to Sp1 and/or Sp1-like proteins and GATA-1 in the promoter and enhancer were occupied in situ in mature and immature erythrocytes. However, the level of protection at Sp1/Sp-1-like binding sites in the H5 enhancer region of mature erythroid cells was generally less than that observed for immature cells, suggesting that for any given mature cell not all of the Sp1/Sp1-like binding sites are occupied. Nevertheless, the results of this study suggest that the enhancer and promoter of the H5 gene in mature erythrocytes should be functional, agreeing with nuclear run-on studies showing transcriptional activity of the H5 gene in mature permeabilized cells.

Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ∼60% of Leri-Weill dyschondrosteosis (LWD) and ∼5–15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ∼286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS.

Mouse cytokeratin EndoA is an intermediate filament subunit of the type II cytokeratin class which initiates expression in trophectoderm cells of blastocyst during embryogenesis. To identify the regulatory elements of the endo A gene, we constructed a series of CAT expression vectors and transfected them into PYS-2 cells. We found an enhancer element locating 1 kb downstream from the endo A gene which acts on both the endo A and SV40 promoters. This enhancer consists of six direct repeated sequences with homology to the PEA3 motif in polyoma virus alpha enhancer core. In undifferentiated F9 embryonal carcinoma cells, expression of the construct containing the enhancer was not detected. These results indicate that one of the regulatory mechanisms of endo A gene expression is the 3' downstream enhancer.

Mutations in components of the Wnt signaling pathway initiate colorectal carcinogenesis by deregulating the beta-catenin transcriptional coactivator. beta-Catenin activation of one target in particular, the c-Myc proto-oncogene, is required for colon cancer pathogenesis. beta-Catenin is known to regulate c-Myc expression via sequences upstream of the transcription start site. Here, we report that a more robust beta-catenin binding region localizes 1.4 kb downstream from the c-Myc transcriptional stop site. This site was discovered using a genome-wide method for identifying transcription factor binding sites termed serial analysis of chromatin occupancy. Chromatin immunoprecipitation-scanning assays demonstrate that the 5' enhancer and the 3' binding element are the only beta-catenin and TCF4 binding regions across the c-Myc locus. When placed downstream of a simian virus 40-driven promoter-luciferase construct, the 3' element activated luciferase transcription when introduced into HCT116 cells. c-Myc transcription is negligible in quiescent HCT116 cells but is induced when cells reenter the cell cycle after the addition of mitogens. Using these cells, we found that beta-catenin and TCF4 occupancy at the 3' enhancer precede occupancy at the 5' enhancer. Association of c-Jun, beta-catenin, and TCF4 specifically with the downstream enhancer underlies mitogen stimulation of c-Myc transcription. Our findings indicate that a downstream enhancer element provides the principal regulation of c-Myc expression.

To the Editor: In the October 2005 issue of the Journal, Benito-Sanz et al. (2005) reported an association of Leri-Weill dyschondrosteosis (LWD [MIM 127300]) with a novel class of heterozygous pseudoautosomal region 1 (PAR1) deletions downstream of SHOX (short-stature homeobox-containing gene [MIM 312865]) in 12 patients with two copies of intact SHOX coding sequences. The deletions were variable in size, with the smallest region of overlapping deletion (SRO) of ∼30 kb between DXYS10086 and rs7067102. The results—in conjunction with the report of Flanagan et al. (2002) describing a monoallelic SHOX expression in the bone marrow fibroblasts taken from the distal radius of a patient with LWD with two copies of normal SHOX coding exons and hemizygosity for a region around DXYS233 downstream of SHOX—suggest the presence of a downstream enhancer for SHOX transcription around the ∼30-kb SRO. Consistent with this, Fukami et al. (2005) found that (1) a 240–350-kb deletion including DXYS233 is present in a heterozygous status in a mother with LWD and two copies of intact SHOX coding exons and (2) the same deletion is present in a hemizygous status in her daughter with Langer mesomelic dysplasia and a mosaic-ring X chromosome missing the PAR1. Here, we report that the putative SHOX enhancer may reside on an ∼800-bp evolutionarily conserved sequence (ECS). First, we analyzed the SHOX 3′ region in five Japanese families in which the proband and one of the parents had variable degrees of LWD and stature from short to low normal in the presence of two copies of intact SHOX-coding exons. The study was approved by the Institutional Review Board Committee at the National Center for Child Health and Development. Genotyping analysis was performed with primers and methods shown in table 1; results indicate that a deletion between SHOX-SNP792 on the 3′ UTR and DXYS85 was shared by the LWD-affected mother and daughter in family A and that a deletion between rs5946324 and rs4504827 was common to the LWD-affected father and daughter in family B (table 2 and fig. 1A). Furthermore, FISH analysis was performed with an RP13-167H21 BAC probe defining a region from rs5946324 to DXYS233 (Ensembl Genome Browser); results show only a single signal in the mother and the daughter of family A and an obviously different signal intensity in the father and the daughter of family B (fig. 2). The results, together with our previous data (Fukami et al. 2005), imply that an ∼40-kb region between rs5946326 and rs4504827 is the SRO in the Japanese patients (SRO-J) (fig. 1A). The SRO-J is in a close agreement with the ∼30-kb SRO in the white patients (SRO-W) (Benito-Sanz et al. 2005), and a region between rs5946326 and rs7067102 is shared by all the patients with SHOX 3′ deletions (fig. 1A). Figure 1 A, PAR1 deletions in the SHOX downstream region. Top, Pedigrees of families A and B. LWD is exhibited by the mother and the daughter in family A and by the father and the daughter in family B. The height of each subject is expressed as an SD score. Bottom, ... Figure 2 Results of the FISH analysis. Red signals indicate the RP13-167H21 region (thick arrows), and green signals represent DXZ1 (arrowheads) or DYZ3 (thin arrow). The RP13-167H21 region is deleted from one of the two X chromosomes of the mother and the daughter ... Table 1 Primer Sequences and PCR Conditions Used for Genotyping[Note] Table 2 Summary of Polymorphism Analyses[Note] Next, we searched the UCSC Genome Browser for the ECSs within the SROs. Seven ECSs (ECS1–ECS7) were present within the SRO-J, whereas ECS6 and ECS7 were found to reside outside the SRO-W (fig. 1B). ECS3–ECS7 were well conserved in chicken and dog, which preserve Shox, and were absent in mouse and rat, which lack Shox (Clement-Jones et al. 2000; Ensembl Genome Browser). ECS4 was also conserved in Fugu and zebrafish, which preserve Shox. By contrast, ECS1 was absent in chicken, and ECS2 was conserved in mouse and rat. Whereas ECS3 and ECS4 were not described in chimpanzee, the sequence analysis remains poor for the Shox 3′ region in chimpanzee, in contrast to the detailed analysis of that region in chicken and dog. These findings suggest that ECS3–ECS5 can be regarded as candidate regions harboring the putative downstream enhancer. In this regard, since ECS3–ECS5 reside between rs5988437 and rs5946533 (UCSC Genome Browser), they should be deleted from the patients in the three families defining the SRO-J (fig. 1A). Thus, we examined the transcription activity of ECS3–ECS5 as well as ECS6 with the dual-luciferase reporter assay system (Promega). Luciferase reporter constructs containing each ECS (ECS3, 861 bp; ECS4, 824 bp; ECS5, 441 bp; ECS6, 634 bp) inserted into the 3′ region of the luciferase gene were created using the pGL3 vector with the SV40 promoter or the human SHOX promoter on exon 2 (−432 to +5 bp) (Blaschke et al. 2003) (fig. 1B). The U2OS osteosarcoma cell line expressing SHOX (Rao et al. 2001) was transfected using lipofectamine (Invitrogen) with each reporter vector together with the pRL-CMV vector used as an internal control for the transfection, and luciferase assays were performed 36 h later. After the experiments were performed five times, the normalized luciferase activity was found to be significantly increased only when the reporter vector with ECS4 and the SHOX promoter was transfected to U2OS cells (empty vs. ECS4; P=.011 by t test) (fig. 1B). This implies that the putative SHOX enhancer resides in ECS4 and interacts with the SHOX promoter on exon 2. Finally, we searched ECS4 for potential binding sites for transcription factors relevant to bone development, using the MATINSPECTOR, TESS, and TFSEARCH programs. The putative binding sites with the maximum core similarity of 1.0 and a matrix similarity >0.75 were identified for HOXA9, HOXB9, PBX1, and MEIS1, as well as for PBX1-HOXA9 and MEIS1-HOXA9 heterodimers, which are known to be involved in limb development (Mercader et al. 1999; Shanmugam et al. 1999; Zakany and Duboule 1999) (fig. 3). Thus, the sequence-specific DNA binding of such a factor(s) might mediate the enhancer activity for SHOX. However, other potential binding sites were also detected for various transcription factors, and the relevance to skeletal development has not been studied or excluded in most of the transcription factors. In addition, the binding sites remain unidentified for many transcription factors. Thus, further studies are necessary to define the enhancer sequence. Figure 3 Nucleotide sequence of ECS4 and putative binding sites for several transcription factors relevant to skeletal development. The human sequence is aligned with the chicken sequence. In summary, the results suggest that the ∼800-bp ECS4 harbors the putative downstream enhancer for SHOX transcription. This information will provide a useful clue for the clarification of the molecular network involved in SHOX-dependent skeletal development.

Acute myeloid leukemias are a highly heterogeneous group of diseases associated with corruption of myeloid cell differentiation, accumulation of immature blasts, and emergence of a transformed leukemia initiating cell (LIC) capable of sustaining leukemic expansion. Genetic and phenotypic properties of LICs are highly variable among AML patients as a consequence of variable cytogenetics, stem/progenitor cell of origin and stage of disease progression. The orphan nuclear receptors NR4A1 and NR4A3 are potent AML tumor suppressors whose deletion in mice leads to rapid AML development. They are also silenced in cytogenetically diverse human LICs, and their anti-leukemic effect is associated with acute transcriptional repression of c-MYC and reprogramming of a core MYC oncogenic gene signature that is common to LICs of distinct cytogenetic backgrounds. c-MYC transcription is controlled at the step of transcriptional elongation, and high levels of c-MYC expression are maintained in many cancers, including AML, by establishment of cancer cell-selective super enhancers that are marked by high levels of the BET bromodomain protein, BRD4, a key regulator of c-MYC transcription elongation together with BRD4 associated components of the Mediator coactivator complex. BRD4 promotes transcription elongation of c-MYC by recruiting the positive transcription elongation factor, P-TEFb, a catalytic component of the super elongation complex (SEC) which phosphorylates RNA PolII at serine 2 to enable release of promoter paused RNA PolII. Here we investigated the transcriptional mechanisms of NR4A1-dependent c-MYC repression. Interrogation of genome-wide NR4A1 binding in Kasumi-1 AML-ETO positive human AML cells revealed NR4A1 occupancy within the 5’ proximal regions of the c-MYC gene and at a downstream enhancer region previously identified as a c-MYC super enhancer in the RN2 mouse model of MLL-AF9/NrasG12D driven AML. Consistent with its classification as a super enhancer, we found strong enrichment of BRD4 and the MED1 component of the Mediator complex at the c-MYC enhancer in Kasumi-1 AML cells in the absence of NR4A1. Interrogation of the effects of acute rescue of NR4A1 expression on the enhancer status of this region and transcriptional elongation of c-MYC revealed that NR4A1 represses c-MYC transcription elongation by interacting directly with the enhancer locus to inhibit recruitment of Mediator complex and BRD4, leading to loss of P-TEFb recruitment to both the enhancer and c-MYC gene and a loss of elongation competent RNA PolII. Together, these data indicate the NR4A1 negatively regulates transcriptional elongation at the c-MYC locus by interfering with factors required for super enhancer maintenance and AML selective overexpression of c-MYC. Since AML cells which rely on c-MYC overexpression are extremely sensitive to BRD4 inhibition, our results predict that strategies directed toward NR4A activation in AML cells may provide an alternative means of targeting c-MYC driven AMLs. Citation Format: Ryan P. Duren, Orla M. Conneely. Transcriptional mechanisms of NR4A orphan nuclear receptor tumor suppression in AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-83. doi:10.1158/1538-7445.AM2014-LB-83

We have isolated and sequenced the pseudo-allelic versions of FoxD5 genes in Xenopus laevis, xlFoxD5a and xlFoxD5b, and the xtFoxD5 gene of Xenopus tropicalis. These genes show a highly conserved structure, they are composed of only one exon, and they exhibit a high degree of sequence conservation within their flanking sequences. The X. tropicalis gene is expressed like its X. laevis orthologues in progenitor cells of the neural floor plate. Serial deletions of the 5'- and 3'-flanking region in combination with reporter gene assays demonstrate that a distal and a proximal upstream element as well as a downstream-located enhancer do mainly contribute to transcriptional activity. The downstream enhancer cooperates with the proximal upstream element and also contributes to the spatial expression. Transgenic animals express enhanced green fluorescent protein in a spatial pattern like xlFoxD5b, when the 5'- and 3'-flanking regions of the xlFoxD5b gene are used to direct transgene expression.

Erythropoietin (Epo) is the humoral regulator of red blood-cell production. Low oxygen tension increases the Epo levels by enhancing transcription, through the hypoxia-inducible factor (HIF)-1, a transcriptional modulator in oxygen-regulated gene expression. In the present work, a cooperative interaction between hypoxia, mediated by the HIF-1 complex, and transforming growth factor-β (TGF-β), mediated by Smad3/4, was revealed in the Epo gene. This cooperation is due to physical interaction between Smad3/4 and HIF-1α. The Smad3/4 binding site is located within the 3′ Epo enhancer, downstream from the HRE consensus, and immediately adjacent to the orphan hepatic nuclear factor receptor (HNF-4). HNF-4 is interacting also with Smad3 and the HIF-1 complex, to potentiate further the cooperative effect between both factors. Moreover, Sp1 has been identified as the factor binding the promoter necessary for the full hypoxia inducibility of the EPO gene. However, this full induction is achieved only if the TGF-β pathway is mediating a cross-talk between promoter (Sp1) and enhancer (HIF-1α) regions through Smad3. We show that Sp1 binding to the proximal promoter is relevant for Epo transcription, and contributes to the Epo induction by hypoxia. A functional cooperation among the transcription factors mediating hypoxia (HIF-1, Sp1), the TGF-β pathway (Smad3/4), and tissue-specific HNF-4 is proposed for the regulation of the Epo gene. In this model, the physical contact between the upstream promoter and the 3′ downstream enhancer is mediated by Sp1 and Smad3 factors, and would occur upon bending of the DNA intervening sequences. Thus, Sp1 would reinforce the promoter/enhancer contact, while Smad3 would stabilize the multifactorial complex by interacting with HIF-1/Sp1/HNF-4 and the coactivator CBP/p300. This model may be extended to other genes where collaboration between TGF-β and hypoxia takes place. © 2003 Elsevier Ltd. All rights reserved., This work was supported by grants from Ministerio de Ciencia y Tecnologı́a (SAF2000-0132), Fondo de Investigacion Sanitaria (PI020200), and Comunidad Autónoma de Madrid (CAM) to C.B.

The expression of skeletal muscle contractile proteins is tightly regulated during embryonic development. In the mouse, the myosin light chain (MLC) 1f/3f gene locus is not activated until E9.5, exclusively in skeletal muscle precursor cells. A potent enhancer downstream of the MLC1f/3f locus confers correct temporal and spatial activation of linked reporter gene in transgenic mouse embryos. To examine roles of the MLC downstream enhancer (MLCE) in its native context of the MLC1f/3f gene locus, we eliminated a 1.5-kb DNA segment containing the enhancer from the mouse genome by targeted deletion, leaving no exogenous sequences at the deletion site. Mouse embryos homozygous for the MLCE deletion were smaller and developmentally delayed, formed no mesoderm by E7.5, and were resorbed almost completely at E8.5. In situ hybridization and RT-PCR analyses of affected mutant embryos at E7.5 revealed ectopic MLC transcripts, whose products would be predicted to interfere with a variety of nonmuscle cell functions determining differentiation of mesoderm. These results suggest that the MLC downstream enhancer and its flanking sequences include negative regulatory elements which block precocious activation of MLC expression in mesodermal precursors during a critical window of development, as well as positive elements which subsequently permit tissue-restricted MLC transcription in differentiating skeletal muscles.

The regulation of chondrogenesis and of the genes expressed as markers of chondrocyte differentiation is poorly understood. The hyaluronan-binding proteoglycan aggrecan is an essential and specific component of cartilage, but the aggrecan proximal promoter is expressed in an unregulated fashion in vitro. DNA comprising the rat aggrecan gene (83 kb including the 30-kb first intron) was surveyed for active elements, which would impart selective expression to the aggrecan promoter in transfection assays in vitro. A 4.7-kb DNA fragment (P3) with cell-specific enhancer activity was discovered approximately 12 kb upstream of the transcription start site; this active DNA fragment is position- and orientation-independent, and strongly stimulates aggrecan promoter expression in chondrocytes, while weakly suppressing transcription in fibroblasts. Most of this activity has been localized to P3-7, a 2.3-kb internal fragment of P3. Another enhancer element (A23), which is not tissue-specific, was discovered about 70 kb downstream of the transcription start site. Several lines of transgenic mice were created using combinations of these DNA elements to drive the lacZ reporter gene. Neither a short (900 bp) nor a long (3.7 kb) promoter alone showed detectable expression in 14.5-day embryos, whereas placing the P3 tissue-specific enhancer together with P0 gave strong expression restricted to embryonic cartilage of transgenic mice. The A23 downstream enhancer in conjunction with P0 did not confer expression. This is the first report of a gene control region which confers authentic tissue-specific regulation of aggrecan in vitro or in vivo and should greatly facilitate understanding the coordinate regulation of chondrocytic genes.

A distal enhancer that specifies apolipoprotein E gene expression in the skin was identified and characterized by in situ hybridization in transgenic mice generated with constructs of the human apolipoprotein E/C-I/C-IV/C-II gene cluster. Transgene constructs containing the enhancer expressed high levels of apolipoprotein E mRNA in the germinative cell layer of the sebaceous gland and in epithelial cells of the hair follicle root sheath. Apolipoprotein E mRNA was also detected in basal epithelial cells of the epidermis. Expression of the human apolipoprotein E transgene at these sites was specified by a unique 1.0 kb enhancer domain located 1.7 kb downstream of the apolipoprotein E gene. No transgene expression was detected in skin epithelial cells in transgenic mice when this enhancer was deleted from the apolipoprotein E gene cluster. The enhancer was used to construct a transgene expression vector that faithfully directed a heterologous cDNA to the normal sites of apolipoprotein E gene expression in epithelial cells of the skin.

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, the tet activator (tTA) can still activate the promoter, even in the presence of sns, demonstrating that sns does not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that sns does not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within sns which are responsible for insulator activity, we have performed in vitro DNase I footprinting and EMSA analysis, and in vivo functional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that sns displays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by sns depends on the assembly of distinct DNA-protein complexes. (C) 2000 Academic Press.

Different mechanisms mediating methylation-dependent repression have been demonstrated. Two of these mechanisms play a role in the context of the granulocyte/macrophage-specific lysozyme gene: direct interference with DNA binding of the transcription factor GA-binding protein and deacetylation of histones. Besides enhancement in the unmethylated state, and transcriptional repression upon DNA methylation, the lysozyme downstream enhancer confers tissue-specific demethylation. Because both demethylation activity and repression ability have been attributed to the methyl-CpG-binding domain-containing protein MBD2, we analyzed this protein. The short form MBD2b binds to the methylated lysozyme enhancer and mediates transcriptional repression. MBD2b is capable of binding to the transcriptional repressor Sin3A. The interaction domain of Sin3A required for binding to MBD2b contains the paired amphipathic helix 3. We identified a minimal functional domain that confers both transcriptional repression as well as the interaction to Sin3A. In contrast to the functionally related proteins MeCP2 and MBD1, the repression domain of MBD2b overlaps with the methyl-CpG-binding domain.

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Em) are the targets of the negative regulator, NF-mNR, found in non-B and early pre-B cells. Expression library screening with NF-mNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-mNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-mNR binding sites within Em with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-mNR preparations and binds NF-mNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Em via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. We propose that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Em enhancer activity by interfering with the function of nuclear matrix-bound transcription activators. The immunoglobulin heavy chain (IgH) locus has been studied extensively as a model of tissue-specific gene expression. Transcription of IgH is controlled by promoters, located 59 of the individual variable (VH) gene segments, and downstream enhancer elements which cooperate to regulate tissue-restricted expression (reviewed in references 56, 66, and 84). The IgH intronic enhancer (Em), which is located in the intron between the joining (J) and constant (C) region gene segments

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.