Direct Repeat Elements Research Articles

Direct and Indirect Functions of TR4 in the Control of Fetal Hemoglobin Silencing

Elevated levels of fetal hemoglobin (HbF) compensate for reduced normal β-globin chains and ameliorate clinical symptoms for patients with beta-globinopathies, such as sickle cell disease (SCD). The embryonic and fetal globin genes, but not the adult β-globin gene, have direct repeat (DR) elements in their promoters, which have been implicated in γ-globin gene repression (Tanabe et al., 2002). We previously purified a DR element-binding protein complex, which we named DRED, and was the first identified HBG gene repressor (Tanabe et al., 2002). DRED activity is conferred by a complex including LSD1, recruited by the nuclear receptors TR2/TR4 (Cui et al., 2011). BCL11A is a well-characterized transcription factor that independently represses the γ-globin genes through binding to their promoters (Liu et al., 2018). We have shown by co-immunoprecipitation that TR4 interacts with BCL11A in HUDEP2 cells. To understand how TR4 acts to repress the γ-globin gene and the relationship of TR4 to BCL11A in γ-globin repression, we characterized the chromatin occupancy of TR4 and BCL11A within the β-globin locus by CUT&RUN (CNR) in HUDEP2 cells. We established HUDEP2 cells bearing TR4 tagged with 3FLAG+3Myc epitopes (FM-TR4) and in the same cells, BCL11A tagged with 3HA+3Myc (HM-BCL11A) epitopes by genome editing. In addition, another HUDEP2 cell line bearing BCL11A tagged with the 3HA+3Myc (HM-BCL11A) epitope as well a cell line bearing TR4 tagged with the 3FLAG+3Myc epitopes (FM-TR4) were generated. The tagged cell lines not only improved the efficiency of antibody targeting, but also facilitated direct comparison of TR4 vs BCL11A chromatin occupancy by binding of the common Myc epitope in FM-TR4 and HM-BCL11A cell lines. Unbiased motif discovery of TR4 CNR binding of these epitopes in all 3 cell lines yielded the motif of a direct repeat consisting of 2 AGGTCA repeat elements with a 1 nt spacer. The exact match of BCL11A motif with one of the direct repeat elements of TR4 motif suggested that BCL11A and TR4 may be compete for binding to the γ-globin promoter. Overall, TR4 and BCL11A showed a similar pattern of occupancy within the globin locus, although of lower intensity. TR4 presented strong interaction with the locus control regions, among which HS2 and HS3 were the highest, consistent with the model of long-distance interaction through chromosomal looping. However, TR4 was found to be significantly less occupied at the γ-globin promoters than BCL11A. To test whether TR4 and BCL11A can bind in vitro to the motifs found in the γ-globin promoters, we performed TR4 and BCL11A electrophoretic mobility shift assays (EMSAs). We showed that TR4 binds to the DR1 element within the γ-globin promoters, which contains the distal TGACCA motif that was bound by BCL11A. Notably, mutation of the TGACCA motif disrupted both BCL11A and TR4 binding, while the other direct motif is indispensable for TR4 binding, supporting overlapping binding of BCL11A and TR4. Interestingly, DNA-TR4 protein complexes were competitively depleted by high concentrations of BCL11A and vice versa, suggesting competitive binding between the two factors. Studies of this mechanism in the erythroid cells are underway. In addition, unbiased motif discovery of TR4 CNR also enriched for KLF1, LRF and NF-Y motifs. Co-IP assays demonstrated interaction between TR4 and KLF1 and LRF, suggesting the presence of a large repressor complex. We also explore genome-wide occupancy of TR4 and discovered strong TR4 interactions at promoters of multiple HbF repressing transcription factors and corepressors, including NFIA and EHMT1, suggesting that the role of TR4 in HbF repression may be partially mediated by regulating expression of various HbF repressor genes. In summary, these data suggest that the function of TR4 in HbF silencing is mediated in part by direct repression of HBG promoters in a competitive manner with BCL11A, and in part by regulating the expression of multiple HbF repressor genes.

Characterization and Vector Competence Studies of Chikungunya Virus Lacking Repetitive Motifs in the 3' Untranslated Region of the Genome.

Using reverse genetics, we analyzed a chikungunya virus (CHIKV) isolate of the Indian Ocean lineage lacking direct repeat (DR) elements in the 3′ untranslated region, namely DR1a and DR2a. While this deletion mutant CHIKV-∆DR exhibited growth characteristics comparable to the wild-type virus in Baby Hamster Kidney cells, replication of the mutant was reduced in Aedes albopictus C6/36 and Ae. aegypti Aag2 cells. Using oral and intrathoracic infection of mosquitoes, viral infectivity, dissemination, and transmission of CHIKV-∆DR could be shown for the well-known CHIKV vectors Ae. aegypti and Ae. albopictus. Oral infection of Ae. vexans and Culex pipiens mosquitoes with mutant or wild-type CHIKV showed very limited infectivity. Dissemination, transmission, and transmission efficiencies as determined via viral RNA in the saliva were slightly higher in Ae. vexans for the wild-type virus than for CHIKV-∆DR. However, both Ae. vexans and Cx. pipiens allowed efficient viral replication after intrathoracic injection confirming that the midgut barrier is an important determinant for the compromised infectivity after oral infection. Transmission efficiencies were neither significantly different between Ae. vexans and Cx. pipiens nor between wild-type and CHIKV-∆DR. With a combined transmission efficiency of 6%, both Ae. vexans and Cx. pipiens might serve as potential vectors in temperate regions.

ZNF802 (JAZF1), a Possible New Therapeutic Target for Treatment of β-Thalassemia

β-Thalassemias are groups of inherited blood disorders caused by reduced or absent production of β-globin chains of adult hemoglobin (HbA; α2β2). The severely affected patients with β-thalassemia require lifelong blood transfusion and chelation therapy. Reactivation of fetal hemoglobin (HbF; α2γ2) could ameliorate the symptoms of patients via increased total hemoglobin production and reduced imbalanced α- to non-α-globin synthesis. Hemoglobin switching during human development requires activities of several transcription factor proteins, including those bind to the globin gene promoters. However, the regulation of fetal to adult hemoglobin switching is not fully understood. Previously, we have carried out the microarray analysis comparing human erythroblasts derived from therapeutic aborted fetal liver and adult peripheral blood. The results showed that ZNF802 mRNA is among the transcripts that up-regulated in adult erythroblast of greater than 2-fold change. ZNF802, also called JAZF1, is a 27 kDa protein containing two zinc finger motifs that acting as transcriptional repressor by binding to the direct repeat (DR) elements on DNA. In this study, quantitative RT-PCR was performed in order to validate the microarray data. We observed 13-fold up-regulation of ZNF802 in adult erythroid cells compared to those cells derived from fetal liver. To further elucidate the role of ZNF802 in reduction of HbF expression in adult erythroids, the lentivirus carrying ZNF802 specific shRNA and non-targeted shRNA were designed and constructed for transduction in erythroblasts. Hematopoietic CD34+ progenitor cells were isolated from β-thalassemia/HbE patients (n=9) and subjected to ex vivo two-phase liquid erythroid culture. The results showed the reduction of greater than 90% of ZNF802 transcript and nearly undetectable level of ZNF802 protein in cells transduced with the ZNF802 shRNA lentivirus. As expected, the level of γ-globin transcript and levels of HbF were increased in ZNF802 knockdown erythroid cells in β-thalassemia/HbE (5-fold changed and HbF 26.9±7.4% vs 18.5±6.8% in non-targeted shRNA (p-value = 0.04), respectively). Interestingly, we found up-regulation of the embryonic ε-globin and ζ-globin transcripts in knockdown ZNF802 cells compared to non-targeted shRNA control (15-fold and 10-fold changed (p-value = 0.01), respectively). To note, the diminishing of ZNF802 effect on the proliferation and differentiation pattern of erythriod cells was not found. Altogether, our findings suggested that ZNF802 may play a role as the transcriptional repressor of γ-globin gene in human adult erythroid cells, thus served as a possible new therapeutic target for treatment of β-thalassemia. The molecular mechanisms underlying the ZNF802 mediated HbF reduction are underway. Disclosures No relevant conflicts of interest to declare.

A putative direct repeat element plays a dual role in the induction and repression of insect vitellogenin-1 gene expression.

Juvenile hormones (JH) regulate wide-ranging physiological and developmental processes in insects. However, molecular mechanisms underlying JH signaling remain to be determined. Vitellogenin (Vg) is primarily an egg-yolk protein, but recently proposed to serve many functions in insects. In the female American cockroach (Periplaneta americana), vitellogenin (Vg) genes are activated by JH III and suppressed by 20-hydroxyecdysone (20E) via cis-regulatory elements in a dose-dependent manner. In the present study, the upstream promoter region (935 bp) of Vg1 was cloned to elucidate the action of these hormones. A luciferase reporter assay identified an 81 bp region in the promoter region of Vg1 (-120 to -39 bp) that we found to be critical for JH III activation and 20E suppression. This 81 bp region contains a direct repeat separated by a 2-nucleotide spacer-designated Vg1HRE- that is similar to the Drosophila ecdysone response element direct repeat 4. Moreover, nuclear proteins isolated from nymphs, males, females, and Sf9 cells successfully bound to Vg1HRE, while binding was outcompeted by a 100-fold excess of cold probe or dephosphorylated nuclear protein extracts. In addition, binding was outcompeted by other ecdysone and JH response elements with similar half-site sequences (direct repeats) but to varying extents. Ultimately, we postulate that JH III indirectly activates Vg expression by interfering with or inhibiting the phosphorylation of nuclear proteins bound to Vg1HRE. Involvement of JH III in both induction of Vg1 and control of nuclear proteins binding to Vg1HRE suggest the latter to play an important role in JH signaling.

The bacterial response regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally.

Despite the importance of maintaining redox homeostasis for cellular viability, how cells control redox balance globally is poorly understood. Here we provide new mechanistic insight into how the balance between reduced and oxidized electron carriers is regulated at the level of gene expression by mapping the regulon of the response regulator ArcA from Escherichia coli, which responds to the quinone/quinol redox couple via its membrane-bound sensor kinase, ArcB. Our genome-wide analysis reveals that ArcA reprograms metabolism under anaerobic conditions such that carbon oxidation pathways that recycle redox carriers via respiration are transcriptionally repressed by ArcA. We propose that this strategy favors use of catabolic pathways that recycle redox carriers via fermentation akin to lactate production in mammalian cells. Unexpectedly, bioinformatic analysis of the sequences bound by ArcA in ChIP-seq revealed that most ArcA binding sites contain additional direct repeat elements beyond the two required for binding an ArcA dimer. DNase I footprinting assays suggest that non-canonical arrangements of cis-regulatory modules dictate both the length and concentration-sensitive occupancy of DNA sites. We propose that this plasticity in ArcA binding site architecture provides both an efficient means of encoding binding sites for ArcA, σ70-RNAP and perhaps other transcription factors within the same narrow sequence space and an effective mechanism for global control of carbon metabolism to maintain redox homeostasis.

Globozoospermia is mainly due to DPY19L2 deletion via non-allelic homologous recombination involving two recombination hotspots

To date, mutations in two genes, SPATA16 and DPY19L2, have been identified as responsible for a severe teratozoospermia, namely globozoospermia. The two initial descriptions of the DPY19L2 deletion lead to a very different rate of occurrence of this mutation among globospermic patients. In order to better estimate the contribution of DPY19L2 in globozoospermia, we screened a larger cohort including 64 globozoospermic patients. Twenty of the new patients were homozygous for the DPY19L2 deletion, and 7 were compound heterozygous for both this deletion and a point mutation. We also identified four additional mutated patients. The final mutation load in our cohort is 66.7% (36 out of 54). Out of 36 mutated patients, 69.4% are homozygous deleted, 19.4% heterozygous composite and 11.1% showed a homozygous point mutation. The mechanism underlying the deletion is a non-allelic homologous recombination (NAHR) between the flanking low-copy repeats. Here, we characterized a total of nine breakpoints for the DPY19L2 NAHR-driven deletion that clustered in two recombination hotspots, both containing direct repeat elements (AluSq2 in hotspot 1, THE1B in hotspot 2). Globozoospermia can be considered as a new genomic disorder. This study confirms that DPY19L2 is the major gene responsible for globozoospermia and enlarges the spectrum of possible mutations in the gene. This is a major finding and should contribute to the development of an efficient molecular diagnosis strategy for globozoospermia.

Nuclear Receptors TR2 and TR4 Recruit Multiple Epigenetic Transcriptional Corepressors That Associate Specifically with the Embryonic β-Type Globin Promoters in Differentiated Adult Erythroid Cells

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal β-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic β-type globin promoters but not to the adult β-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1β, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult β-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult β-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic β-type globin genes through coordinated epigenetic chromatin modifications.

Common architecture of nuclear receptor heterodimers on DNA direct repeat elements with different spacings

Nuclear hormone receptors (NHRs) control numerous physiological processes through the regulation of gene expression. The present study provides a structural basis for understanding the role of DNA in the spatial organization of NHR heterodimers in complexes with coactivators such as Med1 and SRC-1. We have used SAXS, SANS and FRET to determine the solution structures of three heterodimer NHR complexes (RXR-RAR, PPAR-RXR and RXR-VDR) coupled with the NHR interacting domains of coactivators bound to their cognate direct repeat elements. The structures show an extended asymmetric shape and point to the important role played by the hinge domains in establishing and maintaining the integrity of the structures. The results reveal two additional features: the conserved position of the ligand-binding domains at the 5' ends of the target DNAs and the binding of only one coactivator molecule per heterodimer, to RXR's partner.

A novel mutation of −50 (G→A) in the direct repeat element of the β-globin gene identified in a patient with severe β-thalassemia

Dear Editor, β-Thalassemia is a heterogeneous genetic disease associated with defective expression of the β-chain of human hemoglobin. So far, more than 200 mutations have been defined and affect almost every known stage of β-globin gene expression resulting in a reduction (β) or complete absence (β) of β-chain synthesis from the affected allele [1, 2]. In southern China, the carrier rate of β-thalassemia in Guangdong province was 2.54% [3], with five common mutations accounting for 90% of all β-thalassemia individuals. Identification of new mutations and update of the mutation spectrum of thalassemia in one specific ethnic population is always needed for improving genetic counseling and prenatal diagnosis. We here describe a novel promoter mutation of −50 (G→A) within the conserved direct repeat element (DRE) leading to β-thalassemia major when associated with a β-thalassemia mutation. A 3-year-old Chinese boy with his parents was seen at our genetics clinic. The boy suffered from β-thalassemia major and had been transfused every 2 months since the age of 6 months. Molecular analyses performed by reverse dot blots (RDB) at other clinics revealed that he was a heterozygote for the common mutation of codons (CDs) 41–42 (−TTCT) of the β-globin gene, but the other mutational allele was not determined. At this referral, the family was planning for another pregnancy and counseled about prenatal diagnosis. Blood samples were obtained from all three family members. Hematological investigations showed that both of the parents had a classical β-thalassemia trait and the son manifested with severe β-thalassemia (Table 1). Determination of the β-thalassemia mutations was performed using the polymerase chain reaction (PCR)–RDB method which can simultaneously detect 17 types of β-globin gene mutations found in Chinese population. This confirmed the heterozygous presence of the CDs 41–42 (−TTCT) mutation in the son and the father, but the mutation was not found in the mother. Gap PCR was also undertaken in the three family members to exclude the coinheritance of αthalassemia, and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−α, −α, –). To further investigate the underlying mutation in this family, the β-globin gene was amplified by PCR from DNA of the mother, and the product was analyzed by direct nucleotide sequencing using the BigDyeTM Terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer (Applied BioSystems). Direct sequencing of the β-globin gene showed the normal sequence except for the heterozygous G→A mutation at position −50 relative to the Cap site (Fig. 1). This so far undescribed mutation was also revealed in the son by DNA sequencing. Thus the patient was confirmed to be a compound heterozygote with a genotype of −50 (G→A)/CDs 41–42 (−TTCT). In order to evaluate the functional effect of this novel mutation, two other family members were investigated. A semiquantitative reverse transcription PCR assay was used to measure β globin mRNA in the reticulocytes from three heterozygous subjects (the mother, grandmother, and an aunt) and eight normal individuals. These analyses showed a mildly reduced β-globin mRNA level (23.4 %) in heterozygotes compared with that in normal subjects. Ann Hematol (2009) 88:1149–1150 DOI 10.1007/s00277-009-0732-8

Distinct modulatory roles for thyroid hormone receptors TR α and TR β in SREBP1-activated ABCD2 expression

Adrenoleukodystrophy-related protein, a peroxisomal ABC transporter encoded by ABCD2, displays functional redundancy with the disease-associated X-linked adrenoleukodystrophy protein, making pharmacological induction of ABCD2 a potentially attractive therapeutic approach. Sterol regulatory element (SRE)-binding proteins (SREBPs) induce ABCD2 through an SRE overlapping with a direct repeat (DR-4) element. Here we show that thyroid hormone ( T 3) receptor (TR) α and TR β bind this motif thereby modulating SREBP1-dependent activation of ABCD2. Unliganded TR β, but not TR α, represses ABCD2 induction independently of DNA binding. However, activation by TR α and derepression of TR β are T 3-dependent and require intact SRE/DR-4 motifs. Electrophoretic mobility shift assays with nuclear extracts support a direct interaction of TR and SREBP1 at the SRE/DR-4. In the liver, Abcd2 expression is high in young mice (with high T 3 and TR α levels) but downregulated in adults (with low T 3 and TR α but elevated TR β levels). This temporal repression of Abcd2 is blunted in TR β-deficient mice, and the response to manipulated T 3 states is abrogated in TR α-deficient mice. These findings show that TR α and TR β differentially modulate SREBP1-activated ABCD2 expression at overlapping SRE/DR-4 elements, suggesting a novel mode of cross-talk between TR and SREBP in gene regulation.

Regulation of Vascular Endothelial Growth Factor D by Orphan Receptors Hepatocyte Nuclear Factor-4α and Chicken Ovalbumin Upstream Promoter Transcription Factors 1 and 2

Vascular endothelial growth factor D has recently been linked to the control of lymphangiogenesis and lymphatic metastasis. The molecular determinants regulating vegf-D gene transcription, however, have not yet been identified. After isolation of 2 kb of 5'-flanking DNA of the human vegf-D gene, we identified a novel, atypical direct repeat (DR) element consisting of a consensus half-site (AGGTCA) at -125/-119 and a degenerated DR half-site (ATGTTA) at -99/-94 as sufficient and necessary for vegf-D transcription. The vegf-D DR element is bound and activated by the orphan receptors hepatocyte nuclear factor 4 alpha (HNF-4 alpha) and chicken ovalbumin upstream promoter transcription factor (COUP-TF)-1/COUP-TF2. Additionally, chromatin immunoprecipitation assays identified transcriptional coactivators cyclic AMP-responsive element binding protein-binding protein and glucocorticoid receptor interacting protein 1 at the vegf-D DR element and functional assays confirmed their stimulatory effect on the vegf-D promoter. Histone deacetylase inhibition by trichostatin A led to accumulation of acetylated histones H3/H4 at the vegf-D promoter, up-regulation of vegf-D mRNA levels, and transactivation of vegf-D promoter reporter gene constructs in cancer cell lines. This study for the first time describes the molecular determinants in cis and trans controlling vegf-D gene transcription and identifies interaction of HNF-4 alpha and COUP-TF1/COUP-TF2 with a proximal, atypical DR element as indispensable for vegf-D transcription. Moreover, our findings suggest that epigenetic control of histone acetylation represents an important determinant of vegf-D gene expression in cancer cells. These results provide novel insights into the molecular machinery controlling vegf-D gene expression and may add to a better understanding of the regulation of lymphangiogenesis in vascular development and cancer.

The TR2 and TR4 orphan nuclear receptors repress Gata1 transcription

When the orphan nuclear receptors TR2 and TR4, the DNA-binding subunits of the DRED repressor complex, are forcibly expressed in erythroid cells of transgenic mice, embryos exhibit a transient mid-gestational anemia as a consequence of a reduction in the number of primitive erythroid cells. GATA-1 mRNA is specifically diminished in the erythroid cells of these TR2/TR4 transgenic embryos as it is in human CD34(+) progenitor cells transfected with forcibly expressed TR2/TR4. In contrast, in loss-of-function studies analyzing either Tr2- or Tr4-germline-null mutant mice or human CD34(+) progenitor cells transfected with force-expressed TR2 and TR4 short hairpin RNAs (shRNAs), GATA-1 mRNA is induced. An evolutionarily conserved direct repeat (DR) element, a canonical binding site for nuclear receptors, was identified in the GATA1 hematopoietic enhancer (G1HE), and TR2/TR4 binds to that site in vitro and in vivo. Mutation of that DR element led to elevated Gata1 promoter activity, and reduced promoter responsiveness to cotransfected TR2/TR4. Thus, TR2/TR4 directly represses Gata1/GATA1 transcription in murine and human erythroid progenitor cells through an evolutionarily conserved binding site within a well-characterized, tissue-specific Gata1 enhancer, thereby providing a mechanism by which Gata1 can be directly silenced during terminal erythroid maturation.

Embryonic and fetal β-globin gene repression by the orphan nuclear receptors, TR2 and TR4

The TR2 and TR4 orphan nuclear receptors comprise the DNA-binding core of direct repeat erythroid definitive, a protein complex that binds to direct repeat elements in the embryonic and fetal beta-type globin gene promoters. Silencing of both the embryonic and fetal beta-type globin genes is delayed in definitive erythroid cells of Tr2 and Tr4 null mutant mice, whereas in transgenic mice that express dominant-negative TR4 (dnTR4), human embryonic epsilon-globin is activated in primitive and definitive erythroid cells. In contrast, human fetal gamma-globin is activated by dnTR4 only in definitive, but not in primitive, erythroid cells, implicating TR2/TR4 as a stage-selective repressor. Forced expression of wild-type TR2 and TR4 leads to precocious repression of epsilon-globin, but in contrast to induction of gamma-globin in definitive erythroid cells. These temporally specific, gene-selective alterations in epsilon- and gamma-globin gene expression by gain and loss of TR2/TR4 function provide the first genetic evidence for a role for these nuclear receptors in sequential, gene-autonomous silencing of the epsilon- and gamma-globin genes during development, and suggest that their differential utilization controls stage-specific repression of the human epsilon- and gamma-globin genes.

Tap and Dbp5, but not Gag, are involved in DR-mediated nuclear export of unspliced Rous sarcoma virus RNA

All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.

Transcriptional regulation of the human hepatic lipase (LIPC) gene promoter

Hepatic lipase (HL) plays a key role in the metabolism of plasma lipoproteins, and its level of activity requires tight regulation, given the association of both low and high levels with atherosclerosis and coronary artery disease. However, little is known about the factors responsible for HL expression. Here, we report that the human hepatic lipase gene (LIPC) promoter is regulated by hepatocyte nuclear factor 4alpha (HNF4alpha), peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), apolipoprotein A-I regulatory protein-1 (ARP-1), and hepatocyte nuclear factor 1alpha (HNF1alpha). Reporter analysis showed that HNF4alpha directly regulates the LIPC promoter via two newly identified direct repeat elements, DR1 and DR4. PGC-1alpha is capable of stimulating the HNF4alpha-dependent transactivation of the LIPC promoter. ARP-1 displaces HNF4alpha from the DR1 site and blocks its ability to activate the LIPC promoter. Induction by HNF1alpha requires the HNF1 binding site and upon cotransfection with HNF4alpha leads to an additive effect. In addition, the in vivo relevance of HNF4alpha in LIPC expression is shown by the ability of the HNF4alpha antagonist Medica 16 to repress endogenous LIPC mRNA expression. Furthermore, disruption of Hnf4alpha in mice prevents the expression of HL mRNA in liver. The overall effect these transcription factors have on HL expression will ultimately depend on the interplay between these various factors and their relative intracellular concentrations.

Increased expression of transglutaminase-1 and PPARγ after vitamin E treatment in human keratinocytes

In skin, vitamin E acts as the predominant lipophilic antioxidant with a protective function against irradiation and oxidative stress. In addition to that, vitamin E can also modulate signal transduction and gene expression. To study whether the four natural tocopherol analogues (α-, β-, γ-, δ-tocopherol) can influence transcriptional activity by modulating the activity of nuclear receptors, a human keratinocytes cell line (NCTC 2544) was transfected with plasmids containing the luciferase reporter gene under control by direct repeat elements (DR1–DR4), representing binding sites for four different classes of nuclear receptors. In this model, the tocopherols positively modulated only the reporter construct containing a consensus element for peroxisome proliferator-activated receptors (PPARs). The induction was strongest with γ-tocopherol and was most likely the direct consequence of stimulation of PPARγ protein expression in keratinocytes. Vitamin E treatment also led to increased expression of a known PPARγ target gene involved in terminal keratinocytes differentiation, the transglutaminase-1.

Different Configurations of Specific Thyroid Hormone Response Elements Mediate Opposite Effects of Thyroid Hormone and GC-1 on Gene Expression

T3 regulates transcription of the rat sarcoendoplasmic reticulum calcium ATPase in the heart. The T3 effect is mediated by three differently configured T3 response elements (TREs). Here we report the mutation of each individual TRE in the promoter and the contribution of each TRE on gene expression. Mutation of TRE1, a direct repeat element, exerted the strongest T3 response, compared with TRE2 and TRE3, which are inverted palindromes. The isolated TRE2 and TRE3, which showed no response (TRE2) or were weakly positive with T3 (TRE3), became strong negative regulatory elements with the T3 analog GC-1. We found that TRE1 recruits corepressor complexes containing nuclear receptor corepressor and histone deacetylase 3 in the absence of ligand, and steroid receptor coactivator-1-containing coactivator complexes with both T3 and GC-1. TRE3 bound the same corepressor complexes without ligand but showed only a weak association with steroid receptor coactivator-1 with T3 and a strong association with corepressor complexes with GC-1. Thus, GC-1 appears to control cofactor association differentially on these two sarcoendoplasmic reticulum calcium ATPase TREs, which could be the mechanism of ligand-dependent transcriptional activation and repression observed with the isolated TRE1 and TRE3 elements. Because the x-ray crystal structures of GC-1 and T3 complexed with the TR ligand binding domain are superimposable, the results imply that GC-1 and T3 induce differential effects on the receptor that are not evident in the static structures but must occur in the dynamic setting of receptor function. These results have implications for selective modulation of receptor function by agonist ligands.

Adult Stage γ-Globin Silencing Is Mediated by a Promoter Direct Repeat Element

The human beta-like globin genes (5'-epsilon-Ggamma-Agamma-delta-beta-3') are temporally expressed in sequential order from the 5' to 3' end of the locus, but the nonadult epsilon- and gamma-globin genes are autonomously silenced in adult erythroid cells. Two cis elements have been proposed to regulate definitive erythroid gamma-globin repression: the DR (direct repeat) and CCTTG elements. Since these two elements partially overlap, and since a well-characterized HPFH point mutation maps to an overlapping nucleotide, it is not clear if both or only one of the two participate in gamma-globin silencing. To evaluate the contribution of these hypothetical silencers to gamma-globin regulation, we generated point mutations that individually disrupted either the single DR or all four CCTTG elements. These two were separately incorporated into human beta-globin yeast artificial chromosomes, which were then used to generate gamma-globin mutant transgenic mice. While DR element mutation led to a dramatic increase in Agamma-globin expression only during definitive erythropoiesis, the CCTTG mutation did not affect adult stage transcription. These results demonstrate that the DR sequence element autonomously mediates definitive stage-specific gamma-globin gene silencing.

Transcriptional Regulation of the lymphangiogenic factor VEGF-D: critical role of orphan receptors and promoter acetylation

Aims: Vascular endothelial growth factor D (VEGF-D) is a ligand for VEGF receptor 3 (VEGFR–3/flt4) and has been implicated in lymphatic development and metastases. VEGF-D expression correlates with lymphatic tumor spread and poor disease outcome; our own studies identified VEGF-D as novel independent prognostic marker in gastric cancer. Despite this clinical importance, the molecular mechanisms controlling VEGF-D are largely unknown. Aim: Detailed characterization of the mechanisms controlling VEGF-D transcription as a basis for development of new intervention strategies influencing lymphangiogenesis and lymphatic metastases. Methods: After initial localization of the VEGF-D minimal promoter between –135/–85, regulatory promoter elements were identified by 3’/5’ deletions, site-directed mutagenesis and element transfer studies. Nuclear proteins and transcriptional co-factors controlling VEGF-D transcription were identified by EMSA and chromatin immunoprecipitation (ChIP) assays, and confirmed by ectopic expression of wild type and dominant-negative factor mutants. The role of acetylation for VEGF-D transcription was determined by application of HDAC inhibitor trichostatin (TSA). Results: A novel, atypical direct repeat (DR) element located within the –138/–85 region was identified as sufficient and necessary for VEGF-D gene transcription. This DR element comprises a 5’ consensus DR half-site (AGGTCA), a 25 bp spacer and a 3’ degenerated DR sequence, and is controlled through an interplay of the orphan receptors HNF–4α and COUP-TF1/2. Moreover, the transcriptional co-factors Grip–1 and CBP were identified to cooperate with HNF–4α and COUP-TF1/2 in regulation of VEGF-D transcription. Modification of acetylation resulted in potent and time-dependent upregulation of VEGF-D mRNA levels and promoter activity. Conclusions: Our studies for the first time describe the molecular mechanisms controlling VEGF-D gene transcription and show that the molecular machinery regulating the VEGF-D gene clearly differs from the mechanisms regulating other VEGF genes. Moreover, we demonstrate that promoter acetylation is an important determinant of VEGF-D transcription and thereby may influence genetic programs underlying lymphatic proliferation and metastasis

Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of H19 has no effect on imprinting in the Igf2-H19 region.

Igf2 and H19 are reciprocally imprinted genes on mouse distal chromosome 7. They share several regulatory elements, including a differentially methylated region (DMR) upstream of H19 that is paternally methylated throughout development. The cis-acting sequence requirements for targeting DNA methylation to the DMR remain unknown; however, it has been suggested that direct tandem repeats near DMRs could be involved. Previous studies of the imprinted Rasgrf1 locus demonstrate indeed that a direct repeat element adjacent to a DMR is responsible for establishing paternal allele-specific methylation at the DMR and therefore allelic expression of the Rasgrf1 transcript. We identified a prominent and conserved direct tandem repeat 1 kb upstream of the H19 DMR and proposed that it played a similar role in imprinted regulation of H19. To test our hypothesis, we generated mice harboring a 1.7-kb targeted deletion of the direct repeat element and analyzed fetal growth, allelic expression, and methylation within the Igf2-H19 region. Surprisingly the deletion had no effect on imprinting. These results together with deletions of other repeats close to imprinted genes suggest that direct repeats may not be important for the targeting of methylation at the majority of imprinted loci and that the Rasgrf1 locus may be an exception to this rule.