cis-DNA Elements Research Articles

New targets for therapy in prostate cancer: modulation of stromal–epithelial interactions

Changes in genomic and phenotypic expression of progressing prostate tumors and their stroma occur in a dynamic fashion based on bidirectional signaling from stromal–epithelial interactions. These interactions may underlie the ability of prostate cancer cells to survive and proliferate in the prostate and bone. By investigating the phenotypic and genotypic changes of stromal cells adjacent to cancer cells and the reciprocal changes of cancer cells, novel molecular markers may be developed to diagnose cancer earlier before pathologic appearance of cancer cells at the primary site. Attacking epithelial and stromal elements together is a unique approach to both localized and metastatic prostate cancer therapy. Co-targeting both tumor cells and stroma requires identifying a reliable tumor and tissue-specific cis-DNA element, such as osteocalcin (OC) promoter. OC expression is elevated in prostate tumor cells and in prostate and bone stromal cells interdigitating with both localized and metastatic prostate epithelium. We have previously designed an adenovirus-based therapeutic gene vehicle and demonstrated that a replication-competent adenoviral vector (Ad vector) is highly efficient in blocking the growth of cancer cells in culture, including cells without androgen receptor as well as cells that do or do not make prostate-specific antigen. In vivo, intravenous administration of an Ad-OC vector was effective against preexisting human prostate cancer subcutaneous and bone xenografts. The addition of vitamin D 3 enhanced further viral replication at target sites. Co-targeting tumor cells and stroma using systemic Ad vector is a viable and promising option for treatment of both localized and metastatic prostate cancer.

New members of the tomato ERF family show specific expression pattern and diverse DNA-binding capacity to the GCC box element

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit ( LeERF1–4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.

Gene transcription of fgl2 in endothelial cells is controlled by Ets-1 and Oct-1 and requires the presence of both Sp1 and Sp3.

The immune coagulant fgl2/fibroleukin has been previously shown to play a pivotal role in the pathogenesis of murine and human fulminant hepatitis and fetal loss syndrome. Constitutive expression of fgl2 transcripts at low levels are seen in cytotoxic T cells, endothelial, intestinal and trophoblast cells, while specific factors (such as virus and cytokines) are required to induce high levels of fgl2 expression in other cell types including monocytes/macrophages. To address the transcriptional mechanisms that regulate constitutive expression of fgl2, murine genomic clones were characterized and the transcription start site was defined by 5'-RACE and primer extension. A comprehensive assessment of basal fgl2 promoter activity in murine vascular endothelial cells defined a minimal 119 bp region responsible for constitutive fgl2 transcription. A complex positive regulatory domain (PRD) spanning a 39-bp sequence from -87 to -49 (relative to the transcription start site) was identified. Electrophoretic mobility shift assay studies in vascular endothelial cells revealed that the nucleoprotein complexes that form on this positive regulatory domain (PRD) contain Sp1/Sp3 family members, Oct-1, and Ets-1. Heterologous expression studies in Drosophila Schneider cells confirmed that the constitutive expression of this gene is controlled by Ets-1 and requires the presence both of the Sp1 and Sp3 transcription factors. The presence of this complex multicomponent PRD in the fgl2 proximal promoter is consistent with the observation that, in vivo, fgl2 expression is tightly regulated. Moreover, viral induced fgl2 expression also requires the presence of this PRD. These results clearly demonstrate that multiple cis DNA elements in a clustered region work cooperatively to regulate constitutive fgl2 expression and interact with inducible elements to regulate viral-induced fgl2 expression in endothelial cells.

The 5′-Flanking Region of the Murine Epididymal Protein of 17 Kilodaltons Gene Targets Transgene Expression in the Epididymis

A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.

USF-1 and USF-2 trans-repress IL-1beta-induced iNOS transcription in mesangial cells.

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682-1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region -904/-883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the -893/-888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the -893/-888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1beta (IL-1beta) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the -893/-888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1beta-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the -904/-883 region augmented IL-1beta stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at -893/-888 serves to trans-repress basal expression and IL-1beta induction of the iNOS promoter.

Opposing effects of Krüppel-like factor 4 (gut-enriched Krüppel-like factor) and Krüppel-like factor 5 (intestinal-enriched Krüppel-like factor) on the promoter of the Krüppel-like factor 4 gene.

KLF4 (Krüppel-like factor 4 or gut-enriched Krüppel-like factor, GKLF) and KLF5 (Krüppel-like factor 5 or intestinal-enriched Krüppel-like factor, IKLF) are two closely related members of the zinc finger-containing Krüppel-like factor family of transcription factors. Although both genes are expressed in the intestinal epithelium, their distributions are different: Klf4 is primarily expressed in the terminally differentiated villus cells while Klf5 is primarily in the proliferating crypt cells. Previous studies show that Klf4 is a negative regulator of cell proliferation and Klf5 is a positive regulator of cell proliferation. In this study, we demonstrate that Klf5 binds to a number of cis-DNA elements that have previously been shown to bind to Klf4. However, while Klf4 activates the promoter of its own gene, Klf5 suppresses the Klf4 promoter. Moreover, Klf5 abrogates the activating effect of Klf4 on the Klf4 promoter and Klf4 abrogates the inhibitory effect of Klf5 on the same promoter. An explanation of this competing effect is due to physical competition of the two proteins for binding to cognate DNA sequence. The complementary tissue localization of expression of Klf4 and Klf5 and the opposing effect of the two Klfs on the Klf4 promoter activity may provide a basis for the coordinated regulation of expression of the Klf4 gene in the intestinal epithelium.

A new adenoviral helper–dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo

We have developed a new helper-dependent (HD) adenoviral vector FTC that contains 3 cis-acting sequences as stuffer DNA: a human fragment of alphoid repeat DNA, matrix-attachment regions (MARs), and the hepatocyte control region enhancer. To determine the most robust human coagulation factor IX (hFIX) expression cassette in an adenovirus, we first tested different hFIX expression sequences with or without flanking MARs in first-generation adenoviral vectors. After intravenous infusion of the vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct comparison, a first-generation and a gene-deleted vector with identical hFIX expression cassettes were constructed. Both first-generation and HD adenovirus-treated animals demonstrated a threshold effect in a dose-response study. With the administration of 2 x 10(9) transducing particles of either vector, supraphysiological serum levels of hFIX were obtained, with the highest expression (41 000 ng/mL) occurring during the first 2 months after injection. The serum factor IX concentrations, while remaining in the therapeutic range, slowly declined by 95% over a period of 1 year. At this dose, interleukin-6 and tumor necrosis factor-alpha serum concentrations were elevated in animals that received the first-generation but not the HD vector. This study compares the properties of a gene-deleted and first-generation adenovirus with equivalent expression cassettes and suggests that the cis-DNA elements contained in the vector and expression cassette have important effects on gene expression in vivo.

Fibroblast growth factor receptor 4 is a target for the zinc-finger transcription factor Ikaros in the pituitary.

Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of endocrine cell hormonal and proliferative properties, and FGFR4 is differentially expressed in normal and neoplastic pituitary. We therefore examined the functionally important cis-DNA elements and multiprotein complexes implicated in the cooperative control of expression of the human FGFR4 gene in pituitary cells. Using deletional mapping, we defined a 214-bp (-115/+99) promoter that was functional in pituitary GH4 and PRL 235 cells. Overlapping 40- to 50-bp fragments of this minimal promoter were examined by EMSA. Interestingly, fragment C (-64/-26) included potential binding sites for the hematopoietic zinc finger-containing transcription factor Ikaros (Ik) flanked by binding sites for Sp and Ets-type factors. DNA binding by Ik, Sp, and Ets-like factors was confirmed by oligonucleotide competition and supershifting with specific antibodies. Transcriptional regulation of FGFR4 by Ik was demonstrated by cotransfection of Ik1 with or without Sp1 or Ets overexpression and by disruption of the Ik binding site. Although both Ets-1 and Sp1 overexpression stimulated promoter activity, mutation of the Ik-binding site completely eliminated the Ik1 effect. Specific Ik expression was identified by Western blotting of pituitary GH4 and PRL235 cells and localized in primary mouse hormone-producing anterior pituitary cells by immunocytochemistry. Our findings point to a new role for Ik outside the hematopoietic system and suggest a novel transcriptional contribution with Ets and Sp1 in regulation of FGFR4 in the pituitary.

Lung surfactant protein B promoter function is dependent on the helical phasing, orientation and combinatorial actions of cis-DNA elements

Surfactant protein B (SP-B), a hydrophobic protein of lung surfactant, is essential for surfactant function, normal respiration and survival. SP-B is expressed in a cell-type specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally induced. Our previous studies showed that the activity of the rabbit SP-B minimal promoter (−236/+39 bp) is dependent on the binding of an array of transcription factors including Sp1, Sp3, thyroid transcription factor 1, hepatocyte nuclear factor 3 and activating transcription factor/cyclic AMP response element binding protein. The SP-B minimal promoter sequence as well as the spacing and orientations of cis-DNA elements are conserved in human, rabbit and mouse SP-B genes. In the present study, we investigated the importance of spacing and orientation of cis-DNA elements on SP-B promoter function in NCI-H441 cells, a human cell line of Clara cell lineage. Further we investigated the effects of transcription factors on SP-B promoter expression by co-transfection experiments. Results showed that disruptions of helical phasing and orientation of cis-DNA elements reduced SP-B promoter activity indicating that proper alignment and orientation of cis-DNA elements are necessary for SP-B promoter function. Co-transfection experiments showed that transcription factors function in a combinatorial rather than in a synergistic manner to enhance SP-B promoter activity.

Modified HIV-1 based lentiviral vectors have an effect on viral transduction efficiency and gene expression in vitro and in vivo.

Gene transfer using lentiviral vectors has been recently shown to be enhanced with cis-acting elements in a cell-type-dependent manner in vivo. For this reason, the study reported here was designed to modify lentiviral vectors that express lacZ, human factor IX (FIX), or human alpha1-anti-trypsin (AAT) to study the effect of different cis DNA elements on transduction efficiencies. We found that incorporation of the central polypurine tract sequence (cppt) increased transduction efficiency in vitro while increasing the transduction of non-cell-cycling hepatocytes in vivo. C57Bl/6 scid mice that were administered lentiviral vectors devoid of the cppt (2 x 10(8) transducing units (T.U.)/mouse) had 81% of their lacZ-transduced hepatocytes colabeled with the cell cycle marker 5'-bromo-2'-deoxyuridine (BrdU). In contrast, inclusion of the cppt reduced the colabeling in mouse hepatocytes by 50%. Further modifications in the lentiviral vectors were performed to enhance viral titer and gene expression. We found that the inclusion of a matrix attachment region (MAR) from immunoglobulin-kappa (Igkappa) significantly increased the transduction efficiency, as measured by transgene protein expression and proviral DNA copy number, compared with vectors without Igkappa MAR. In vitro studies using human hepatoma cells demonstrated a significant increase (two- to fourfold) in human AAT and human FIX production when the Igkappa MAR was incorporated. In vivo transduction of partially hepatectomized C57Bl/6 mice given an optimized lentiviral vector containing the cppt and Igkappa MAR (2 x 10(8) T.U./mouse) resulted in sustained therapeutic levels of serum FIX (approximately 65 ng/ml). Our study demonstrates the importance of cis-acting elements to enhancing the transduction ability of lentiviral vectors and the expression of vector transgenes.

Regulation of gene transcription in the epididymis.

The epididymis exhibits region-specific as well as cell-specific patterns of gene expression within the epithelium. The spatial and temporal patterns of gene expression originate during development and are critical to the formation and maintenance of a fully functional epididymis. Despite the number of mechanisms reported to contribute to the regulation of eukaryotic gene expression, little is known about the specific mechanisms involved in the control of epididymal gene expression. This review will outline some of the cis-DNA elements and associated transcription factors that have been identified in the epididymis, in addition to discussing the potential role of co-regulator molecules and changes in chromatin structure as critical control points of gene expression. Although gene expression can be controlled at several points, discussion will focus on gene regulation at the transcriptional level. The role of post-transcriptional control, with particular attention to mRNA stability, will also be discussed.

Regulation of gene transcription in the epididymis

The epididymis exhibits region-specific as well as cell-specific patterns of gene expression within the epithelium. The spatial and temporal patterns of gene expression originate during development and are critical to the formation and maintenance of a fully functional epididymis. Despite the number of mechanisms reported to contribute to the regulation of eukaryotic gene expression, little is known about the specific mechanisms involved in the control of epididymal gene expression. This review will outline some of the cis-DNA elements and associated transcription factors that have been identified in the epididymis, in addition to discussing the potential role of co-regulator molecules and changes in chromatin structure as critical control points of gene expression. Although gene expression can be controlled at several points, discussion will focus on gene regulation at the transcriptional level. The role of post-transcriptional control, with particular attention to mRNA stability, will also be discussed.

Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription.

Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5' portion of the human annexin A5 gene, from bp -1275 to +79 relative to the most 5' of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp -202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5' flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp -717 to -1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35 kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.

Functional analysis of the human annexin A5 gene promoter: a downstream DNA element and an upstream long terminal repeat regulate transcription

Human annexin A5 is a ubiquitous protein implicated in diverse signal transduction processes associated with cell growth and differentiation, and its gene regulation is an important component of this function. Promoter transcriptional activity was determined for a wide 5′ portion of the human annexin A5 gene, from bp −1275 to +79 relative to the most 5′ of several discrete transcription start points. Transfection experiments carried out in HeLa cells identified the segment from bp −202 to +79 as the minimal promoter conferring optimal transcriptional activity. Two canonical Sp1 sites in the immediate 5′ flanking region of a CpG island were required for significant transcription. Strong repressive activity in the distal promoter region between bp −717 to −1153 was attributed to the presence of an endogenous retroviral long terminal repeat, homologous with long terminal repeat 47B. The downstream sequence from bp position +31 to +79 in untranslated exon 1 was also essential for transcription, as its deletion from any of the plasmid constructs abolished activity in transfection assays. Electrophoretic mobility-shift assays, Southwestern-blot analysis and affinity chromatography were used to identify a protein doublet of relative molecular mass 35kDa that bound an octanucleotide palindromic sequence in exon 1. The DNA cis-element resembled an E-box, but did not bind higher molecular mass transcription factors, such as upstream stimulatory factor or activator protein 4. The discovery of a downstream element crucial for annexin A5 gene transcription, and its interaction with a potentially novel transcription factor or complex, may provide a clue to understanding the initiation of transcription by TATA-less, multiple start site promoters.

Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy.

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the ss-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.

Cloning and characterization of 5′-flanking region of mouse prostacyclin synthase gene

To gain an insight into the mechanisms of prostacyclin expression, a genomic DNA clone harboring 2.0 kb of the 5′-flanking sequence of the mouse prostacyclin synthase (PGIS) gene was isolated. The 5′-flanking region did not possess a TATA box, but contained a GC-rich region and several consensus cis DNA elements. The major product of the primer extension analysis suggested that the transcription of the gene started from 72 bases upstream of the translational initiation codon. To analyze the PGIS promoter activity, the 2.0 kb fragment was fused to the luciferase gene and transient transfection assays were conducted with cultured rat vascular smooth muscle cells (VSMC). The fragment showed significant promoter activity in the cells. Analysis of a series of 5′-deletion constructs showed that the 5′-flanking regions spanning bases −371 to −285 and −229 to −119 were important for the basal transcriptional activity of the mouse PGIS gene. Gel mobility shift assays revealed that DNA–protein complexes were formed with the nuclear extracts from VSMC, and that the formation of these complexes was inhibited by excess consensus Sp1 oligonucleotide. Prior incubation of anti-Sp1 antibody with nuclear extracts in this assay resulted in supershift of the band for the DNA–protein complex. In addition, mutation of two Sp1 recognition motifs residing at bases −297 to −289 and −197 to −192 markedly reduced the basal PGIS promoter activity and retarded the band in a gel mobility shift assay. These results indicated that binding of one Sp1 to two Sp1 sites on the promoter region activated the basal transcription of the PGIS gene.

Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia.

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5' flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3' flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1-GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for investigating the organization of primitive eukaryotic vesicular transport.

Differential Expression of the Adipocyte Amino Acid Transporter Is Transactivated by SP1 and SP3 during the 3T3-L1 Preadipocyte Differentiation Process

It was identified that a Sp1-like cis-element in the adipocyte amino acid transporter gene (AAAT) promoter is the major cis-motif for its induced expression during the 3T3-L1 preadipocyte differentiation process. Electrophoretic mobility shift analysis of this cis DNA element showed that the transcription factors binding to this sequence were Sp1 and Sp3. Protein analysis of Sp1 and Sp3 in nuclear extracts from 3T3-L1 cells at various differentiation stages indicated that these two transcription factors existed in noninduced 3T3-L1 preadipocytes, but they did not bind to the AAAT promoter element with high affinity. They were activated after differentiation induction. It was further demonstrated that dephosphorylation of Sp1 increased its binding affinity to this inducible AAAT promoter element.

Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1.

A central question in vertebrate transcriptional regulation is how cis-regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human beta-globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to Maf recognition elements (MARE). Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human beta-globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain-dependent manner. Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis-regulatory elements in order to regulate gene expression.

Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5' of RAG2.

RAG1 and RAG2 are essential for V(D)J recombination and lymphocyte development. These genes are thought to encode a transposase derived from a mobile genetic element that was inserted into the vertebrate genome 450 million years ago. The regulation of RAG1 and RAG2 was investigated in vivo with bacterial artificial chromosome (BAC) transgenes containing a fluorescent indicator. Coordinate expression of RAG1 and RAG2 in B and T cells was found to be regulated by distinct genetic elements found on the 5' side of the RAG2 gene. This observation suggests a mechanism by which asymmetrically disposed cis DNA elements could influence the expression of the primordial transposon and thereby capture RAGs for vertebrate evolution.