Cell–cell interaction in prostate gene regulation and cytodifferentiation

To examine the role of intercellular interaction on cell differentiation and gene expression in human prostate, we separated the two major epithelial cell populations and studied them in isolation and in combination with stromal cells. The epithelial cells were separated by flow cytometry using antibodies against differentially expressed cell-surface markers CD44 and CD57. Basal epithelial cells express CD44, and luminal epithelial cells express CD57. The CD57+ luminal cells are the terminally differentiated secretory cells of the gland that synthesize prostate-specific antigen (PSA). Expression of PSA is regulated by androgen, and PSA mRNA is one of the abundant messages in these cells. We show that PSA expression by the CD57+ cells is abolished after prostate tissue is dispersed by collagenase into single cells. Expression is restored when CD57+ cells are reconstituted with stromal cells. The CD44+ basal cells possess characteristics of stem cells and are the candidate progenitors of luminal cells. Differentiation, as reflected by PSA production, can be detected when CD44+ cells are cocultured with stromal cells. Our studies show that cell–cell interaction plays an important role in prostatic cytodifferentiation and the maintenance of the differentiated state.

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

“TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

Efficient studies of long-distance Bmp5 gene regulation using bacterial artificial chromosomes

The regulatory regions surrounding many genes may be large and difficult to study using standard transgenic approaches. Here we describe the use of bacterial artificial chromosome clones to rapidly survey hundreds of kilobases of DNA for potential regulatory sequences surrounding the mouse bone morphogenetic protein-5 (Bmp5) gene. Simple coinjection of large insert clones with lacZ reporter constructs recapitulates all of the sites of expression observed previously with numerous small constructs covering a large, complex regulatory region. The coinjection approach has made it possible to rapidly survey other regions of the Bmp5 gene for potential control elements, to confirm the location of several elements predicted from previous expression studies using regulatory mutations at the Bmp5 locus, to test whether Bmp5 control regions act similarly on endogenous and foreign promoters, and to show that Bmp5 control elements are capable of rescuing phenotypic effects of a Bmp5 deficiency. This rapid approach has identified new Bmp5 control regions responsible for controlling the development of specific anatomical structures in the vertebrate skeleton. A similar approach may be useful for studying complex control regions surrounding many other genes important in embryonic development and human disease.

DNA bending by an adenine–thymine tract and its role in gene regulation

To gain insight into the structural basis of DNA bending by adenine–thymine tracts (A-tracts) and their role in DNA recognition by gene-regulatory proteins, we have determined the crystal structure of the high-affinity DNA target of the cancer-associated human papillomavirus E2 protein. The three independent B-DNA molecules of the crystal structure determined at 2.2-Å resolution are examples of A-tract-containing helices where the global direction and magnitude of curvature are in accord with solution data, thereby providing insights, at the base pair level, into the mechanism of DNA bending by such sequence motifs. A comparative analysis of E2–DNA conformations with respect to other structural and biochemical studies demonstrates that (i) the A-tract structure of the core region, which is not contacted by the protein, is critical for the formation of the high-affinity sequence-specific protein–DNA complex, and (ii) differential binding affinity is regulated by the intrinsic structure and deformability encoded in the base sequence of the DNA target.

CDP/cut is the DNA-binding subunit of histone gene transcription factor HiNF-D: a mechanism for gene regulation at the G1/S phase cell cycle transition point independent of transcription factor E2F.

Transcription of the genes for the human histone proteins H4, H3, H2A, H2B, and H1 is activated at the G1/S phase transition of the cell cycle. We have previously shown that the promoter complex HiNF-D, which interacts with cell cycle control elements in multiple histone genes, contains the key cell cycle factors cyclin A, CDC2, and a retinoblastoma (pRB) protein-related protein. However, an intrinsic DNA-binding subunit for HiNF-D was not identified. Many genes that are up-regulated at the G1/S phase boundary are controlled by E2F, a transcription factor that associates with cyclin-, cyclin-dependent kinase-, and pRB-related proteins. Using gel-shift immunoassays, DNase I protection, and oligonucleotide competition analyses, we show that the homeodomain protein CDP/cut, not E2F, is the DNA-binding subunit of the HiNF-D complex. The HiNF-D (CDP/cut) complex with the H4 promoter is immunoreactive with antibodies against CDP/cut and pRB but not p107, whereas the CDP/cut complex with a nonhistone promoter (gp91-phox) reacts only with CDP and p107 antibodies. Thus, CDP/cut complexes at different gene promoters can associate with distinct pRB-related proteins. Transient coexpression assays show that CDP/cut modulates H4 promoter activity via the HiNF-D-binding site. Hence, DNA replication-dependent histone H4 genes are regulated by an E2F-independent mechanism involving a complex of CDP/cut with cyclin A/CDC2/ RB-related proteins.

A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation.

The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.

DNA sequence insertion and evolutionary variation in gene regulation.

Current evidence on the long-term evolutionary effect of insertion of sequence elements into gene regions is reviewed, restricted to cases where a sequence derived from a past insertion participates in the regulation of expression of a useful gene. Ten such examples in eukaryotes demonstrate that segments of repetitive DNA or mobile elements have been inserted in the past in gene regions, have been preserved, sometimes modified by selection, and now affect control of transcription of the adjacent gene. Included are only examples in which transcription control was modified by the insert. Several cases in which merely transcription initiation occurred in the insert were set aside. Two of the examples involved the long terminal repeats of mammalian endogenous retroviruses. Another two examples were control of transcription by repeated sequence inserts in sea urchin genomes. There are now six published examples in which Alu sequences were inserted long ago into human gene regions, were modified, and now are central in control/enhancement of transcription. The number of published examples of Alu sequences affecting gene control has grown threefold in the last year and is likely to continue growing. Taken together, all of these examples show that the insertion of sequence elements in the genome has been a significant source of regulatory variation in evolution.

Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation.

Glutamate dehydrogenase (GDH) is ubiquitous to all organisms, yet its role in higher plants remains enigmatic. To better understand the role of GDH in plant nitrogen metabolism, we have characterized an Arabidopsis mutant (gdh1-1) defective in one of two GDH gene products and have studied GDH1 gene expression. GDH1 mRNA accumulates to highest levels in dark-adapted or sucrose-starved plants, and light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The Arabidopsis gdh-deficient mutant allele gdh1-1 cosegregates with the GDH1 gene and behaves as a recessive mutation. The gdh1-1 mutant displays a conditional phenotype in that seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen. These results suggest that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess. This notion is further supported by the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.

Quantifying Eukaryotic Gene Regulation in Hormone Response and Disease.

Quantifying the function of mammalian enhancers at the genome or population scale has been longstanding challenge in the field of gene regulation. Studies of individual enhancers have provided anecdotal evidence on which many foundational assumptions in the field are based. Genome-scale studies have revealed that the number of sites bound by a given transcription factor far outnumber the genes that the factor regulates. In this dissertation we describe a new method, chromatin immune-enriched reporter assays (ChIP-reporters), and use that approach to comprehensively test the enhancer activity of genomic loci bound by the glucocorticoid receptor (GR). Integrative genomics analyses of our ChIP-reporter data revealed an unexpected mechanism of glucocorticoid (GC)-induced gene regulation. In that mechanism, only the minority of GR bound sites acts as GC-inducible enhancers. Many non-GC-inducible GR binding sites interact with GC-induced sites via chromatin looping. These interactions can increase the activity of GC-induced enhancers. Finally, we describe a method that enables the detection and characterization of the functional effects of non-coding genetic variation on enhancer activity at the population scale. Taken together, these studies yield both mechanistic and genetic evidence that provides context that informs the understanding of the effects of multiple enhancer variants on gene expression.

Functional analysis of the 5′ flanking region of the human G6PC3 gene: regulation of promoter activity by glucose, pyruvate, AMP kinase and the pentose phosphate pathway

G6PC3 is a widely expressed isoform of glucose-6-phosphatase, found in many foetal and adult tissues. Mutations in this gene cause developmental abnormalities and severe neutropenia due to abolition of glucose recycling between the cytoplasm and endoplasmic reticulum. Low G6PC3 expression as a result of promoter polymorphisms or dysregulation could produce similar outcomes. Here we investigated the regulation of human G6PC3 promoter activity. HeLa and H4IIE cells were transiently transfected with G6PC3 promoter coupled to the firefly luciferase gene, and promoter activity was measured by dual luciferase assay. Activity was highest in a 453 bp segment of the G6PC3 promoter, from − 455 to − 3 relative to the transcriptional start site. This promoter was unresponsive to glucostatic hormones. Its activity increased significantly between 1 and 5.5 mM glucose, and was not elevated further by glucose concentrations up to 25 mM. Pyruvate increased its activity, but β-hydroxybutyrate and sodium acetate did not. Promoter activity was reduced by inhibitors of hexokinase, glyceraldehyde phosphate dehydrogenase and the oxidative branch of the pentose phosphate pathway, but not by a transketolase inhibitor. Deletion of two adjacent Enhancer-boxes (− 274 to − 279 and − 299 to − 304) reduced promoter activity and abolished the glucose effect, suggesting they could function as a glucose response element. Deletion of an additional downstream 140 bp (− 140 to − 306) restored activity, but not the glucose response, suggesting the presence of repressor elements in this region. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) reduced promoter activity, showing dependence on AMP-kinase. Regulation of the G6PC3 promoter is thus radically different to that of the hepatic isoform, G6PC. It is sensitive to carbohydrate, but not to fatty acid metabolites, and at much lower physiological concentrations. Based on these findings, we speculate that reduced G6PC3 expression could occur during hypoglycemic episodes in vivo, which are common in utero and in the postnatal period. If such episodes lower G6PC3 expression they could place the foetus or infant at risk of impaired immune function and development, and this possibility requires further examination both in vitro and in vivo.

Implication of DNA methylation and PAX5 factor in the transcriptional regulation of hTERT, the human telomerase reverse transcriptase gene

RESUME La télomérase est une enzyme dite "d'immortalité" qui permet aux cellules de maintenir la longueur de leurs télomères, ce qui confère une capacité de réplication illimitée aux cellules reproductrices et cancéreuses. A l'inverse, les cellules somatiques normales, qui n'expriment pas la télomérase, ont une capacité de réplication limitée. La sous-unité catalytique de la télomérase, hTERT, est définie comme le facteur limitant l'activité télomérasique. Entre activateurs et répresseurs, le rôle de la méthylation de l'ADN et de l'acétylation des histones, de nombreux modèles ont été suggérés. La découverte de l'implication de CTCF dans la régulation transcriptionnelle de hTERT explique en partie le mécanisme de répression de la télomérase dans la plupart des cellules somatiques et sa réactivation dans les cellules tumorales. Dans les cellules télomérase-positives, l'activité inhibitrice de CTCF est bloquée par un mécanisme dépendent ou non de la méthylation. Dans la plupart des carcinomes, une hyperméthylation de la région 5' de hTERT bloque l'effet inhibiteur de CTCF, alors qu'une petite région hypométhylée permet un faible niveau de transcription du gène. Nous avons démontré que la protéine MBD2 se lie spécifiquement sur la région 5' méthylée de hTERT dans différentes lignées cellulaires et qu'elle est impliquée dans la répression partielle de la transcription de hTERT dans les cellules tumorales méthylées. Par contre, nous avons montré que dans les lymphocytes B normaux et néoplasiques, la régulation de hTERT est indépendante de la méthylation. Dans ces cellules, le facteur PAX5 se lie sur la région 5' de hTERT en aval du site d'initiation de la traduction (ATG). L'expression exogène de PAX5 dans les cellules télomérase-négatives active la transcription de hTERT, alors que la répression de PAX5 dans les cellules lymphomateuses inhibe la transcription du gène. PAX5 est donc directement impliqué dans l'activation de l'expression de hTERT dans les lymphocytes B exprimant la télomérase. Ces résultats révèlent des différences entre les niveaux de méthylation de hTERT dans les cellules de carcinomes et les lymphocytes B exprimant la télomérase. La méthylation de hTERT en tant que biomarqueur de cancer a été évaluée, puis appliquée à la détection de métastases. Nous avons ainsi montré que la méthylation de hTERT est positivement corrélée au diagnostic cytologique dans les liquides céphalorachidiens. Nos résultats conduisent à un modèle de régulation de hTERT, qui aide à comprendre comment la transcription de ce gène est régulée par CTCF, avec un mécanisme lié ou non à la méthylation du gène hTERT. La méthylation de hTERT s'est aussi révélée être un nouveau et prometteur biomarqueur de cancer. SUMMARY Human telomerase is an "immortalizing" enzyme that enables cells to maintain telomere length, allowing unlimited replicative capacity to reproductive and cancer cells. Conversely, normal somatic cells that do not express telomerase have a finite replicative capacity. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors, and the role of DNA methylation and histone acetylation, an abundance of hTERT regulatory models have been suggested. The discovery of the implication of CTCF in the transcriptional regulation of hTERT in part explained the mechanism of silencing of telomerase in most somatic cells and its reactivation in neoplastic cells. In telomerase-positive cells, the inhibitory activity of CTCF is blocked by methylation-dependent and -independent mechanisms. In most carcinoma cells, hypermethylation of the hTERT 5' region has been shown to block the inhibitory effect of CTCF, while a short hypomethylated region allows a low transcription level of the gene. We have demonstrated that MBD2 protein specifically binds the methylated 5' region of hTERT in different cell lines and is therefore involved in the partial repression of hTERT transcription in methylated tumor cells. In contrast, we have shown that in normal and neoplastic B cells, hTERT regulation is methylation-independent. The PAX5 factor has been shown to bind to the hTERT 5'region downstream of the ATG translational start site. Ectopic expression of PAX5 in telomerase-negative cells or repression of PAX5 expression in B lymphoma cells respectively activated and repressed hTERT transcription. Thus, PAX5 is strongly implicated in hTERT expression activation in telomerase-positive B cells. These results reveal differences between the hTERT methylation patterns in telomerase-positive carcinoma cells and telomerase-positive normal B cells. The potential of hTERT methylation as a cancer biomarker was evaluated and applied to the detection of metastasis. We have shown that hTERT methylation correlates with the cytological diagnosis in cerebrospinal fluids. Our results suggest a model of hTERT gene regulation, which helps us to better understand how hTERT transcription is regulated by CTCF in methylation-dependant and independent mechanisms. Our data also indicate that hTERT methylation is a promising new cancer biomarker.

Cis-, trans-, transcriptional regulation of the Arabidopsis thaliana gene AtTrxo1 and its response upon germination and to salt

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in redox regulation of many enzymes in different cell compartments. Among them, mitochondrial Trxo has been described to have a response in plants grown under salinity but there is scarce information about its functional role in abiotic stress or its gene regulation. In this work, the transcriptional regulation of the mitochondrial AtTrxo1 gene has been studied for the first time, by identifying functionally relevant cis- elements in its promoter: two conserved motives were found as positive and one as negative regulators. Using them as baits for the screening of an arrayed yeast library containing Arabidopsis Transcription Factors (TF) ORFs, two TFs were selected that are now being validated at the molecular level. We have also studied the response of T-DNA insertion mutant plants for AtTrxo1 to salt stress. The K.O. AtTrxo1 mutants presented several phenotypic changes including the time required to reach 50% germination under salinity, without affecting the final germination percentage.

Positive and negative regulation of endogenous genes by designed transcription factors

Gene regulation by imposed localization was studied by using designed zinc finger proteins that bind 18-bp DNA sequences in the 5′ untranslated regions of the protooncogenes erbB-2 and erbB-3. Transcription factors were generated by fusion of the DNA-binding proteins to repression or activation domains. When introduced into cells these transcription factors acted as dominant repressors or activators of, respectively, endogenous erbB-2 or erbB-3 gene expression. Significantly, imposed regulation of the two genes was highly specific, despite the fact that the transcription factor binding sites targeted in erbB-2 and erbB-3 share 15 of 18 nucleotides. Regulation of erbB-2 gene expression was observed in cells derived from several species that conserve the DNA target sequence. Repression of erbB-2 in SKBR3 breast cancer cells inhibited cell-cycle progression by inducing a G1 accumulation, suggesting the potential of designed transcription factors for cancer gene therapy. These results demonstrate the willful up- and down-regulation of endogenous genes, and provide an additional means to alter biological systems.

Social insect genomes exhibit dramatic evolution in gene composition and regulation while preserving regulatory features linked to sociality.

Genomes of eusocial insects code for dramatic examples of phenotypic plasticity and social organization. We compared the genomes of seven ants, the honeybee, and various solitary insects to examine whether eusocial lineages share distinct features of genomic organization. Each ant lineage contains ∼4000 novel genes, but only 64 of these genes are conserved among all seven ants. Many gene families have been expanded in ants, notably those involved in chemical communication (e.g., desaturases and odorant receptors). Alignment of the ant genomes revealed reduced purifying selection compared with Drosophila without significantly reduced synteny. Correspondingly, ant genomes exhibit dramatic divergence of noncoding regulatory elements; however, extant conserved regions are enriched for novel noncoding RNAs and transcription factor-binding sites. Comparison of orthologous gene promoters between eusocial and solitary species revealed significant regulatory evolution in both cis (e.g., Creb) and trans (e.g., fork head) for nearly 2000 genes, many of which exhibit phenotypic plasticity. Our results emphasize that genomic changes can occur remarkably fast in ants, because two recently diverged leaf-cutter ant species exhibit faster accumulation of species-specific genes and greater divergence in regulatory elements compared with other ants or Drosophila. Thus, while the "socio-genomes" of ants and the honeybee are broadly characterized by a pervasive pattern of divergence in gene composition and regulation, they preserve lineage-specific regulatory features linked to eusociality. We propose that changes in gene regulation played a key role in the origins of insect eusociality, whereas changes in gene composition were more relevant for lineage-specific eusocial adaptations.