WO2002099131A1 - Element de liaison de facteur de transcription - Google Patents

Element de liaison de facteur de transcription Download PDF

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WO2002099131A1
WO2002099131A1 PCT/IL2001/000517 IL0100517W WO02099131A1 WO 2002099131 A1 WO2002099131 A1 WO 2002099131A1 IL 0100517 W IL0100517 W IL 0100517W WO 02099131 A1 WO02099131 A1 WO 02099131A1
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transcription factor
factor binding
cells
binding element
promoter
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PCT/IL2001/000517
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Karl L. Skorecki
Maty Tzukerman
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Rappaport Family Institute For Research In The Medical Sciences
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to novel transcription factor binding elements, to the correlation between transcription factor binding and promoter activity, specifically between transcription factor binding and TERT promoter activity, to constructs comprising the transcription factor binding element useful in regulation of telomerase activity, and to use of said elements for characterization of transcription factors.
  • Eukaryotic telomeric DNA is composed of repetitive stretches of guanine-rich sequences (TTAGGG in mammalian cells), which together with an associated set of proteins, form a DNA-protein complex, thought to maintain the integrity of chromosome ends (Blackburn et al, 1992; Greider, 1991; Levy et al, 1992). Shortening of telomere ends, with progressive rounds of cell-division, has been proposed to serve as a mitotic clock by which cell divisions are counted, eventuating in a state of replicative arrest, known as cellular senescence (Allsopp et ⁇ /, 1992; Harley, 1991; Harley et al, 1990).
  • telomere shortening involves telomerase and associated telomeric proteins.
  • Telomerase is a ribonucleoprotein enzyme with reverse transcriptase activity, capable of extending chromosome ends with specific telomeric DNA sequences, by using a portion of its RNA component as a template (Autexier and Greider, 1996; Kim et al, 1994; Lundblad and Wright, 1996).
  • the protein (TERT) and mRNA (TER) components of the telomerase ribonucleoprotein are each encoded at a separate genetic locus, and are under independent regulatory control.
  • telomere activity is achieved at least in part, through rate-limiting mechanisms governing expression of the human TERT (hTERT) (Greider, 1999; Nakayama et al, 1998).
  • the 5 '-flanking region of the hTERT gene has been characterized in part using sequence analysis, transient transfection of promoter-reporter constructs, as well as electrophoretic mobility shift and DNAse footprint analysis (Cong et al, 1999; Horikawa et al, 1999; Takakura et al, 1999; Wick et al, 1999). These studies have identified a core promoter extending from -283nt upstream of the transcription initiation site to +78nt of the first exon, containing several putative transcription factor-binding elements. The finding of multiple putative c-Myc binding elements was considered of particular interest, in view of the role of c-Myc in cell proliferation and transformation.
  • telomerase activity is readily detected in male and female germ-line cells, ensuring maintenance of chromosome integrity through successive generations. Activation of telomerase has also been implicated as a major mechanism for attainment of cellular immortalization in the molecular pathogenesis of most but not all malignant tumors. In contrast, most normal adult human somatic cells lack significant telomerase activity. Even in rapidly proliferating normal (non-transformed) tissues, only low levels of telomerase activity are detected post-natally, representing the contribution of the small subset of stem cells which replenish the rapidly proliferating population (Vasiri et al, 1994). However, as a notable exception, telomerase is active in human lymphocytes during development, differentiation, activation and aging (Weng et al, 1997, Liu et al, 1999).
  • telomere shortening and telomerase have been investigated with respect to the process of aging, cellular senescence, and neoplastic transformation, there has been a relative paucity of information with respect to its role during embryonic and fetal development, as well as cellular differentiation.
  • Expression of telomerase activity has been detected at the earliest gestational stages during human embryonic development, with variable down-regulation of telomerase activity during subsequent fetal development (Ulaner and Giudice, 1997; Wright et al, 1996; Youngren et al, 1998).
  • telomere activity has been detected in fetal, neonatal, and adult gonadal tissues, but not in mature spermatazoa or oocytes (Ulaner and Giudice, 1997). Telomerase activity was also detectable at high levels in human blastocysts, which were obtained from patients who had undergone in vitro fertilization, and in some, but not all, human somatic tissues during early but not later stages of prenatal development (Youngren et al, 1998).
  • telomere activity was lower or absent in the corresponding neonatal or adult tissues, including placental tissue at birth.
  • telomere activity is restricted to a limited number of somatic stem cells, which permit cell renewal during post-natal tissue loss and regeneration.
  • this level of telomerase activity is not sufficient to prevent eventual exhaustion of telomeres in renewing tissues with a rapid cellular turnover.
  • lysates of telomerase negative tissues do not inhibit the activity of telomerase positive cells taken during various stages of gestational development, suggesting that suppression of telomerase activity, is due to loss of telomerase, rather than to the presence of an inhibitor (Youngren et al, 1998).
  • telomerase activity is associated with a marked reduction in telomerase activity in at least two different cell lines, as first reported by Sharma et al. (1995). Subsequent studies have confirmed these results but did not determine whether this reduction could be attributed to inhibition of the promoter (Holt et al, 1996, Holt et al, 1997, Horikawa et al, 1998, Tanaka et al, 1998).
  • Telomerase reverse transcriptase transcriptional regulatory sequences have previously been disclosed to be useful in viral constructs for use in the treatment of cancer (WO 00/46355).
  • oncolytic viruses are described, in which a toxin or genetic element essential for viral replication is placed under control of the TERT promoter.
  • the virus replicates preferentially in cells expressing TERT, and selectively lyse cancer cells.
  • the current invention is based on a study that identified a novel transcription factor binding element, involved in the transcriptional regulation of TERT (Tzukerman et al., Mol. Biol. of the Cell 11, 4381-4391, 2000). That disclosure however did not teach the utility of the novel element.
  • the present invention relates to a novel nucleotide sequence for binding transcription factors, specifically a transcription factor binding element.
  • the transcription factor binding element of the invention is useful in the regulation of telomerase expression.
  • the transcription factor binding element of the invention may further be useful in polynucleotide constructs comprising the transcription factor binding element or a promoter comprising the transcription factor binding element. Vectors comprising said constructs may be used in control of cell differentiation or cell immortalization.
  • the invention also relates to the use of constructs comprising the nucleotide sequence in controlling the development of cell lines from stem cells.
  • the invention further relates to the use of the transcription factor binding element for the isolation of transcription factors.
  • the transcription factor binding element which is demonstrated in accordance with an embodiment of the invention, in the human and mouse TERT promoter region, is found to be involved in telomerase promoter activity in association with differentiation in cell differentiation model systems.
  • the present invention provides a system for testing molecular mechanisms useful in control of telomerase expression. It further provides a system for characterizing the factors that bind to the novel element.
  • the present invention especially provides methods for regulation of telomerase activity, by using constructs comprising the transcription factor binding element.
  • the nucleotide sequence of the invention comprises the sequence CGTGGGAXG, wherein X is A or G.
  • the nucleotide sequence is preferably a 5' CGTGGGAXG 3' sequence.
  • the factors binding the nucleotide sequence of the invention may be proteins, specifically transcription factors from nuclear extracts. According to a preferred embodiment of the invention, the binding of factors to the nucleotide sequence of the invention will be stringent. According to one more preferred embodiment of the invention, the factors bind to the sequence in a reaction lasting 20 minutes at 4°C.
  • the factors are nuclear extract factors from undifferentiated cells, such as undifferentiated human embryonic stem (hES) cells.
  • the factors are nuclear factors from undifferentiated hES cells grown on a cell feeder layer as exemplified by but not limited to an MEF feeder layer.
  • nucleic acid construct comprising a transcription factor binding element comprising the nucleotide sequence CGTGGGAXG, wherein X is A or G.
  • a construct encoding a promoter element comprising the nucleic acid sequence CGTGGGAXG, wherein X is A or G, a heterologous gene operatively linked to the promoter element, wherein the expression of the heterologous gene is under the control of the promoter sequences.
  • the heterologous gene may be selected from the the group consisting of a reporter gene or a selectable marker or any structural or functional gene that it is desired to express under control of the promoter of choice.
  • a vector comprising a nucleic acid construct, comprising a transcription factor binding element comprising the nucleotide sequence CGTGGGAXG, wherein X is A or G.
  • a construct encoding a promoter element comprising the nucleic acid sequence CGTGGGAXG, wherein X is A or G, a heterologous gene operatively linked to the promoter element, within a vector useful for transfection of host cells, wherein the expression of the heterologous gene is under the control of the promoter sequences.
  • nucleic acid construct and/or a vector comprising the nuclic acid construct may also be provided, in accordance with an embodiment of the invention, in a composition with at least one pharmaceutically acceptable carrier and/or excipient.
  • a method for controlling the development of cell lines or progenitors of cell lineages from stem cells comprises the step of expressing, preferably by transfection, a promoter comprising the nucleotide sequence CGTGGGAXG, wherein X is A or G, in the stem cells.
  • the promoter comprising the nucleotide sequence of the invention may be expressed through a vector comprising a construct comprising the promoter.
  • the nucleotide sequence of the invention may act to bind factors which act as either activators or as repressors.
  • Figure 1 show results of a TRAP assay performed using extracts from hES cells at different stages of differentiation
  • Figure 2 A is a schematic illustration of a pGL3 -basic vector containing hTERT promoter fragments and a luciferase reporter gene;
  • Figure 2B shows the relative luciferase activity in HEK293 and NHF cells that have been tranfected with the constructs illustrated in Fig. 2 A
  • Figure 2C shows the relative luciferase activity in F9 cells that have been tranfected with the constructs illustrated in Fig. 2A, before and after differentiation;
  • Figure 3A shows results of EMSA with nuclear extracts from F9 cells, hES cells and HL-60 cells before and after differentiation, that were incubated with 32 P- labeled oligonucleotide corresponding to position -177 to- 146 of the hTERT (oligo-1);
  • Figure 3B shows results of EMSA with nuclear extracts from F9 cells, hES cells and HL-60 cells before and after differentiation, that were incubated with 32 P- labeled oligonucleotide corresponding to position +32 to +58 (oligo-2);
  • Figure 4 shows results of EMSA with nuclear extracts from undifferentiated F9 cells and from F9 cells following induction of differentiation that were incubated with P-labeled oligonucleotide corresponding to oligo-1 or oligo-2 with or without competitor;
  • Figure 5 A schematically shows sequences of mutated oligonucleotide used for competition in EMSA analysis for mapping the transcription factor binding sites included in oligo-2;
  • Figure 5B shows results of EMSA with F9 nuclear extracts that were incubated with 3 P-labeled oligonucleotide without or with excess of unlabelled oligo-1 (-177), oligo-2 (+32), or the mutated sequences M1-M8 indicated in Fig.
  • Figure 6A shows the relative luciferase activity in F9 cells that were transiently cotransfected with pGL-3 construct containing as insert either the wild-type hTERT core promoter or the mutant core promoter sequences Ml, M3 or M4 as indicated in Fig. 5A; and
  • Figure 6B shows the relative luciferase activity in HEK cells that were transiently cotransfected with pGL-3 construct containing as insert either the wild-type hTERT core promoter or the mutant core promoter sequences Ml, M3 or M4 as indicated in Fig. 5 A.
  • TERT promoter A distinct and novel element was identified within the TERT promoter.
  • the element underwent time-dependent reduction in transcription factor binding with differentiation. Site directed mutagenesis of this novel element, revealed a correlation between transcription factor binding and promoter activity. Since this sequence had not been previously described as a transcription factor binding element, it was given a tentative new designation (MT-box).
  • the utility of this element includes:
  • hES cells human embryonic stem cells
  • hTERT over-expressing hES cells i.e. insulin secreting cells, beta progenitor cells, neuronal cells, cardiomyocytes.
  • promoter regions including enhancers are characterized by their ability to bind to RNA polymerase and other activating proteins, and generally contain recognition sites for the various proteins.
  • One of the most essential elements is the promoter used to express an exogenous or heterologous gene.
  • exogenous protein and heterologous protein can be used interchangeably.
  • the transcription factor binding element of the invention has been found in the context of the telomerase promoter, however, it can be utilized as an element in constructs of nucleic acids that comprise another promoter of choice.
  • the construct may further comprise a protein of choice that is operably linked downstream to the promoter of choice.
  • different cell differentiation experimental model systems human embryonic stem cells (hES), mouse F9 cells, and human HL-60 promyelocytic cells
  • hES human embryonic stem cells
  • mouse F9 cells mouse F9 cells
  • human HL-60 promyelocytic cells were used to determine the relationship between the reduction in telomerase activity following differentiation, and regulation of the promoter for the hTERT gene.
  • Promoter constructs of three different lengths were subcloned into the PGL3 -basic luciferase reporter vector.
  • the novel element in accordance with an embodiment of the invention was identified within the core promoter, and also underwent time-dependent reduction in transcription factor binding with differentiation.
  • the element specifically binds protein from nuclear extracts of undifferentiated telomerase positive cells; but not from the corresponding telomerase negative cells, following induction of differentiation. Site directed mutagenesis of this element, revealed a correlation between transcription factor binding and promoter activity.
  • EXAMPLES The examples are focussed on the behavior and role of the TERT promoter in differentiation, using different cell culture model systems. Differentiation is associated with a marked reduction in telomerase activity but it has not been determined whether this reduction could be attributed to inhibition of the promoter (Holt et al, 1996, Holt et al, 1997, Horikawa et al, 1998, Tanaka et al, 1998). In the present invention this finding is confirmed and extended to human embryonic stem cell differentiation. It is shown that this reduction is associated pari passu with a corresponding marked reduction in promoter activity. In the case of HL-60 cells, differentiation is associated with a loss of proliferative capacity.
  • telomere inhibition has also been shown during mouse fetal development (Prowse and Greider 1995). It was also found, in accordance with the present invention, in the mouse derived F9 cell line that differentiation in vitro was also associated with inhibition of the hTERT promoter, which was accompanied by a similar pattern of reduced transcription factor binding.
  • telomere binding As previously reported in telomerase positive cancer cell lines, it was found that a core region extending 283 nt upstream of the translation initiation site, is sufficient to give maximum promoter activity. Larger promoter fragments, extending further 5 '-upstream result in inhibition compared to the core fragment.
  • EMSA results in accordance with the present invention, revealed that loss of promoter activity with differentiation was associated with inhibition of transcription factor binding to both E-boxes contained within the core-promoter. Although c-Myc can bind to E-box elements, super-shift experiments did not identify the transcription factor as containing c-Myc.
  • H9 cell line of human embryonic stem cells (hES) (Thomson et al, 1998), were maintained in the undifferentiated state by propagation in culture on a feeder layer of irradiated primary mouse embryonic fibroblasts (MEF). MEFs were prepared according to Robertson (Robertson, 1987), and plated on gelatin coated six-well plates. hES cells were grown in knockout DMEM supplemented with 20% serum replacement, 1% nonessential amino acids, 0.1 mM 2- mercaptoethanol (all from GIBCO/BRL), lmM glutamine (Biological Industries, Israel) and 4ng/ml human recombinant bFGF (PEPRO TECH INC.).
  • Undifferentiated hES cells spontaneously differentiate when passaged in the absence of MEF's and formed multicellular aggregates, from which emerged multiple identifiable differentiated cell types.
  • undifferentiated hES cells were disaggregated and transferred in suspension into bacterial grade petri dishes (Greiner) for induction of synchronous differentiation that results in acquisition of a configuration of simple embryoid bodies (EBs).
  • F9 cells were induced to differentiate by adding l ⁇ M RA to the medium for seven days. After seven days cells were collected and transferred in suspension into bacterial grade petri dishes to form EBs. Differentiation was assessed by microscopic observation of changes in cellular morphology.
  • HL-60 cells were induced to differentiate by exposure to 1 " .3% DMSO (Sigma) for seven days. Differentiation was assessed by monitoring the CDl lb (M01) integrin expression profile by FACS analysis using CDl lb (LeuTM- 15) monoclonal antibody (Becton Dickinson).
  • TRAP Assay Telomerase activity was measured by a modified telomerase repeat amplification protocol using the TRAPeze telomerase detection kit (Oncor, Gaithersburg, MD, USA) (Kim et al, 1994). Total cellular extracts were prepared from undifferentiated and differentiated cells according to the manufacturer's instructions.
  • hTERT Promoter-Luciferase Reporter Constructs A human genomic library (Clontech) in EMBL3 (7.5xl0 6 phages) was screened using a labeled 450 bp fragment corresponding to the 5' hTERT cDNA as a probe. The sequence of the 5.8 Kb promoter fragment was subjected to computer analysis [Wisconsin Package Version 8.0, Genetics Computer Group (GCG), Madison, WI].
  • Mutated Core Promoter-Luciferase Reporter Constructs Site directed mutagenesis was performed using the QuikChangeTM kit (Stratagene) to introduce mutations into the E-box and the MT-box motifs of the hTERT core promoter-luciferase construct (283bp promoter fragment).
  • the mutated oligonucleotide primers that were used for disruption of the proximal E-box and the MT-box motifs were as follows: 5'GTCCTGCTGCGATCGTGGGAA GCCCTGGC3' for Ml (mutantl), 5'GTCCTGCTGCGCACGATGGAAGCC CTGGC3' for M3, 5'GTCCTGCTGCGCACGTGGCAAGCCCTGGC3' forM4.
  • HEK293 cells (6xl0 4 ), F9 cells (2.5xl0 4 ) and HNF cells (5xl0 4 ) were plated 24h prior to transfection in 24-well plates in the appropriate medium. Cotransfections were performed using FuGENE 6 transfection reagent (Boehringer Mannheim) with 0.07 ⁇ g promoter-luciferase reporter construct, 0.14 ⁇ g pCMV- ⁇ galactosidase expression vector for calibration of transfection efficiencies. Transfection mixtures were adjusted with pBluescript DNA to a total of 0.3 ⁇ g per well. At 24h after transfection the medium was changed and the cells were incubated for an additional 48h. Cell extracts were prepared and luciferase or ⁇ - galactosidase activities were measured.
  • Electrophoretic Mobility Shift Assay (EMSA). Nuclear extracts were prepared from the various cell lines as previously described (Schreiber et al, 1998). Ten micrograms of protein were incubated with 3 OOng poly(dl-dC) for 10 minutes at 4°C in a 20 ⁇ l reaction volume containing 25mM Hepes pH 7.9, 50mM KC1, 0.5mM PMSF, lmM DTT and 10% glycerol with or without 200-fold molar excess of unlabeled competitor DNA. After incubation, lxl 0 5 cpm of 32 P-labeled probe was added, and the reaction was incubated for an additional 20 minutes at 4°C.
  • the DNA-protein complexes were separated by electrophoresis on a 5.3% polyacrylamide gel and visualized by autoradiography.
  • samples were preincubated with 5 ⁇ g antiserum against c-Myc (Santa Cruz Biotechnology) for 1 h at 4°C.
  • sequences of the oligonucleotides used as probes were:
  • telomere differentiation is associated with suppression of telomerase activity.
  • three cell culture model systems for differentiation were utilized: human embryonic stem cells (hES), mouse embryonal carcinoma F9 cells, and HL-60 human promyelocytic leukemia cells.
  • hES cells are defined as being derived from pluripotent cells of the early mammalian embryo, with the capacity for unlimited proliferation in vitro in the undifferentiated state, when grown on a feeder layer of mouse embryonic fibroblasts (MEF). These cells maintain the ability to differentiate following propagation in the absence of the feeder layer, and as a result display the outgrowth of cells deriving from each of the major developmental lineages.
  • hES cells can also be induced to form embryoid bodies (EB's), when grown on bacterial grade petri dishes.
  • F9 teratocarcinoma cells can be induced to differentiate when
  • HL-60 cells can be induced to differentiate to mature granulocyte-like cells, that cease proliferating and express the CDl lb antigen, following exposure to 1.3% DMSO for 7 days. Telomerase activity was measured before and at varying time points after induction of differentiation in these three model systems. As shown in Fig. 1, pluripotent hES cells are strongly telomerase positive. TRAP assay was performed using extracts from hES cells cultured on MEF cells (day 1) and from hES cells that were allowed to differentiate for 7,9,11 and 14 days.
  • TRAP activity was measured in extracts of MEF, buffer control (be), and of telomerse positive cells supplied in the kit (pc).
  • telomerase activity was measured using increasing amount of extracts; 1, 5, 10 and 50ug, to ensure that the assay was performed in the linear range.
  • hTERT promoter A human genomic library (7.5x10 6 phages) was screened using a 450bp hTERT cDNA fragment as a probe.
  • Fig. 2A three sub-fragments of varying sizes were sub-cloned in the pGL3 -basic vector upstream to the luciferase reporter gene: 5790nt (full-length fragment), 3345nt (intermediate fragment), and 283nt (core promoter) upstream to the luciferase reporter gene: 5790nt (full-length fragment), 3345nt (intermediate fragment), and 283nt (core promoter) upstream to the
  • telomere negative normal human fibroblasts and telomerase positive HEK 293 cells are shown in Fig. 2B.
  • the highest level of promoter activity was consistently observed using the core promoter fragment (46-fold) compared to the control level.
  • oligo-1 The oligonucleotide used for the distal E-box, designated as oligo-1, corresponded to the promoter sequence from -177 to -145, as follows: 5'GGACCGCGCTCCCCACGTGGCGGAGGGACTGGG3'.
  • oligo-2 corresponded to the promoter sequence from +32nt to +61nt, with the following sequence: 5'GTCCTGCTGCGCACGTGGGAAGCCCTGGC3'.
  • Fig. 3 A shows the effect of differentiation on transcription factor binding to the distal E-box using oligo-1.
  • a clear band shift was observed using nuclear extracts from telomerase positive, undifferentiated F9, hES, and HL-60 cell lines. This band shift was markedly reduced using nuclear extracts from the corresponding telomerase negative, differentiated cell lines. Specificity of binding was confirmed using competition with excess cold oligonucleotide. No supershift was observed using antibodies to c-Myc.
  • the proximal E-box was examined next using the corresponding labelled oligo- 2 in the EMSA. In this case, two distinct shifted bands were noted. The upper band appears similar to the band shift noted using oligo-1, containing the distal E-box. However, in addition, there appeared another distinct faster-migrating band in all three cell lines in the undifferentiated state (see Fig. 3B). In all three cell lines, induction of differentiation was associated with a marked reduction in the intensity of this band as well. Furthermore, in F9 cells, accompanying the reduction in intensity, there appeared an additional faster-migrating band which could potentially represent a degradative or covalently modified product derived from the binding protein(s).
  • binding competition experiments were performed using undifferentiated F9 nuclear extracts and labeled proximal oligo-2, in which either excess unlabelled oligo-1 or excess unlabelled oligo-2, (containing the distal or proximal E-box consensus motifs respectively) were added to the reaction mix. As shown in Fig.
  • nuclear extracts from undifferentiated F9 cells (lanes 1,3,4,9,11,12) and from F9 cells following induction of differentiation (lanes 2,5,6,10,13,14) were incubated with P-labeled oligonucleotide corresponding to position - 177 to - 146 (oligo- 1) or with P-labeled oligonucleotide corresponding to position +32 to +58 (oligo-2) of the hTERT promoter. Incubation was performed with or without competitor (150 fold excess of cold oligonucleotide) as indicated. As control, nuclear extract from telomerase negative cells NHF was used (lanes7 and 15).
  • c-Myc in vitro tanscription- translation product was used to examine binding to the oligo-1 E-box motif (lane 8). Arrowheads indicate specific retarded bands. As can be seen in Fig. 4 either excess unlabelled oligo-1 or oligo-2 reduced the upper gel-shift band. In contrast, only oligo-2 competed with itself in binding to the faster migrating transcription factor(s).
  • oligonucleotides were generated, in which individual nucleotide pairs were systematically replaced. Each of the mutated oligonucleotides was used as an unlabelled competitor in EMSA reactions using undifferentiated F9 nuclear extracts, with labeled wild-type oligo-2. As shown in Fig. 5B, the mutations in mutants Ml, M2, and M3 involve the E-box motif itself (CACGTG). These unlabelled mutant oligonucleotides failed to compete with binding to labeled oligo-2.
  • Mutant oligonucleotides M4 and M5 failed to compete with binding at the lower gel-shift band, indicating that the two nucleotides AG, represent the 3 '-limit of the novel binding element.
  • hTERT promoter sequence corresponding to oligo-2 includes two overlapping binding sites as indicated in Fig. 5A; namely, an E- box with sequence CACGTG and an overlapping but distinct binding element with sequence CGTGGGAAG. These results were also confirmed using mutant oligonucleotides as the labeled probes (unpublished results). Since this sequence has not been previously described as a transcription factor binding element, it was given a tentative new designation (MT-box).
  • HEK cells (correspondingly) were transiently cotransfected with pGL-3 construct containing as insert either the wild-type hTERT core promoter or the mutant core promoter sequences Ml, M3 or M4 as indicated. Cells were cotransfected with the ⁇ - galactosidase expression vector (CMV- ⁇ -gal) for calibration of transfection efficiency.
  • CMV- ⁇ -gal ⁇ - galactosidase expression vector
  • the Ml mutation which interferes with transcription factor binding to the proximal E-box results in enhanced promoter activity (173% compared to the core promoter activity), possibly reflecting augmented binding at the MT element upon interference with binding to the overlapping E-box.
  • mutation M3 which abrogates binding to both elements, yields the lowest level of promoter activity (42% of core promoter activity). Residual promoter activity with the M3 mutation, could represent activity emanating from the distal E-box at -177bp.
  • the M4 mutation which eliminates binding only at the MT element yields promoter activity similar to the wild-type core promoter (89%).
  • telomeres Shortening of chromosome ends (telomeres) is the result of incomplete DNA replication during each S-phase of the cell cycle.
  • preservation of the integrity of chromosome ends, and hence maintenance of genome stability, requires the activity of the ribonucleoprotein enzyme, telomerase.
  • This enzyme is activated in the earliest stages of embryonic development, and then its activity is suppressed during subsequent stages of fetal development. This suppression accompanies the transition of cells from a pluripotent to a differentiated state, during the process of organogenesis. Accordingly, the mechanisms for the suppression of telomerase activity are likely to be of key importance in regulation of tissue differentiation and organogenesis during fetal development.
  • telomere activity is enabled by the impact of ectopically expressing hTERT during differentiation. Selected cell lineages derived from hTERT over-expressing hES cells.
  • pluripotent human embryonic stem cells are capable of differentiating into many cell types, they or their derivatives can be used for research and medical applications, including cellular transplantation.
  • a major objective of this invention is to modulate the differentiation of stem cells including hES cells so that a desired population of committed precursors or fully differentiated cells can be obtained.
  • telomere length and integrity may be utilized.
  • Telomerase in particular may be used to maintain telomere length and integrity and thereby extend the proliferation capacity of the cells. In those instances where ectopic over-expression of hTERT does not adversely influence the differentiation pattern, it is possible to generate a fully differentiated desired lineage of telomerase positive cells.
  • hTERT over- expressing hES cells are allowed to differentiate under specific conditions chosen for selection and enrichment for production of the selected cell lineage that it is desired to obtain. As is commonly known in the art these conditions include the use of varied cell growth factors, growth supplements, antioxidants or any other selected modifications to the culture medium that are known to predispose the cells to commit to a particular cell lineage.
  • the hTERT coding region is constructed to reside in a single cassette with an IRES and hygromycin or other antibiotic resistance selection marker.
  • this vector carries a neomycin (or other antibiotic) selection marker under the control of a constitutive promoter, including for example PGK promoter or another mammalian promoter.
  • the construct is transfected into undifferentiated hES cells and neomycin resistant clones are selected. In the subsequent step, these clones are allowed to differentiate and hygromycin resistant clones are selected. Virtually, all the transfected cells, which express the selection marker, are expected to express telomerase and most likely differentiate into the desired cell lineage that has the ability to activate the specific cell specific promoter at the very early stages of this differentiation pathway.
  • Non-limiting examples of specific cell lineages that can be selected according to the principles and methods of the present invention include, but are not limited to, progenitor cells in the pathway of beta cells, mature beta cells, neuronal cells, cardiomyocytes, and any other cell lineage that it is desired to obtain.
  • Non-limiting examples of cell lineage specific promoters will include: PDX-1, NGN-3 promoters which are active in progenitors of beta islets, or nestin promoter which is active in neuronal cells, insulin promoter for mature beta cells, MHC promoter (alpha-cardiac myosin heavy chain) for producing cardiomyocytes.
  • Quality control of differentiated cells derived as above from hES cells will be carried out using appropriate parameters, including but not limited to: ultrastructural characterization using electron microscope, electrophysiological profile, metabolic profile, or any other suitable parameter for testing the cells selected.
  • hTERT gene coding sequence driven by a powerful promoter such as the ⁇ -actin gene promoter or PGK gene promoter, are stably transfected with a selection marker, to overcome the disadvantage of low proliferation capacity and limited life span. This strategy is to promote proliferation of specific cell lineages that might be useful for cell transplantation.
  • the final clonal population is examined for all the criteria mentioned above. In parallel, this clonal population is examined for telomerase activity, telomere length, extension of life span and lack of tumorigenic properties as outlined above.
  • EMSA EMSA we have identified a novel motif within the hTERT regulatory sequence, which binds a nuclear protein(s) of undifferentiated hES, F9 and HL-60 cells. This binding is reduced upon induction of differentiation.
  • the nuclear protein or proteins which binds to this DNA sequence do not appear to be one of the known transcription factors already evaluated in previous studies of the hTERT promoter (cMyc, AP2, SP1), and may represent another as yet unknown transcription factor complex.
  • cMyc, AP2, SP1 cMyc, AP2, SP1
  • the one-hybrid assay is an in vivo genetic assay used for isolating novel genes encoding proteins that bind to a target, s-acting regulatory element or other short, DNA binding sequences.
  • To conduct the one-hybrid assay we insert the specific oligonucleotides already identified using EMSA, in tandem copies upstream of the HIS3 and LacZ reporter gene promoters (presented on separate vectors). Subsequently, the reporters are integrated into the yeast genome to create two new yeast reporter strains that are transformed with a library of hybrid proteins consisting of the GAL4 activation domain and protein of interest.
  • the Lambda gtl 1 cDNA expression library is able to express high level of desired products as proteins. Cloning into the lacZ gene can result in the expression of foreign DNA as part of the ⁇ -galactosidase fusion protein. Taking advantage of this feature, we screen the library with labeled oligo-1 that includes the overlapping E-box and MT-box binding sites and with oligo-1 /Ml that includes only a proper MT-box binding site.
  • the origin of the cDNA expression library is chosen following extraction of nuclear extract from a parallel organ, and examination for the presence of MT binding protein in the tissue, using EMSA.
  • Affinity chromatography The purification of the proteins that bind to the oligonucleotides utilizes by standard DNA affinity chromatography methodologies. We are attempting to purify the protein by using the same buffers and large quantities of ES cell nuclear extracts as used for the EMSA. If needed, we partially purify the protein before applying it to a DNA affinity column by using procedures such as ammonium sulfate or PVP precipitation, followed by gel filtration on an S-300 column or by heparin agarose chromatography.
  • Biotin/Streptavidin Affinity System Oligo-1 oligonucleotide is labeled with biotin and is used as a probe in DNA-protein binding reaction in solution together with nuclear extracts from hES cells.
  • the biotin groups are used for immobilization of the DNA-protein complex on streptavidin bound magnetic particles that provide a convenient way for efficient isolation and purification of the complex.
  • the complex of magnetic beads-streptavidin-biotin-DNA-protein are subjected to incubation in increasing salt concentration to elute the protein from the complex.
  • this method is in progress using F9 nuclear extract, and results in an MT-binding protein enriched solution. The presence of this protein in the solution was examined using EMSA with ⁇ - 32 P labeled oligo-1. Larger amounts of this protein solution will be further analyzed and separated using FPLC for purification of the MT- binding protein.
  • telomerase catalytic subunit hTERT organization of the gene and characterization of the promoter.
  • Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation. Oncogene 18, 1219-1225.
  • telomere catalytic subunit by a gene on human chromosome 3 that induces cellular senescence. Mol. Carcinog. 22, 65-72.
  • telomerase reverse transcriptase gene hTERT
  • Nuc. Acid. Res. 28, 669-677 Liu, K., Schoonmaker, M.M., Levine, B.L., June, C.H., Hodes, R.J., and Weng, N.P. (1999).
  • telomere catalytic subunit gene the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization.
  • telomerase catalytic subunit hTERT
  • telomere reverse transcriptase hTERT
  • telomeres extends the lifespan of immortal x normal cell hybrids.

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Abstract

L'invention concerne une nouvelle séquence de nucléotide pour des facteurs de liaison, en particulier un élément de liaison de facteur de transcription. L'invention concerne également l'utilisation de cette séquence de nucléotide dans la régulation de l'expression de la télomérase, en contrôlant le développement des souches cellulaires à partir des cellules souches embryonnaires et dans l'isolation de facteurs de transcription spécifiques qui se lient à cet élément.
PCT/IL2001/000517 2001-06-05 2001-06-05 Element de liaison de facteur de transcription WO2002099131A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072787A2 (fr) * 2001-03-13 2002-09-19 Sierra Sciences, Inc. Répresseurs d'expression de la télomérase et leurs procédés d'utilisation

Citations (1)

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US5972605A (en) * 1994-07-07 1999-10-26 Geron Corporation Assays for regulators of mammalian telomerase expression

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5972605A (en) * 1994-07-07 1999-10-26 Geron Corporation Assays for regulators of mammalian telomerase expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TZUKERMAN ET AL.: "Identification of a novel transcription factor binding element involved in the regulation by differentiation of the human telomerase (hTERT) promoter", MOLECULAR BIOLOGY OF THE CELL, vol. 11, December 2000 (2000-12-01), pages 4381 - 4391, XP002906990 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002072787A2 (fr) * 2001-03-13 2002-09-19 Sierra Sciences, Inc. Répresseurs d'expression de la télomérase et leurs procédés d'utilisation
WO2002072787A3 (fr) * 2001-03-13 2005-05-06 Sierra Sciences Inc Répresseurs d'expression de la télomérase et leurs procédés d'utilisation

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