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Molecular and Cellular Biology, February 2003, p. 831-841, Vol. 23, No. 3
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.3.831-841.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Genomics Institute of the Novartis Research Foundation, San Diego, California 92121
Received 24 June 2002/ Returned for modification 1 August 2002/ Accepted 10 October 2002
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ABSTRACT |
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INTRODUCTION |
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The antiviral activities of CDKIs were first observed in studies describing their interaction with positive transcription elongation factor b (P-TEFb), a factor required for human immunodeficiency virus (HIV) replication (32, 54). P-TEFb, composed of CDK9 and a cyclin subunit derived from one of three different genes (cyclin T1, T2, or K), controls RNA polymerase II-dependent transcription elongation by phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (41). Most importantly, P-TEFb is a cellular cofactor required for Tat-dependent transactivation of the HIV genome (54). One study used a combination of in vitro and in vivo assays to screen more than 100,000 compounds for inhibitors of HIV Tat transactivation (32). All the compounds identified, including 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB), were found to inhibit P-TEFb (32). These results suggest that CDK9 represents a potential target for the development of novel HIV therapeutics. Flavo has been recently identified as the most potent P-TEFb inhibitor (6, 7). Infection of cultured cells by HIV has been shown to be potently inhibited by Flavo (50% inhibitory concentration [IC50] = 8 nM) without significant toxic effects (6). Surprisingly, at 30 and 100 nM Flavo (the conditions which completely block HIV Tat transactivation and replication), negligible effects on cellular transcription were observed (7). It is possible that specific sets of cellular genes are inhibited by low intracellular concentrations of Flavo and that some of these genes might be involved in HIV replication. If such hypersensitive genes do exist, they could represent ideal targets for development of novel anti-HIV therapeutics. Thus far, no such cellular genes have been identified. Other CDKIs, including purvalanol A (Purv), roscovitine (Ros), and olomoucine (Olo), have also been shown elsewhere to block HIV basal and Tat-activated transcription, but much less potently than Flavo (53).
Ros and Olo were also shown previously to block herpes simplex virus (HSV) replication and transcription (45, 46). It has been shown previously that Ros and Olo inhibit human CDK7 and CDK9 effectively (IC50 = 0.6 µM) (20, 53). These results again raise the possibility that Ros and Olo block HSV by blocking transcription of specific cellular genes. It is important to determine if such CDKI-hypersensitive genes indeed exist and to examine the possible involvement of these cellular targets in viral transcription and replication. Furthermore, it should be very interesting to determine if CDKIs could be utilized as broad-spectrum antiviral reagents by examining their effects on other viruses.
In this paper, we examine the effects of CDKIs on murine leukemia virus (MLV), which is implicated in oncogenesis and used in the development of gene therapy strategies (19, 52). We found that three CDKIs, including Flavo, Purv, and methoxy-roscovitine (MeO-Ros), were able to block Moloney MLV transcription. We observed that both viral and cellular transcription were inhibited by CDKIs. Cellular genes whose expression is dramatically inhibited by CDKIs were identified by microarray technology. Since our goal was to identify cellular genes affecting viral transcription, we further investigated the subset of CDKI-affected cellular genes involved in transcription regulation. One cellular homeodomain gene, the pre-B-cell leukemia transcription factor 1 (Pbx1) gene, was identified as an important regulator of MLV transcription. We found a PBX1 binding sequence, PBX consensus element (PCE), TGATTGAC, that was perfectly conserved in the U5 region of the Moloney MLV long terminal repeat (LTR) and 14 other murine retroviruses. The PCE was first identified by Knoepfler and Kamps (25). Previous work has demonstrated that PBX1 binds the PCE as a heterodimer with other homeodomain factors such as MEIS1 or PREP1 (5, 25). Selection studies with degenerate oligonucleotides have shown that PBX1 binds to the first half of the PCE (i.e., TGAT) and that MEIS1 or PREP1 contacts the second half (i.e., TGAC) (24). Gel shift assays demonstrated the interaction between the PCE and PBX1-PREP1 proteins, whereas mutations in the PCE resulted in complete inhibition of viral transcription. In addition, overexpression of Pbx1 and Prep1 increased MLV transcription and partially eliminated the inhibitory effects of CDKIs. Furthermore, we demonstrated that transfection of antisense oligonucleotides and short interfering RNAs (siRNAs) directed against Pbx1 resulted in a significant inhibition of MLV transcription. We conclude that the homeodomain proteins PBX1 and PREP1 are involved in the Moloney MLV replication cycle through regulation of viral transcription.
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MATERIALS AND METHODS |
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Plasmids. Two similar MLV-based retroviral vectors were used to measure MLV transcription, MLV-LTR-Luc and WZL-Luc. The first, MLV-LTR-Luc, used exclusively in retroviral infection assays, was derived from pBabe-neo (34), wherein (i) the simian virus 40 promoter was replaced with the internal ribosome entry site (IRES) of hepatitis C virus, (ii) the neomycin resistance gene was changed to the luciferase gene from the pGL2 control plasmid (Promega), and (iii) the U3 region of the 5' LTR was replaced with the strong cytomegalovirus (CMV) promoter for higher-titer virus production. MLV-LTR-Luc was used in the Moloney MLV infection assay to generate viral supernatants (see below). During retroviral replication, the wild-type U3 region is recovered before integration so that measurements of MLV transcription are dependent on wild-type LTR (U3-R-U5)-driven luciferase expression. The second vector, WZL-Luc, used exclusively in transient-transfection assays, was also derived from pBabe-neo (34), wherein (i) the simian virus 40 promoter was replaced with the IRES of encephalomyocarditis virus and (ii) the firefly luciferase gene from the pGL2 control plasmid (Promega) was cloned upstream of the IRES. In the resulting configuration, WZL-Luc, the MLV LTR drives luciferase expression, and the IRES maintains expression of the neomycin resistance gene. Therefore, in both MLV-LTR-Luc, which measures LTR-driven transcription from integrated proviral sequences, and the WZL-Luc vector, which measures LTR-driven transcription from unintegrated, transfected plasmid, luciferase expression is driven by identical MLV LTRs. The mammalian expression vector for human NFAT1 has been described previously (37). CMV-PBX1a and CMV-PBX1b were obtained from Michael L. Cleary. pSV-SPORT-MEIS1 and pSV-SPORT-PREP1, containing the cDNAs for mouse Meis1 and human Prep1, respectively, were provided by F. Bradley Hillgartner. The coding sequences of Meis1 and Prep1 were subcloned into pcDNA6 (Invitrogen) to generate CMV-MEIS1 and CMV-PREP1. The pCITE-PBX1a, pCITE-MEIS1, and pCITE-PREP1 constructs were generated by cloning the coding sequences of Pbx1a, Meis1, and Prep1 into pCITE plasmids (Novagen). The pCITE constructs were used to synthesize translated PBX1a-MEIS1-PREP1 protein in vitro.
Moloney MLV infection assay. The production of MLV supernatants was conducted as previously described (39) with a retroviral producer cell line transfected with MLV-LTR-Luc. 3T3 or 293 cells were grown to 50 to 80% confluence in 96-well plates and incubated with individual CDKIs for 30 min. MLV-LTR-Luc supernatants were then added and incubated for 24 h. Luciferase activity was measured with the Bright-Glo assay system (Promega), and the activity was determined with an Acquest Ultra-HTS system (LJL Biosystems, Inc.). For the overexpression assays, 293 cells were transfected with the indicated expression vectors (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT) and a CMV-ß-galactosidase (ß-Gal) internal control plasmid for 24 h with Fugene 6 reagent as described in the manufacturer's manual (Roche). MLV-LTR-Luc supernatants were then added and incubated with cells for another 48 h. Luciferase and ß-Gal activities were measured with the dual-light system (Applied Biosystems). ß-Gal activity was used to normalize luciferase activity to account for differences in transfection efficiency. The IC50s were determined with the GraphPad Prism program (GraphPad Software, Inc.).
Cell proliferation assay. 3T3 cells were incubated with CDKIs at indicated concentrations for 24 h. Effects of the CDKIs on proliferation were determined with the cell proliferation enzyme-linked immunosorbent assay system, version 2, based on the measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells (Amersham Pharmacia Biotech, Inc.).
Moloney MLV transcription assay. Two different approaches were utilized to examine the effects of CDKIs on MLV transcription. 3T3 or 293 cells were grown to 50 to 80% confluence in 96-well plates and transfected with WZL-Luc vector DNA with Fugene 6 or Lipofectamine Plus reagent as described in the manufacturer's manual (Invitrogen). Three hours posttransfection, CDKIs were added at the indicated concentrations for another 18 to 24 h. Effects of CDKIs on unintegrated MLV LTR-driven transcription were measured by assaying luciferase activity. Another approach with cells stably expressing integrated MLV LTR proviral sequences was used to measure viral transcription. 293 cells were incubated with MLV supernatants (MLV-LTR-Luc, as described previously) for 18 h to allow viral entry and integration. The medium was removed, and the cells were washed with phosphate-buffered saline. The MLV-LTR-Luc-integrated 293 cells were treated with CDKIs for 24 h followed by luciferase activity measurements. For the overexpression assays, 293 cells were cotransfected with the WZL-Luc retroviral vector, the indicated expression vectors (i.e., CMV-PBX1a, -PBX1b, -MEIS1, -PREP1, or -NFAT), and a CMV-ß-Gal internal control plasmid for 24 h. Detection of ß-Gal and luciferase activity was performed as described above.
Microarray procedure. Microarray analysis was performed essentially as previously described (31). Briefly, murine NIH 3T3 cells were treated with 10 µM Purv, 10 µM MeO-Ros, 0.3 µM Flavo, or vehicle (DMSO) for 6 h (final concentration of DMSO in the medium, 0.1%) followed by total cellular RNA isolation with the RNeasy Mini kit (Qiagen). Five micrograms of total RNA was used to synthesize cDNA that was then used as a template to generate biotinylated cRNA. cRNA was fragmented and added to Affymetrix (Santa Clara, Calif.) MGU74Av2 chips (containing probes for over 6,000 mouse genes) as described in the standard protocol outlined in the gene chip expression analysis technical manual (Affymetrix). After sample hybridization, microarrays were washed and scanned with a laser scanner (Agilent, Palo Alto, Calif.). Each treatment condition was represented with duplicate arrays as total RNA preparation and cDNA and cRNA synthesis, and array hybridizations were performed in parallel from duplicate drug treatments.
Microarray data analysis. Regulated probe sets were chosen from Affymetrix MAS 4.0 output files by filtering for differences in the average difference values for each CDKI and vehicle (DMSO). Filtering was done by two methods. The first method involved using Student's t test for each probe set with the average difference values of the duplicates. Probe sets with differences between drug and vehicle were chosen if P values were less than 0.01, the fold change in expression was at least an absolute value of 2.0, and the average difference values were at least a value of 200 from at least two of the four chips. The second filter also selects for probe sets with a fold change of at least 2.0 and average difference values of at least 200 from at least two of the four chips but also requires that the standard deviation of the duplicate values divided by the mean of the duplicate values be no more than 10% for either condition. Results from both filtering methods were combined, and comparisons between the different CDKIs were made.
Quantitative RT-PCR. Oligonucleotide probe and primers for Pbx1 (the probe, forward, and reverse primers corresponded to regions 565 to 589, 532 to 552, and 604 to 623, respectively, after the start codon of Pbx1; accession no. NM_002585) were purchased from Applied Biosystems. Reverse transcription-PCR (RT-PCR) was done with the TaqMan One-Step RT-PCR Master Mix reagents kit as described by the manufacturer (Applied Biosystems). Amplification and detection of specific products were performed with the ABI Prism 7700 sequence detection system (Applied Biosystems).
Western blots.
3T3 cells were treated with 10 µM Purv, 10 µM MeO-Ros, 0.3 µM Flavo, or vehicle (DMSO) for 6 h. Cells were lysed in a solution containing 10 mM Tris (pH 7.6), 150 mM NaCl, 2 mM MgCl2, 1% Triton X-100, 15% glycerol, 0.1% of a saturated solution of phenylmethanesulfonyl fluoride in isopropanol, and protease inhibitor cocktail tablets (Roche). The cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immobilized on nitrocellulose filters. PBX1 proteins were detected by Western blot analysis and quantitated by the NIH Java-based image processing program (NIH Image/J). The polyclonal antibodies against PBX1a-PBX1b and against 
-tubulin were obtained from Santa Cruz Biotechnology, Inc. A monoclonal antibody against the PBX1b isoform was kindly provided by M. L. Cleary (47).
Gel mobility shift assay.
The U5 region oligonucleotides, which contained wild-type PCE (5'-GGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGG [sequences of PCE are underlined]), mutant PCE 1mt (5'-GGGAGGGTCTCCTCTGAGCCCCTGACTACCCGTCAGCGGG [sequences of PCE mutant are underlined]), or mutant PCE 2mt (5'-GGGTCTCCTCTGAGTGATCCCCTACCCGTCAGCGGGGGTC), were used as the DNA probes. Double-stranded oligonucleotides were prepared by combining equal amounts of complementary single-stranded DNAs in a solution containing 10 mM Tris (pH 8.0) and 1 mM EDTA followed by heating to 95°C for 2 min and then cooling to room temperature. DNA binding reactions and 32P labeling of oligonucleotides were performed with gel shift assay systems (Promega). Anti-PBX1, anti-MEIS1 (Santa Cruz Biotechnology, Inc.), anti-PREP1 (Santa Cruz Biotechnology, Inc.), or anti--tubulin antibodies were incubated with nuclear extracts of 293 cells for 10 min before the 32P-labeled probe was included. PBX1a, MEIS1, and PREP1 proteins were produced in vitro from T7 expression plasmids (i.e., pCITE plasmids) with a coupled reticulocyte lysate system (TNT Quick Coupled transcription-translation systems; Promega). DNA binding reactions were performed at 4°C for 30 min when in vitro-translated proteins were used. DNA and DNA-protein complexes were resolved on 6% nondenaturing polyacrylamide gels at room temperature in 0.3x TBE (27 mM Tris-borate [pH 8.3], 0.6 mM EDTA). Following electrophoresis, the gels were dried and exposed to X-ray film.
PCE mutants. To generate mutations in the PCE of the Moloney MLV LTR, site-directed mutagenesis was performed according to the Stratagene manual (QuikChange site-directed mutagenesis kit) with a WZL plasmid as the template. The primers used to create the PCE mutants are as follows: PCE 1mt (5'-GGGAGGGTCTCCTCTGAGCCCCTGACTACCCGTCAGCGGG and 5'-CCCGCTGACGGGTAGTCAGGGGCTCAGAGGAGACCCTCCC), PCE 2mt (5'-GGGTCTCCTCTGAGTGATCCCCTACCCGTCAGCGGGGGTC and 5'-GACCCCCGCTGACGGGTAGGGGATCACTCAGAGGAGACCC), and PCE 12mt (5'-GGGAGGGTCTCCTCTGAGCCCCCCCCTACCCGTCAGCGGGGGTC and 5'-GACCCCCGCTGACGGGTAGGGGGGGGCTCAGAGGAGACCCTCCC). Luciferase sequences (obtained from the BamHI and SalI digestions of WZL-Luc) were cloned into the PCE-mutated WZL plasmids (i.e., PCE 1mt, PCE 2mt, and PCE 12mt). Mutations were confirmed by DNA sequencing. 293 cells were transiently transfected with wild-type or mutant WZL-Luc, and luciferase activity was determined 48 h posttransfection.
Antisense oligonucleotides and siRNAs. Two sets of sense and antisense oligonucleotides were utilized to inhibit PBX1 protein synthesis: 61Pbx (sense) and as61Pbx (antisense) corresponded to region 61 to 80 after the start codon (accession number NM_002585) and 421Pbx and as421Pbx targeted positions 421 to 440 from the start codon. The sense oligonucleotides served as controls. In addition, a pcDNA3.1 reverse primer (5'-TAGAAGGCACAGTCGAGG) was used as another control. 293 cells were cotransfected with 0.035 µg of WZL-Luc and 250 nM oligonucleotides. Luciferase activity was measured 24 h after transfection. An siRNA targeting positions 220 to 243 after the start codon of Pbx1 was also generated (Dharmacon Research, Inc.). The siRNA of Renilla luciferase was used as a control (14). 293 cells were transfected with 0.02 µg of siRNAs for 24 h. Ten microliters of MLV-LTR-Luc viral supernatants was added to the cells and incubated for another 24 h. Luciferase activity was measured as described previously.
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RESULTS |
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300 nM and when concentrations of Purv and MeO-Ros were 10 µM (data not shown).
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CDKIs inhibit transcription of Moloney MLV. CDK7 and CDK9 are essential transcription factors involved in transcription initiation and elongation, respectively. Flavo was previously identified as the most potent CDK9 inhibitor, which associates with CDK9 at a 1:1 stoichiometric ratio even at enzyme concentrations in the low-nanomolar range (6, 7). In addition, it has been shown previously that Ros blocks CDK7 and CDK9 effectively (32, 53). These observations led us to examine if viral transcription driven by the Moloney MLV LTR could be blocked by the CDKIs. The results of the MLV infection assay (Fig. 2) show that the CDKIs inhibit MLV infection, resulting in decreased levels of luciferase activity. However, the MLV infection assay cannot distinguish between inhibition of events leading up to proviral integration into host chromatin and inhibition of retroviral LTR-driven transcription. To determine if the CDKIs might inhibit the latter, two different experimental approaches were utilized. First, murine NIH 3T3 cells were transiently transfected with a WZL-Luc retroviral construct that contains luciferase coding sequences under the control of the Moloney MLV LTR. A decrease in activity of the reporter gene was detected in response to treatment with all three CDKIs (Fig. 3A). The IC50s for inhibition of viral transcription by the CDKIs (Flavo IC50 = 0.11 µM, Purv IC50 = 2.1 µM, and MeO-Ros IC50 = 6.8 µM) closely approximated the IC50s affecting viral infection (Fig. 2 and 3A). Identical experiments were performed with 293 cells, and similar IC50s were obtained (data not shown). Another set of experiments was performed to determine if the CDKIs could inhibit MLV LTR-driven transcription from an integrated provirus. Cells containing stably integrated MLV-LTR-Luc proviruses were utilized. As shown in Fig. 3B, all three CDKIs blocked transcription with similar inhibitory efficiencies as those determined for Fig. 3A. The results suggest that the CDKIs specifically block transcription of Moloney MLV, and not necessarily the processes upstream of viral integration.
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Utilizing microarray analysis, we attempted to identify cellular genes selectively deregulated by CDKIs. Briefly, 3T3 cells were treated with 10 µM Purv, 10 µM MeO-Ros, and 0.3 µM Flavo and then total RNAs were isolated for analysis. Under these conditions, 76 to 99% of MLV transcription was blocked whereas 56 to 92% of the cells were still replicating without significant cellular toxicity (Fig. 2 and data not shown). The microarray data were analyzed as described in Materials and Methods. In agreement with these observations, the results showed that the majority of cellular transcripts were not significantly affected (85 to 89% of cellular RNAs had less than twofold changes; data not shown). We found that 261 cellular genes were significantly affected by Purv, MeO-Ros, and Flavo. Fifty-seven out of 261 genes were affected by two or three CDKIs and were classified as putative MLV regulatory genes. These candidates include 25 known genes (Table 1) and 32 expression sequences-RIKEN cDNAs (data not shown). Since our goal in this study was to identify cellular genes affecting viral transcription, we focused only on the candidate genes involved in regulation of transcription.
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DISCUSSION |
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However, in the treatment of retroviruses, a window does exist where the CDKIs block HIV-MLV very effectively with no significant effects on cellular proliferation or transcription (Fig. 2) (6, 7). It has been hypothesized that CDKIs block viral replication through the inhibition of specific cellular genes. In this paper, we test this idea by utilizing a microarray approach to identify cellular targets of Flavo, Purv, and MeO-Ros and to further investigate the possible involvement of these cellular genes in MLV transcription. Since CDKIs have broad effects on cellular transcription, it is likely that most cellular genes are nonspecifically inhibited by the CDKIs and not involved in MLV transcriptional regulation. To identify cellular cofactors required for MLV transcription, we focused only on genes that were dramatically affected by two or three CDKIs. Although each CDKI exhibits specificity in blocking specific CDKs, different CDKIs might utilize similar mechanisms to block MLV transcription. Based on this assumption, the genes commonly inhibited by CDKIs could represent candidate genes involved in MLV transcription. Using this simple strategy, we were able to reduce the number of putative cellular genes potentially targeted by CDKIs. Identification of PBX1 as a cellular cofactor for MLV transcription suggests that this approach indeed works. The strategy should be useful in identifying cellular targets involved in HIV, HSV, and cancers that are blocked by CDKIs, which could represent novel drug targets (6, 7, 45, 49).
There are at least three different mechanisms utilized by the CDKIs in the inhibition of MLV: (i) induction of cell arrest by blocking the CDKs controlling the cell cycle, (ii) inhibition of viral transcription through direct inhibition of CDK7-CDK9, and (iii) indirect down-regulation of the cellular gene PBX1 through the inhibition of CDK7-CDK9. Our results demonstrate that PBX1 binds to the PCE present in the U5 region of Moloney MLV LTR (Fig. 5B) and functions as a cellular regulator for MLV transcription, as supported by overexpression and gene knockdown experiments (Fig. 7 and 8). Three candidate genes (those for serine/threonine protein phosphatase PP1- catalytic subunit, serine/threonine-protein kinase PLK, and prohibitin) identified by microarray are required for cell division (8, 38, 43), which could help to explain how CDKIs induce cell cycle arrest. In addition to CDKs and PBX1, there might be more cellular genes involved in MLV transcription. HMG I-C, for example, is involved in HIV and MLV integration in vitro (28), and its gene is one of the candidate genes identified in this study. There are three members in the high-mobility group protein family: HMG I, HMG Y, and HMG I-C (3). It has been shown previously that HMG I modulates the binding of transcription factors to the LTR of HIV (22). It is possible that HMG I-C also functions as a regulator of HIV-MLV transcription. Furthermore, it is likely that both murine and human retroviruses require the same cellular proteins during viral replication. It should be interesting to determine the possible involvement of the other MLV candidate genes in HIV infection.
The LTR of MLV is divided into three regions termed U3, R, and U5 regions. The U3 region contains the viral promoter and transcriptional enhancers. It has been shown previously that both the pathogenic potential and disease specificity of MLV are dependent on the enhancer sequences in the U3 regions (11, 12, 27, 29). Several binding sites for cellular proteins and transcription factors have been identified, including a binding site for leukemia virus factor b, a viral core-like element, the consensus motif for nuclear factor 1, and the glucocorticoid response element (15). The R region is used by reverse transcriptase to mediate translocation of a DNA intermediate during viral DNA synthesis (30). In addition, the transcriptional initiation and postinitiation of MLV are also regulated by the R region (9, 10). On the other hand, our understanding of the functions mapping to the U5 region remains incomplete. Previous studies suggest that the U5 region might be involved in RNA packaging and reverse transcription (35). In addition, it has been reported previously that no enhancer is identified in the U5 region by computer-assisted sequence analysis (40).
The discovery of the involvement of homeodomain proteins PBX1 and PREP1 in MLV transcription is novel. The PBX1 binding motif is the first enhancer identified in the U5 region of MLV. The strong sequence conservation of U5 region compared with the U3 region suggests that the PCE and PCE-associated proteins might be key regulators in retroviral transcription. Gel shift assays confirmed the association between the PCE and PBX1-PREP1 proteins (Fig. 5B and C). No association between the PCE and MEIS1 (or PBX1-MEIS1) proteins was detected in gel shift assays (Fig. 5B and C). However, overexpression of MEIS1 proteins indeed resulted in increased MLV transcription (Fig. 7A). It has been shown previously that the Drosophila Meis homolog, Hth, induces nuclear translocation of PBX1 proteins (1). It is possible that overexpression of MEIS1 causes more PBX1 proteins to be translocated into the nucleus and therefore induces MLV transcription. Mutations in the PCE indicate that the MEIS1-PREP1 tetramer (TGAC) is more important than the PBX1 tetramer (TGAT) in regulating viral transcription (Fig. 6C, PCE 1mt versus PCE 2mt). It has been shown previously that PBX1 interacts and binds DNA with HOX proteins to form trimeric protein complexes (PBX1-MEIS1-HOX or PBX1-PREP1-HOX) (2, 51). One study showed that PBX1 could function as a non-DNA-binding partner in the trimeric complexes (50). Future work will be required to investigate the association between PBX1-PREP1 complexes and the U5 region of the LTR in detail and the possible requirement of HOX proteins in MLV transcription.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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