Open Access

Selective inhibition of c-Myb DNA-binding by RNA polymers

BMC Biochemistry20045:15

DOI: 10.1186/1471-2091-5-15

Received: 23 February 2004

Accepted: 04 November 2004

Published: 04 November 2004

Abstract

Background

The transcription factor c-Myb is expressed in hematopoietic progenitor cells and other rapidly proliferating tissues, regulating genes important for proliferation, differentiation and survival. The DNA-binding domain (DBD) of c-Myb contains three tandemly arranged imperfect repeats, designated Myb domain R1, R2 and R3. The three-dimensional structure of the DBD shows that only the second and third Myb domains are directly involved in sequence-specific DNA-binding, while the R1 repeat does not contact DNA and only marginally affects DNA-binding properties. No structural information is available on the N-terminal 30 residues. Since deletion of the N-terminal region including R1 plays an important role in oncogenic activation of c-Myb, we asked whether this region confers properties beyond DNA-binding to the neighbouring c-Myb DBD.

Results

Analysis of a putative RNA-binding function of c-Myb DBD revealed that poly(G) preferentially inhibited c-Myb DNA-binding. A strong sequence-selectivity was observed when different RNA polymers were compared. Most interesting, the poly(G) sensitivity was significantly larger for a protein containing the N-terminus and the R1-repeat than for the minimal DNA-binding domain.

Conclusion

Preferential inhibition of c-Myb DNA binding by poly(G) RNA suggests that c-Myb is able to interact with RNA in a sequence-selective manner. While R2 and R3, but not R1, are necessary for DNA-binding, R1 seems to have a distinct role in enhancing the RNA-sensitivity of c-Myb.

Background

The transcription factor c-Myb is regulating genes involved in proliferation and differentiation during hematopoiesis in vertebrates (reviewed in [1, 2]). c-Myb is expressed at high levels in hematopoietic progenitor cells, but becomes down-regulated when the cells reach terminal differentiation. The critical role of c-Myb in the development of hematopoietic cells is emphasized by the embryonic lethality observed in mice with a c-myb null mutation, caused by failure of fetal hepatic hematopoiesis [3]. c-Myb expression has also been detected in other rapidly proliferating tissues such as hair follicles and immature epithelial cells from colon, respiratory tract, skin and retina [4, 5]. c-Myb is essential for early T cell development [6] and several c-Myb target genes play an important role during T cell development, like CD4, TCRγ, TCRδ and RAG-2 [1, 7]. The best characterized c-Myb target gene is chicken mim-1, which is encoding a secretable component of granules found in normal promyelocytes [8, 9].

The Myb family of proteins is defined by the presence of a well-conserved DNA-binding domain (DBD) composed of Myb repeats [10]. Each repeat consists of about 50 amino acids with three regularly spaced tryptophans forming a hydrophobic core [1113]. The c-Myb DBD contains three tandem imperfect Myb repeats (R1, R2 and R3), located in the N-terminus of the protein. Determination of the structure of the c-Myb DBD has revealed that each repeat folds into three well-defined helices forming a helix-turn-helix-related structural motif, where the last helix in R2 and R3 makes specific DNA contacts (recognition helices) [1315]. The c-Myb protein harbours two functional domains in addition to the DBD: a central activation domain and a C-terminal negative regulatory domain. The viral counterpart of the chicken c-myb gene, v-myb, found in the AMV and E26 viruses, has deletions in both ends, leading to a v-Myb protein lacking the N-terminus, most of the first Myb repeat (R1) and a large part of the C-terminal negative regulatory domain (reviewed in [16]).

The c-Myb DBD binds specifically to the sequence PyAAC(T/G)G, termed the Myb recognition element (MRE) [1719]. The minimal sequence-specific DBD consists of the two carboxy-terminal Myb repeats, R2R3 [20, 21]. The role of the first Myb repeat, R1, is not fully understood. It has been shown to be dispensable for specific DNA-binding [20, 22], but bears striking similarities to the other repeats regarding sequence and structure [23]. Some groups have reported that R1 stabilizes the protein-DNA complex [24, 25] and it has been proposed to allow for more flexibility in the downstream region of the Myb recognition sequence [26]. According to the recent three-dimensional structure of the c-Myb DBD, the R1 repeat does not contact DNA directly [15]. However, a long-distance electrostatic interaction is suggested to stabilize the protein-DNA complex. Its free position in the complex makes it possible that it could be involved in other functions as well. This possibility is supported by the fact that v-myb-like truncation of the N-terminus of c-Myb (until the end of R1) has been shown to be sufficient for oncogenic transformation of chicken bone marrow cells [27]. The R1 repeat could serve as a special regulatory module, either acting as a target for molecular interactions or being subject to post-translational modifications.

In the present work we address whether the N-terminal region including the R1 repeat confers properties beyond DNA-binding to c-Myb DBD. We sought evidence for a putative RNA-binding function, analogous with several other transcription factors harbouring dual DNA-RNA binding properties. The motivation for investigating RNA-interaction was the design of the c-Myb DBD, built of repeating modules, a design resembling the structural logic of zinc finger proteins. Some well-studied zinc fingers have been found to use subsets of the repeats as RNA- or DNA-binding units. The classical example is the Xenopus zinc finger protein TFIIIA, which acts as a DNA-binding activator of 5S ribosomal RNA genes [28, 29]. TFIIIAalso forms a stable complex with 5S rRNA in Xenopus oocytes [30, 31]. The nine zinc fingers of TFIIIA contribute differentially to DNA- and RNA-binding; the N-terminal triplet (1–3) dominating the DNA recognition event while the middle triplet (4–6) is more important for RNA-binding [32, 33]. Another example of a zinc finger with similar dual properties is the Wilms tumor suppressor gene 1 (WT1) [3436]. One splice variant (the +KTS isoform) seems to be a better RNA-interacting form that co-localizes with splicing proteins in nuclear speckles, whereas another variant (the -KTS isoform) interacts stronger with DNA and co-localizes with transcription factors [34, 35].

The capability of specific interaction with both DNA and RNA is not restricted to the zinc finger family of proteins. The homeodomain protein bicoid of Drosophila acts both as a transcription factor, activating zygotic segmentation genes during blastoderm formation, and as a regulator of mRNA translation by binding to the mRNA of another homeodomain transcription factor, caudal [37, 38]. Interestingly, the homeodomain bears structural similarities to the Myb domain, especially with respect to the presence of a helix-turn-helix-related motif [12, 13, 15, 21]. A final example is p53, which has been reported to bind to both single-stranded DNA and RNA in addition to its established role as a sequence-specific DNA-binding protein (reviewed in [39]). Murine p53 and human Cdk4 translation have actually been shown to be regulated by p53 mRNA binding.

Based on these occurrences of dual nucleic acid interactions, we asked whether a similar design was found in c-Myb. In particular, we raised the question whether the N-terminal region including R1 might be implicated in RNA-binding rather than DNA-binding. As a test of our proposed RNA-binding function of c-Myb, we investigated the effect of different homoribopolymers on c-Myb DNA-binding. The homoribopolymer polyguanylic acid (poly(G)) strongly inhibited the sequence-specific DNA-binding of c-Myb, while poly(A), poly(C) and poly(U) did not, indicative of an RNA-binding activity. The same phenomenon, although weaker, was observed for A- and B-Myb. An order-of-addition experiment indicated that poly(G) bound directly to c-Myb in competition with DNA. Interestingly, the DBD construct containing the N-terminus and R1 was significantly more sensitive to poly(G) than the minimal DBD. Thus, the N-terminus including R1 seems to be important for the RNA-sensitivity of c-Myb DBD.

Results

Differential binding to homoribopolymers has been used as evidence for RNA-binding activity of various proteins [4045]. To investigate whether c-Myb and its R1 repeat is involved in RNA-binding we first examined the effects of homoribopolymers on c-Myb DNA-binding. The sequence-specific DNA-binding was analyzed by the electrophoretic mobility shift assay (EMSA) using two purified recombinant human c-Myb protein domains, NR1R2R3 (amino acid 1–192) and R2R3 (amino acid 89–192), in mixture. The assay was performed in the presence of increasing amounts of the homoribopolymers poly(A), poly(C), poly(G) and poly(U) (Fig. 1A). The DNA-binding of both proteins was strongly inhibited by poly(G) but not by the other polymers. NR1R2R3 was most sensitive, being inhibited when as little as 1 ng poly(G) was added. We also tested two artificial RNAs, poly(I*C) and poly(I). The former is a duplex RNA, and the latter is a variant of poly(G) less prone to forming unusual structures, but retaining some of the pairing properties of guanosines. The duplex did not affect DNA-binding, while poly(I) caused some inhibition at high concentrations (Fig. 1B). To confirm that the observed inhibition by poly(G) was indeed due to the added RNA, we carried out the poly(G) inhibition experiment in the presence and absence of RNase T1, an endoribonuclease that specifically cuts RNA at the 3'-end of guanosine residues. As shown in Fig. 2, DNA-binding was no longer inhibited by poly(G) after RNase T1 treatment, confirming the RNA-dependence of the inhibition.
Figure 1

Inhibition of c-Myb DNA-binding by homoribopolymers. Panel A: NR1R2R3 (40 fmol) and R2R3 (20 fmol) in mixture were incubated with 30 fmol MRE-containing DNA probe and 1 ng (lane 4,7,10 and 13), 10 ng (lane 5, 8, 11 and 14) and 100 ng (lane 6, 9, 12 and 15) of homoribopolymers poly(A) (lane 4–6), poly(C) (lane 7–9), poly(G) (lane 10–12) and poly(U) (lane 13–15). The binding reactions were incubated for 15 minutes at 25°C and subsequently analyzed by EMSA and phosphorimaging. Lane 1, 2 and 3 show binding reactions without homoribopolymer addition, with NR1R2R3 alone in lane 1, R2R3 in lane 2 and the mixture of both in lane 3. Panel B: NR1R2R3 and R2R3 were combined in a binding reaction as in panel A, but now in the presence of 1 ng, 10 ng or 100 ng of the ribopolymers poly(G) (lanes 3–5), poly(I) (lanes 6–8), poly(I-C) (lanes 9–11) and 100 ng poly(A) (lane 12), respectively. Lane 1 shows free probe, whereas lane 2 shows binding reaction without ribopolymer addition.

Figure 2

RNase treatment relieves poly(G)-mediated inhibition of c-Myb DNA-binding. NR1R2R3 and R2R3 were incubated with the MRE-containing DNA probe in the presence of 20 ng poly(G) homoribopolymer (lanes 3 and 4). The sample shown in lane 4 was in addition incubated with 2000 U RNase T1. To allow for RNase T1 mediated degradation of poly(G), all samples were incubated for 30 minutes at 37°C prior to addition of Myb-protein mixture. Binding reactions were subsequently incubated for 15 minutes at 25°C and analyzed by EMSA and phosphorimaging. Lanes 1 and 2 show free probe and binding reaction without homoribopolymer addition, respectively.

To better compare the sensitivity of the two proteins, we titrated the poly(G) inhibition (Fig. 3). The DNA-binding of NR1R2R3 was significantly more sensitive to poly(G) competition than R2R3, indicating that the N-terminus including the R1 repeat was important for the inhibitory effect.
Figure 3

Titration of the poly(G) inhibition. NR1R2R3 (40 fmol) and R2R3 (20 fmol) were incubated separately with 20 fmol MRE-containing radiolabelled probe and increasing amounts of poly(G). The binding reactions were incubated for 15 minutes at 25°C and subsequently analyzed by EMSA and phosphorimaging. The intensities of the complex bands were quantified by phosphorimaging software and plotted as percentage of the complex band intensity when no homoribopolymers was added.

Different plausible mechanisms for the observed poly(G) inhibition of c-Myb sequence-specific DNA-binding may be operating. Poly(G) might bind to c-Myb in direct competition with DNA. Or, the binding of poly(G) to c-Myb might be allosteric, inducing structural changes in c-Myb that reduces its DNA-affinity. A third explanation might be that poly(G) in a subtle way interacts with the DNA-probe and blocks its specific interaction with c-Myb. To clarify the mechanism of inhibition, we studied the importance of the order of addition of probe and homoribopolymers to the binding reaction (Fig. 4). Three situations were investigated, designated R, S and D in Fig. 4: (R) RNA was mixed with the proteins before addition of probe, (S) RNA and probe were mixed and added simultaneously to the proteins, (D) Probe (DNA) was mixed with protein before addition of RNA. The reasoning was that if the inhibition is allosteric of nature, the magnitude of inhibition should be independent of the order of addition. If probe-interference is the mechanism, the strongest inhibition should be observed with the simultaneous addition of RNA and probe. If a competitive binding of poly(G) to c-Myb is the case, the strongest inhibition should be expected with the addition of RNA to protein first and the weakest inhibition when the probe was added first. The last scenario was in fact what we observed, indicating that c-Myb binds to poly(G) in direct competition with DNA-binding (Fig. 4). No inhibition at all was observed when 100 ng of poly(A) was added. It is noteworthy that the relative sensitivity of the two Myb forms also changed as a function of the order of addition. Pre-incubation with RNA enhanced the difference between NR1R2R3 and R2R3 significantly, the first being fully inhibited while the latter seemed to be almost unaffected. In contrast, pre-incubation with DNA allowed the two forms to bind with similar efficiency. This supports the notion that the N-terminal region including the R1 repeat plays an important role in conferring RNA-sensitivity to the protein.
Figure 4

Order-of-addition analysis. A mixture of 40 fmol NR1R2R3 and 20 fmol R2R3 was incubated with 30 fmol MRE-containing DNA probe and 1 ng poly(G) (lane 4–6), 10 ng poly(G) (lane 7–9) and 100 ng poly(A) (lane 10–12). In the cases marked R (for "RNA first", lane 4, 7 and 10), the proteins were incubated with homoribopolymers for 15 minutes at 25°C before addition of DNA-probe and further incubation for 15 minutes at 25°C. The cases marked S (for ``simultaneous addition'', lane 5, 8 and 11) represents reactions where homoribopolymers and DNA probe were mixed before addition of protein and 15 minutes of incubation. Finally, the cases marked D (for ``DNA first'', lane 6, 9 and 12) show reactions where the protein were incubated with DNA first and then homoribopolymers were added. The reactions in lane 1–3 contained no homoribopolymers, NR1R2R3 protein alone in lane 1 and R2R3 protein alone in lane 2. The binding reactions were subsequently analyzed by EMSA and phosphorimaging.

The homoribopolymer experiments reported above were performed with 15 minutes of incubation at 25°C. To exclude that the poly(G) effect was just a consequence of slower complex formation, we repeated the experiment with longer incubation times up to 90 minutes (results not shown). No change in the inhibition pattern was observed. This argues against the possibility that poly(G) only reduces the rate of c-Myb/DNA complex formation and indicates that the complex with RNA is highly stable.

We then asked whether the poly(G) inhibition was specific for c-Myb or if other Myb proteins exhibited the same properties. Purified NR1R2R3 forms of the vertebrate Myb relatives A- and B-Myb together with c-Myb were analyzed by EMSA in the presence of different concentrations of poly(G) and poly(A). As shown in Fig. 5, the DNA-binding of A- and B-Myb was clearly inhibited by poly(G), although the effect was somewhat weaker than for c-Myb. A-Myb behaved most similar to c-Myb, consistent with the close resemblance between these two transcription factors [46].
Figure 5

Comparison of A-, B- and c-Myb's poly(G) inhibition. A-, B- and c-Myb NR1R2R3 (40 fmol each) were incubated separately with 20 fmol radiolabelled MRE-containing DNA probe, 0.5 ng poly(G) (lane 2),1 ng poly(G) (lane 3), 5 ng poly(G) (lane 4) and 100 ng poly(A) (lane 5). Homoribopolymers and proteins were first incubated for 15 minutes at 25°C. Then the DNA-probe was added to the mixtures and subjected to a new incubation of the same time and temperature. The binding reactions were analyzed by EMSA and autoradiography. The extra incubation step without probe was added to the experimental setup because it enhanced the inhibitory effect (lanes marked R in Fig. 3).

Discussion

The DBD of c-Myb contains three tandemly arranged pseudo-repeats, R1, R2 and R3, among which R2 and R3 together are responsible for sequence-specific DNA-binding. The function of the first repeat, R1, has remained elusive in particular because R1 does not directly contact DNA. Its involvement in the process of oncogenic activation of Myb suggests a specific biological role of R1. In this report we have focused on the N-terminal region including the R1 repeat of c-Myb, trying to find a function beyond DNA-binding. A homoribopolymer inhibition assay revealed a strong preferential inhibition of sequence-specific DNA-binding by poly(G), suggesting that c-Myb can interact with RNA in a sequence-selective fashion. The most interesting observation was the finding that the RNA-interference function of c-Myb was highly dependent on the N-terminal region including R1. Upon exposure to RNA before DNA, the protein domain containing this region was severely inhibited by low concentrations of poly(G) while the protein domain lacking this region was almost unaffected. What the precise biological role of this novel RNA-interaction is, remains to be elucidated.

Central to our examination of the RNA-binding hypothesis is a homoribopolymer inhibition assay. Differential binding to homoribopolymers has been exploited as evidence for RNA-binding activity of several proteins, like the Ets-related transcription factor PU.1 [41], the neuronal KH domain containing protein Nova-1 [43], the chloroplast ribosomal protein CS1 [44], and the recently cloned RRM domain containing Ciona intestinalis protein RGC [45]. Whether homoribopolymer binding reflects a sequence-specific RNA binding or a more general RNA-binding, like in the case of polypyrimidine-tract or poly(A) binding proteins, requires further analysis in each case.

In addition to the data presented above, the SELEX (systematic evolution of ligands by exponential enrichment) technology was applied to search for more sequence-specific RNA patterns recognized by c-Myb. The SELEX procedure did produce specific patterns, confirming the proper behaviour of the experiment, but the selected RNAs did not seem to mimic the homoribopolymer effect in terms of inhibitory efficiency and content of G-bases (results not shown). This does not argue against RNA-binding per se, but indicates that c-Myb may not interact in a strictly sequence-specific fashion with RNA. Rather, we believe that the inhibitory RNA-effect seen in the homoribopolymer experiments reflects an R1 -dependent RNA-interaction where G-rich RNA interacts more avidly than other RNAs. Why G-rich species are so much more potent inhibitors is not obvious. G-rich RNA molecules have special folding capacities that could be recognized by c-Myb. This is illustrated by the fact that poly(G) has been reported to fold into several unique structures, including single, double and four-stranded helices [4749]. To investigate the possibility that poly(dG) had a similar effect on c-Myb DNA binding as poly(G), we added DNA oligonucleotides containing deoxy-guanine stretches of varying length (1 to 5) to EMSA reactions. However, we were not able to correlate the presence of poly(dG) stretches to any inhibitory effects (results not shown). Neither did we observe any strong inhibition when poly(I*C) or poly(I) was added. It is quite probable therefore that unique structural properties of poly(G) are critical to the mechanism of inhibition.

The poly(G) inhibition was surprisingly strong. Due to the undefined length of the homoribopolymers, it was not evident how to precisely determine their molar concentrations. A fictitious poly(G)-length of 23 nucleotides, which is the length of the probe, gives an RNA-concentration of about 6 nM when 1 ng is added to the binding reactions. Consequently, a six-fold estimated molar excess of poly(G) compared to the probe concentration (1 nM) was sufficient to completely abolish sequence-specific DNA-binding (Fig. 3).

We also analyzed the effects of a series of mononucleotides (results not shown). Interestingly, specific nucleotide triphosphates did in fact inhibit DNA-binding of c-Myb with differences resembling the pattern observed with ribopolymers in the present work. GTP produced the most prominent effect among the nucleotides, while CTP or UTP had little or no inhibitory effect. However, since the inhibition was observed first in the mM concentration range, significantly higher than the amount of poly(G) producing the same level of inhibition, the GTP phenomenon seems to be only a weak reflection of the strong inhibition we see with poly(G). Still, it is intriguing that we observe the same specificity suggesting some type of specific interactions between guanosines and the DNA-binding domain of c-Myb.

The reported results represent evidence for an RNA-binding function of c-Myb. The dependence on the N-terminal region including R1 is not total, in the sense that without this region all RNA-interference disappears. Rather, the presence of this region seems to enhance the sensitivity to RNA-mediated inhibition several fold, making it possible to find conditions where a Myb DBD containing this region becomes fully inhibited while a minimal DBD remains more or less unaffected (Fig. 4). At higher concentrations of poly(G) RNA, however, both proteins become inhibited, suggesting that other parts of the DBD are involved too. An appealing hypothesis would be that the flexible second repeat were involved in interactions with both types of macromolecules, cooperating with R1 for RNA-binding and with R3 for DNA-binding. This would explain why this repeat appears to be a more flexible protein domain than the other repeats. It is noteworthy that a DNA-bound protein seems to be resistant to RNA-mediated inhibition and that an RNA-associated protein does not bind DNA even after prolonged incubation. We have previously shown that c-Myb R2R3 undergoes a conformational change upon binding to DNA [14, 50, 51]. It is possible that the DNA-induced conformation is resistant to RNA-interference and that RNA induces another conformation that is unable to bind DNA; in other words that the two nucleic acids lock the protein in two distinct conformations.

It could be argued that we have shown mainly experiments of the RNA-interference type, not directly demonstrating RNA-binding. We have, however, several lines of evidence indicating that RNA-interference occurs through direct binding of c-Myb DBD to RNA. c-Myb NR1 R2 R3 was observed to interact with RNA in a North-Western experiment where R2R3 did not, and c-Myb NR1R2R3 bound to RNA-linked beads (results not shown).

The identification of an increasing number of proteins capable of both DNA- and RNA-binding challenges the established picture of DNA-bound regulators with functions confined to promoter-activation, and suggests a broader function for some transcription factors [39]. The precise physiological role of the novel RNA-binding property of c-Myb remains to be elucidated. An interesting possibility to investigate is whether c-Myb plays a role beyond transcriptional activation in biological processes involving RNA, like splicing, capping, polyadenylation, nuclear export or transport of RNA. A role in one or several of these processes will fit the forthcoming model of a coupling between transcription and the post-transcriptional fate of mRNA [52, 53].

Conclusions

We have obtained evidence that c-Myb DNA-binding is preferentially inhibited by poly(G) RNA, indicative of a sequence-selective RNA binding function. The N-terminus of c-Myb, including the R1 repeat, was shown to contribute substantially to this RNA-sensitivity. This finding suggests a more specific function of the enigmatic first Myb repeat than having a stabilizing effect on DNA-binding only.

Methods

Homoribopolymers

All homoribopolymers were purchased from Sigma Aldrich. Purities were higher than 98% for all polymers. The length distributions of the homoribopolymers were determined by agarose gel electrophoresis and UV-shadowing. When compared to a dsDNA ladder, poly(A), poly(C), poly(G)and poly(U) migrated with a distribution corresponding to the following ranges: 200–1600 bp, 250–1200 bp, 100–700 bp and 250–700 bp, respectively (results not shown). RNase T1 was purchased from Ambion, Inc.

Expression and purification of recombinant proteins

The following DBD subdomains were expressed in E. coli (strain BL21 (DE3) LysS) using the T7 system [54]: Human c-Myb residue 1–192 (NR1R2R3) and 89–192 (R2R3), human A-Myb 1–187 (NR1R2R3) and B-Myb 1–183 (NR1R2R3). Expression and purification were performed as previously described [46].

Electrophoretic mobility shift assay

Sequence-specific DNA-binding was examined by electrophoretic mobility shift assay (EMSA) as previously described [55]. The binding reactions were performed in 20 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 10% glycerol, 0.1 mM DTT, 0.005% Triton X-100 and 50 mM NaCl in a total volume of 20 μl. RNAguard (Amersham Biosciences) RNAse inhibitor (6–20 U) was added to each reaction to avoid degradation of the homoribopolymers.

The sequence of the MRE-containing DNA probe was from the mim-1 promoter: 5'-GCATTATAACGGTTT TTTAGCGC-3'. Double-stranded DNA oligonucleotides were 32P- labelled by T4 polynucleotide kinase according to the specifications of the manufacturer (Ready-To-Go T4 Polynucleotide kinase, Amersham Biosciences). Radiolabelled probe was purified on G-25 MicroSpin columns (Amersham Biosciences) or by polyacrylamide gel electrophoresis with subsequent gel extraction.

Phosphorimaging

EMSA gels were transferred to 3 MM paper and dried for 90 minutes at 80°C in a vacuum gel drier. Radiolabelled bands were detected with Molecular Imaging Screen BI (Bio-Rad) and analyzed with a Bio-Rad GS-250 PhosphorImager. Quantitation of band intensities was performed with the Molecular Analyst 2.0.1 software (Bio-Rad).

Declarations

Acknowledgements

This work was supported by The Norwegian Research Council, Norwegian Cancer Society and Anders Jahres Foundation. We thank Christophe Orvain for performing North-western analysis of RNA-binding, which supported the concept of c-Myb having RNA-binding properties.

Authors’ Affiliations

(1)
Department of Molecular Biosciences, University of Oslo
(2)
Department of Haematology and Oncology, Rogaland Central Hospital

References

  1. Oh IH, Reddy EP: The myb gene family in cell growth, differentiation and apoptosis. Oncogene. 1999, 18: 3017-3033. 10.1038/sj.onc.1202839.View ArticlePubMedGoogle Scholar
  2. Ganter B, Lipsick JS: Myb and oncogenesis. Adv Cancer Res. 1999, 76: 21-60.View ArticlePubMedGoogle Scholar
  3. Mucenski ML, McLain K, Kier AB, Swerdlow SH, Schreiner CM, Miller TA, Pietryga DW, Scott WJJ, Potter SS: A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. 1991, 65: 677-689. 10.1016/0092-8674(91)90099-K.View ArticlePubMedGoogle Scholar
  4. Ess KC, Witte DP, Bascomb CP, Aronow BJ: Diverse developing mouse lineages exhibit high-level c-Myb expression in immature cells and loss of expression upon differentiation. Oncogene. 1999, 18: 1103-1111. 10.1038/sj.onc.1202387.View ArticlePubMedGoogle Scholar
  5. Sitzmann J, Noben-Trauth K, Klempnauer KH: Expression of mouse c-myb during embryonic development. Oncogene. 1995, 11: 2273-2279.PubMedGoogle Scholar
  6. Allen RD, Bender TP, Siu G: c-Myb is essential for early T cell development. Genes Dev. 1999, 13: 1073-1078.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Wang QF, Lauring J, Schlissel MS: c-Myb binds to a sequence in the proximal region of the RAG-2 promoter and is essential for promoter activity in T-lineage cells. Mol Cell Biol. 2000, 20: 9203-9211. 10.1128/MCB.20.24.9203-9211.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Ness SA, Marknell A, Graf T: The v-myb oncogene product binds to and activates the promyelocyte- specific mim-1 gene. Cell. 1989, 59: 1115-1125. 10.1016/0092-8674(89)90767-8.View ArticlePubMedGoogle Scholar
  9. Bischoff KM, Pishko EJ, Genovese KJ, Crippen TL, Holtzapple CK, Stanker LH, Nisbet DJ, Kogut MH: Chicken mim-1 protein, P33, is a heterophil chemotactic factor present in Salmonella enteritidis immune lymphokine. J Food Prot. 2001, 64: 1503-1509.PubMedGoogle Scholar
  10. Lipsick JS: One billion years of Myb. Oncogene. 1996, 13: 223-235.PubMedGoogle Scholar
  11. Kanei-Ishii C, Sarai A, Sawazaki T, Nakagoshi H, He DN, Ogata K, Nishimura Y, Ishii S: The tryptophan cluster: a hypothetical structure of the DNA-binding domain of the myb protooncogene product. J Biol Chem. 1990, 265: 19990-19995.PubMedGoogle Scholar
  12. Frampton J, Gibson TJ, Ness SA, Doderlein G, Graf T: Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis. Protein Eng. 1991, 4: 891-901.View ArticlePubMedGoogle Scholar
  13. Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H, Sarai A, Ishii S, Nishimura Y: Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices. Cell. 1994, 79: 639-648. 10.1016/0092-8674(94)90549-5.View ArticlePubMedGoogle Scholar
  14. Jamin N, Gabrielsen OS, Gilles N, Lirsac PN, Toma F: Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study. Eur J Biochem. 1993, 216: 147-154.View ArticlePubMedGoogle Scholar
  15. Tahirov TH, Sato K, Ichikawa-Iwata E, Sasaki M, Inoue-Bungo T, Shiina M, Kimura K, Takata S, Fujikawa A, Morii H, Kumasaka T, Yamamoto M, Ishii S, Ogata K: Mechanism of c-Myb-C/EBP beta cooperation from separated sites on a promoter. Cell. 2002, 108: 57-70. 10.1016/S0092-8674(01)00636-5.View ArticlePubMedGoogle Scholar
  16. Lipsick JS, Wang DM: Transformation by v-Myb. Oncogene. 1999, 18: 3047-3055. 10.1038/sj.onc.1202745.View ArticlePubMedGoogle Scholar
  17. Biedenkapp H, Borgmeyer U, Sippel AE, Klempnauer KH: Viral myb oncogene encodes a sequence-specific DNA-binding activity. Nature. 1988, 335: 835-837. 10.1038/335835a0.View ArticlePubMedGoogle Scholar
  18. Ording E, Bergholtz S, Brendeford EM, Jamin N, Gabrielsen OS: Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis. Oncogene. 1996, 13: 1043-1051.PubMedGoogle Scholar
  19. Berge T, Bergholtz SL, Andersson KB, Gabrielsen OS: A novel yeast system for in vivo selection of recognition sequences: defining an optimal c-Myb-responsive element. Nucleic Acids Res. 2001, 29: E99-10.1093/nar/29.20.e99.PubMed CentralView ArticlePubMedGoogle Scholar
  20. Howe KM, Reakes CF, Watson RJ: Characterization of the sequence-specific interaction of mouse c-myb protein with DNA. EMBO J. 1990, 9: 161-169.PubMed CentralPubMedGoogle Scholar
  21. Gabrielsen OS, Sentenac A, Fromageot P: Specific DNA binding by c-Myb: evidence for a double helix-turn-helix- related motif. Science. 1991, 253: 1140-1143.View ArticlePubMedGoogle Scholar
  22. Ramsay RG, Ishii S, Gonda TJ: Increase in specific DNA binding by carboxyl truncation suggests a mechanism for activation of Myb. Oncogene. 1991, 6: 1875-1879.PubMedGoogle Scholar
  23. Ogata K, Morikawa S, Nakamura H, Hojo H, Yoshimura S, Zhang R, Aimoto S, Ametani Y, Hirata Z, Sarai A: Comparison of the free and DNA-complexed forms of the DNA-binding domain from c-Myb. Nat Struct Biol. 1995, 2: 309-320. 10.1038/nsb0495-309.View ArticlePubMedGoogle Scholar
  24. Tanikawa J, Yasukawa T, Enari M, Ogata K, Nishimura Y, Ishii S, Sarai A: Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain. Proc Natl Acad Sci U S A. 1993, 90: 9320-9324.PubMed CentralView ArticlePubMedGoogle Scholar
  25. Ording E, Kvavik W, Bostad A, Gabrielsen OS: Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein. Eur J Biochem. 1994, 222: 113-120.View ArticlePubMedGoogle Scholar
  26. Ganter B, Chao ST, Lipsick JS: Transcriptional activation by the myb proteins requires a specific local promoter structure. FEBS Lett. 1999, 460: 401-410. 10.1016/S0014-5793(99)01373-3.View ArticlePubMedGoogle Scholar
  27. Grässer FA, Graf T, Lipsick JS: Protein truncation is required for the activation of the c-myb proto- oncogene. Mol Cell Biol. 1991, 11: 3987-3996.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Pelham HR, Brown DD: A specific transcription factor that can bind either the 5S RNA gene or 5S RNA. Proc Natl Acad Sci U S A. 1980, 77: 4170-4174.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Honda BM, Roeder RG: Association of a 5S gene transcription factor with 5S RNA and altered levels of the factor during cell differentiation. Cell. 1980, 22: 119-126. 10.1016/0092-8674(80)90160-9.View ArticlePubMedGoogle Scholar
  30. Picard B, Wegnez M: Isolation of a 7S particle from Xenopus laevis oocytes: a 5S RNA-protein complex. Proc Natl Acad Sci U S A. 1979, 76: 241-245.PubMed CentralView ArticlePubMedGoogle Scholar
  31. Guddat U, Bakken AH, Pieler T: Protein-mediated nuclear export of RNA: 5S rRNA containing small RNPs in xenopus oocytes. Cell. 1990, 60: 619-628. 10.1016/0092-8674(90)90665-2.View ArticlePubMedGoogle Scholar
  32. Christensen JH, Hansen PK, Lillelund O, Thogersen HC: Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene. FEBS Lett. 1991, 281: 181-184. 10.1016/0014-5793(91)80388-J.View ArticlePubMedGoogle Scholar
  33. Theunissen O, Rudt F, Guddat U, Mentzel H, Pieler T: RNA and DNA binding zinc fingers in Xenopus TFIIIA. Cell. 1992, 71: 679-690. 10.1016/0092-8674(92)90601-8.View ArticlePubMedGoogle Scholar
  34. Hastie ND: Life, sex, and WT1 isoforms--three amino acids can make all the difference. Cell. 2001, 106: 391-394. 10.1016/S0092-8674(01)00469-X.View ArticlePubMedGoogle Scholar
  35. Little M, Holmes G, Walsh P: WT1: what has the last decade told us?. Bioessays. 1999, 21: 191-202.View ArticlePubMedGoogle Scholar
  36. Caricasole A, Duarte A, Larsson SH, Hastie ND, Little M, Holmes G, Todorov I, Ward A: RNA binding by the Wilms tumor suppressor zinc finger proteins. Proc Natl Acad Sci U S A. 1996, 93: 7562-7566. 10.1073/pnas.93.15.7562.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Rivera-Pomar R, Niessing D, Schmidt-Ott U, Gehring WJ, Jackle H: RNA binding and translational suppression by bicoid. Nature. 1996, 379: 746-749. 10.1038/379746a0.View ArticlePubMedGoogle Scholar
  38. Dubnau J, Struhl G: RNA recognition and translational regulation by a homeodomain protein. Nature. 1996, 379: 694-699. 10.1038/379694a0.View ArticlePubMedGoogle Scholar
  39. Cassiday LA, Maher LJ: Having it both ways: transcription factors that bind DNA and RNA. Nucleic Acids Res. 2002, 30: 4118-4126. 10.1093/nar/gkf512.PubMed CentralView ArticlePubMedGoogle Scholar
  40. Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN, Reddy ES: The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains. Oncogene. 1994, 9: 3087-3097.PubMedGoogle Scholar
  41. Hallier M, Tavitian A, Moreau-Gachelin F: The transcription factor Spi-1/PU.1 binds RNA and interferes with the RNA-binding protein p54nrb. J Biol Chem. 1996, 271: 11177-11181. 10.1074/jbc.271.19.11177.View ArticlePubMedGoogle Scholar
  42. Drabkin HA, West JD, Hotfilder M, Heng YM, Erickson P, Calvo R, Dalmau J, Gemmill RM, Sablitzky F: DEF-3(g16/NY-LU-12), an RNA binding protein from the 3p21.3 homozygous deletion region in SCLC. Oncogene. 1999, 18: 2589-2597. 10.1038/sj.onc.1202601.View ArticlePubMedGoogle Scholar
  43. Buckanovich RJ, Yang YY, Darnell RB: The onconeural antigen Nova-1 is a neuron-specific RNA-binding protein, the activity of which is inhibited by paraneoplastic antibodies. J Neurosci. 1996, 16: 1114-1122.PubMedGoogle Scholar
  44. Shteiman-Kotler A, Schuster G: RNA-binding characteristics of the chloroplast S1-like ribosomal protein CS1. Nucleic Acids Res. 2000, 28: 3310-3315. 10.1093/nar/28.17.3310.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Piscopo A, Branno M, Aniello F, Corrado M, Piscopo M, Fucci L: Isolation and characterization of the cDNA for a Ciona intestinalis RNA binding protein: spatial and temporal expression during development. Differentiation. 2000, 66: 23-30. 10.1046/j.1432-0436.2000.066001023.x.View ArticlePubMedGoogle Scholar
  46. Bergholtz S, Andersen TO, Andersson KB, Borrebaek J, Luscher B, Gabrielsen OS: The highly conserved DNA-binding domains of A-, B- and c-Myb differ with respect to DNA-binding, phosphorylation and redox properties. Nucleic Acids Res. 2001, 29: 3546-3556. 10.1093/nar/29.17.3546.PubMed CentralView ArticlePubMedGoogle Scholar
  47. Henderson E, Hardin CC, Walk SK, Tinoco IJ, Blackburn EH: Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs. Cell. 1987, 51: 899-908. 10.1016/0092-8674(87)90577-0.View ArticlePubMedGoogle Scholar
  48. Sen D, Gilbert W: A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature. 1990, 344: 410-414. 10.1038/344410a0.View ArticlePubMedGoogle Scholar
  49. Saenger W: Principles of nucleic acid structure. 1984, New York, Springer-Verlag, 1st editionView ArticleGoogle Scholar
  50. Myrset AH, Bostad A, Jamin N, Lirsac PN, Toma F, Gabrielsen OS: DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein. EMBO J. 1993, 12: 4625-4633.PubMed CentralPubMedGoogle Scholar
  51. Zargarian L, Le Tilly V, Jamin N, Chaffotte A, Gabrielsen OS, Toma F, Alpert B: Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb. Biochemistry. 1999, 38: 1921-1929. 10.1021/bi981199j.View ArticlePubMedGoogle Scholar
  52. Ladomery M: Multifunctional proteins suggest connections between transcriptional and post-transcriptional processes. Bioessays. 1997, 19: 903-909.View ArticlePubMedGoogle Scholar
  53. Bentley D: Coupling RNA polymerase II transcription with pre-mRNA processing. Curr Opin Cell Biol. 1999, 11: 347-351. 10.1016/S0955-0674(99)80048-9.View ArticlePubMedGoogle Scholar
  54. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW: Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990, 185: 60-89.View ArticlePubMedGoogle Scholar
  55. Gabrielsen OS, Matre V, Bergholtz S: Protein-Oligonucleotide interactions, Encyclopedia of Analytical Chemistry Edited by: Meyers RA. 2000, Chichester, John Wiley % Sons Ltd, 5997-6017.Google Scholar

Copyright

© Nordgård et al; licensee BioMed Central Ltd. 2004

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement