©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Sequence-specific Single-stranded DNA Binding Protein That Suppresses Transcription of the Mouse Myelin Basic Protein Gene (*)

Susan Haas (1)(§), Andrzej Steplewski (1), Linda D. Siracusa (2)(¶), Shohreh Amini (1), Kamel Khalili (1)(**)

From the (1) Molecular Neurovirology Section, Jefferson Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology and the (2) Jefferson Cancer Institute, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.


INTRODUCTION

Myelin basic protein (MBP)() is a peripheral membrane protein that is required for maintenance of the compact multilamellar membrane structure of mature myelin. This protein is produced only by specialized myelin-forming cells, i.e. oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system (reviewed in Ref. 1). Myelin formation occurs postnatally in the mouse, such that MBP is first detected at 7 days postnatal, increases dramatically to a peak at 18 days, and decreases to about 20% of peak levels in the mature animal (2) . Steady-state levels of MBP mRNA correspond well with the observed pattern of protein accumulation (3) , and direct measurement of MBP RNA synthesis by nuclear run-on transcription assay demonstrates that MBP gene expression is determined primarily at the level of transcript synthesis (4-6).

Functional dissection of the MBP 5`-flanking region has identified multiple elements important for cell type-specific transcription in transient transfection and cell-free in vitro transcription systems (7, 8, 9) . Distal promoter elements, between nucleotides -93 and -209 relative to the transcription start site, stimulate MBP transcription and encompass binding sites for the CTF/NF1 and thyroid hormone receptor transcription factors (10, 11) . A proximal promoter element of the MBP gene, termed MB1 and located at nucleotides -14 to -50 relative to the transcription start site, has been shown to participate in cell type-specific transcription of the MBP gene (12-14). A developmentally regulated nuclear protein derived from mouse brain has recently been identified that binds the MB1 element and stimulates MBP gene transcription (15) .

It has long been assumed that promoter and enhancer binding factors recognize their cognate sites on duplex DNA (4, 16) , although some transcription factors have the capacity to bind in a sequence-specific manner to single-stranded DNA. The myogenic determination factor MyoD has been shown to bind specifically to the muscle-specific enhancer of the mouse creatine kinase M gene, whether the element is presented in duplex or single-stranded form (17) . The estrogen receptor, a nuclear protein that acts as a receptor for the hormone estradiol and modulates transcription of genes containing its cognate estrogen responsive element, binds the estrogen responsive element only when presented in single-stranded form (18) or in the presence of an associated single-stranded DNA binding protein (19) . Other novel transcription factors have been identified that possess sequence-specific single-stranded DNA binding activity, interacting with elements in a number of muscle-specific genes (17, 20) and in the regulatory region of the human neurotropic JC virus (21) . Perhaps it should be emphasized that the importance of this kind of interaction in transcription of the respective genes has been well established.

The promoter regions of several genes are unusually sensitive to nucleases, indicating that these regions assume unusual non-B DNA conformations. As first demonstrated by Weintraub and Groudine (22) , the enzyme DNase I preferentially digests genes that are transcriptionally active. In this respect, analysis of the chicken adult -globin gene indicates that its 5`-flanking region is highly sensitive to cleavage by nuclease S1 (23) and by bromoacetaldehyde (24), suggesting that this promoter region contains unpaired or single-stranded DNA bases.

Since the torsional stress of DNA bending may be relieved by localized melting of the double helix (25) , the site-specific bends observed in the promoter regions of some eukaryotic genes may indicate the presence of single-stranded DNA in these elements. In this context, it has been demonstrated that several transcription factors can bind to bent DNA and/or induce DNA bending after forming DNA-protein complexes (26-31). Of particular interest is the recent finding that the general transcription factor TFIID, which binds to the TATA box of many pol II promoters, has an affinity for its site on single-stranded DNA and induces localized bending of the DNA molecule (27, 32) . Despite its lack of a consensus TATA motif, the MB1 element has been reported to interact with TFIID (13) , leading to the assumption that this element may exhibit a non-B DNA conformation.

In this study, we have investigated the potential role of sequence-specific single-stranded DNA binding proteins in mediating the transcriptional activity of MBP through the MB1 element. Using the in situ filter detection method (33) , we have identified a mouse brain cDNA encoding Myelin Expression Factor-2 (Myef-2), a protein which binds specifically to the noncoding strand of the MB1 element of the MBP gene. We have also characterized the DNA binding activity, tissue distribution, and developmental regulation of the Myef-2 gene product and assessed the transcriptional activity of Myef-2 at the molecular level.


MATERIALS AND METHODS

Library Screening by in Situ Detection of DNA Binding Activity

The 15-day postnatal mouse brain cDNA library cloned in gt-11 (kindly provided by Dr. A.T. Campagnoni, UCLA) was screened as described (33) for clones encoding DNA binding proteins. The probe was generated by end labeling of the single-stranded MB1 oligonucleotide (5`-TCAGAGGGCCTGTCTTTGAAGGTGTTGTCCTCCCT-3`) using T4 polynucleotide kinase and [P]ATP (34) . Recombinant phage (400, 0) were screened with the MB1 probe, and the Myef-2 cDNA was one of two clones isolated.

Subcloning and DNA Sequencing

Myef-2 phage was grown by the plate lysis method (34) , and DNA was purified using commercially available Qiagen columns. Phage DNA was digested with EcoRI to release a cDNA fragment of approximately 2.5 kb in size. Plasmid pBlue-Myef-2 was generated by insertion of the 2.5-kb Myef-2 cDNA into the EcoRI sites of pBluescript KS (Stratagene). The Myef-2 cDNA was sequenced from pBlue-Myef-2 using the dideoxy chain termination method (35) . Sequencing proceeded from both ends until the sequences overlapped, and sequences were verified by sequencing of the opposite strand. Sequence analysis was performed on GCG sequence analysis software through the Jefferson Cancer Institute shared computer facility.

For eukaryotic expression, an initiator methionine codon (AUG) and the proper flanking sequences (36) were engineered into a synthetic double-stranded oligonucleotide (coding strand, 5`-TCGACGCCGCCATGG-3`; noncoding strand, 5`-AGCTCCATGGCGGCG-3`) and inserted into pBlue-Myef-2 between the SalI and HindIII sites directly upstream of the 5`-end of the Myef-2 cDNA. The cDNA containing the initiator codon was excised by digestion with SalI and XbaI and cloned directly downstream of the cytomegalovirus (CMV) immediate early promoter to generate the eukaryotic expression vector pCMV-Myef-2. In pCMV-Myef-2, the initiator codon and vector sequences encode the heptapeptide MELDIEF, which is fused to the N terminus of the Myef-2 protein.

Expression of Recombinant Myef-2 Protein in Bacteria and Southwestern Blot Analysis

Maltose-binding/Myef-2 fusion protein and maltose-binding/lacZ fusion protein were produced in Escherichia coli HB101 using the plasmids pMAL-Myef-2 and pMAL-cRI, respectively, according to the procedure described by the supplier (New England BioLabs). For Southwestern blot analysis, approximately 100 µg of proteins were fractionated on a 10% SDS-polyacrylamide gel, and the proteins were transferred to nitrocellulose filters by a transblot apparatus (Bio-Rad Laboratories) using transfer buffer containing 192 mM glycine, 25 mM Tris-base, and 20% methanol for 3 h at 400 mA. The transferred proteins were denatured in buffer containing 50 mM Tris (pH 8.0), 7 M guanine HCl, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol for 1 h at room temperature. The nitrocellulose filters were immersed in 50 mM Tris (pH 8.0), 100 mM NaCl, 2 mM dithiothreitol, 2 mM EDTA, 0.1% Nonidet P-40, and 0.25% Blotto for 24 h at 4 °C prior to incubation with 10 cpm/100 ng of P-end-labeled single-stranded probes in TN50 buffer containing 20 mM Tris (pH 8.0), 1 mM EDTA, and 100 mM NaCl. After incubation for 1 h, filters were washed five times in TN50 buffer, dried, and exposed 3-5 h at -70 °C.

RNA Isolation and Northern Blot Analysis

Total RNA was isolated from mouse tissues by the guanidine isothiocyanate/cesium chloride method (37) . Total RNA samples (20 µg/lane) were separated on a formaldehyde-agarose gel, stained with ethidium bromide, and photographed. Gels were then transferred to nylon membrane (Hybond N, Amersham Corp.). Myef-2 and MBP (gift of Jeffrey Green, Frederick Cancer Research and Development Center, Frederick, MD) cDNA probes were labeled by nick translation with [P]dCTP, hybridized at a concentration of 2.5 10 cpm/ml at 65 °C in a solution containing 5 SSC, 2 Denhardt's solution, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA, and washed under high stringency conditions (65 °C, 0.1 SSC, 0.1% SDS). Blots were quantitated by laser densitometry of autoradiograms.

Chromosomal Localization

To identify useful restriction fragment length polymorphisms (RFLPs) for mapping the MYEF2 locus, genomic DNAs from AEJ/Gn and Mus spretus mice were digested with 14 different restriction enzymes and analyzed by Southern blot hybridization as described (38) , The Myef-2 cDNA detected multiple fragments in both species, some of which were polymorphic. To simplify the RFLP analysis, hybridization with a 253-bp BamHI fragment derived from the 3`-end of the Myef-2 cDNA was performed, detecting a 13-kb HindIII fragment in M. spretus and a 4.3-kb HindIII fragment in AEJ/Gn. DNA from N progeny of an (AEJ/Gn M. spretus) AEJ/Gn interspecific backcross (38) were subjected to Southern blot hybridization with the Myef-2 probe and scored for the presence or absence of the 13-kb M. spretus band. Results using the full-length and 253-bp Myef-2 probes were in complete agreement. RFLP mapping of probes that identify the B2m, II1a, and Avp loci has been described (38) . A probe flanking the MPMV28 integration site (gift of Wayne N. Frankel, The Jackson Laboratory, Bar Harbor, ME and Jonathan P. Stoye, NMRC, Mill Hill, London, United Kingdom) was also used in the interspecific backcross; the MPMV28 locus was previously mapped in other crosses (39, 40) . The simple sequence length polymorphism identified by the D2Mit19 oligomer pair (41) was mapped by polymerase chain reaction analysis as described (42) .

Site-directed Mutagenesis

The 423-bp HindIII-BamHI DNA fragment of the mouse MBP gene upstream of the regulatory sequences (-402 to +21) was cloned into the HindIII-BamHI site of the M13 mP19 and mutagenized as previously described (43) . The mutagenic oligonucleotide contained altered nucleotides in the Myef-2 binding site within the MB1 domain as illustrated in Fig. 3D. After sequencing of the candidate mutated clones, the HindIII-BamHI fragment was removed from the double-stranded phage DNA and placed at 5`-position of the CAT gene in the pBL-CAT promoter plasmid.


Figure 3: Binding of Myef-2 to single-stranded DNA derived from the proximal promoter element (MB1) of the MBP gene. A, Coomassie Blue staining of protein extracts from IPTG-treated E. coli containing control pMAL-cRI (lane1) and recombinant pMAL-cRI-Myef-2 (lane2) on 10% SDS-polyacrylamide gel electrophoresis. The arrow indicates position of the Myef-2 fusion protein. B, Southwestern analysis of bacterial extracts as shown in panelA with the probes representing the non-coding strand of MB1 or its various regions MB1 and MB1 sequences. C, Coomassie Blue staining and Southwestern analysis of proteins derived from E. coli containing pMal-cRI-Myef-2, which were grown in the absence (lane1) or presence (lane2) of IPTG. An arrow indicates the position of Myef-2, and the asterisk points to the unrelated band appearing in both extracts upon binding to MB1dl. D, primary structure of the oligonucleotides used in this study. The underlined nucleotides represent the binding site for Myef-2, and the dottedunderlinednucleotides indicate the position of the sequence with similarity to the Myef-2 binding site.



Transient Transfection and CAT Assay

The reporter plasmid pMB402 contains mouse MBP promoter/enhancer sequences, from nucleotide -402 to +21 relative to the transcription start site, cloned directly upstream of the CAT coding region as described (15) . Transfections were performed by the calcium phosphate precipitation method (44) . At 48 h post-transfection, cellular proteins were harvested, and an equal amount of protein extract was used to analyze for CAT activity. Each experiment was repeated multiple times to ensure transfection efficiency and reproducibility of the results.


RESULTS

Molecular Cloning Identifies Myef-2, a Novel DNA Binding Protein

The proximal cis-acting element of the MBP gene, termed MB1, directs cell type-specific transcription of a heterologous promoter in cultured cells and is expressed in a tissue-specific manner in vitro(7, 12, 13, 14) . Since tissue-specific and developmentally regulated DNA binding proteins are known to interact with the MB1 region (15, 45) , we set out to isolate cDNA clones encoding such DNA binding proteins using the in situ filter detection method. A gt11 cDNA library prepared from mouse brain RNA was screened with a single-stranded oligonucleotide corresponding to the noncoding strand of the MB1 element. We chose to use the single-stranded DNA probe since our preliminary bandshift assay revealed a strong and specific interaction of nuclear proteins from brain with the critical proximal regulatory element of MBP encompassing the MB1 sequence. The result of this screening was isolation of partial Myef-2 cDNA clone. Sequence analysis of the Myef-2 cDNA (Fig. 1) predicts that it encodes a novel protein of 436 amino acids, consistent with its apparent molecular mass in SDS-polyacrylamide gel electrophoresis of 46 kDa (data not shown). The predicted protein is basic in nature (pI = 9.9).


Figure 1: Primary sequence of the Myef-2 cDNA. Sequence determination of the Myef-2 cDNA was by the method of Sanger et al. (35). Sequences were verified by sequencing of the second strand. Translation of the Myef-2 cDNA predicts a protein of 435 amino acids, which is shown below the nucleotide sequences.



The Myef-2 protein contains two copies of an RNA recognition motif (RRM), previously shown to be responsible for sequence-specific binding to both RNA and single-stranded DNA (24, 46) . Of all RRM-containing proteins, Myef-2 shares the greatest degree of homology with heterogeneous ribonucleoprotein M4 (hnRNP M4) (47) . The N-terminal (amino acids 1-203) and C-terminal (amino acids 350-435) regions of Myef-2 share some homology with hnRNP M4, while the central region (amino acids 204-349) is more divergent (Fig. 2). The N-terminal RRM, denoted RRM1 and found within the N-terminal homology region (amino acids 78-162), is 87% identical to the corresponding RRM in hnRNP M4. The C-terminal RRM, denoted RRM2 (amino acid 350-435), shares 70% amino acid sequence identity with its corresponding sequence in hnRNP M4. RRM1 and RRM2 demonstrate a lesser degree of homology to RRMs found in a wide variety of RNA and single-stranded DNA binding proteins (24, 46) . Sequence analysis demonstrates that Myef-2 contains no other commonly recognized DNA binding motifs, such as zinc finger, homeobox, POU, or helix-loop-helix domains. Presumably, the RRMs are responsible for the sequence-specific single-stranded DNA binding activity of Myef-2, and their presence suggests that this protein may also bind RNA.


Figure 2: Sequence homology between Myef-2 and hnRNP M4 proteins. Top, schematic diagram of the structure of the Myef-2 and hnRNP M4 proteins. RRMs in both proteins are shown as openboxes, and homologous regions are shown with dottedlines. Sequence homology is greatest within the RRM1 and RRM2 motifs. Bottom, amino acid sequence comparison of RRM1 and RRM2 to homologous regions in hnRNP M4 is shown, and identical residues are marked with lines between the two sequences. The most highly conserved residues within the previously characterized RRM consensus are underlined (20).



Myef-2 Binds Specifically to the Noncoding Strand of the Proximal (MB1) Element of the MBP Promoter

To study the DNA binding properties of Myef-2, the protein was expressed in E. coli using the bacterial expression vector pMAL-cRI. This vector produces the protein of interest as a fusion with maltose binding protein. The ability of the Myef-2 fusion protein to bind the MB1 sequence was examined by Southwestern blot assay. The control protein that consists of maltose binding/lacZ fusion was produced in bacteria containing pMAL-cRI. As shown in Fig. 3, lane2, the maltose binding/Myef-2 fusion protein bound to MB1 oligonucleotide probe appeared in the IPTG-treated cells (panelB). This binding was not observed in the extract containing the control maltose binding/lacZ fusion protein (Fig. 3B, lane1), which demonstrates specificity of MB1-Myef-2 interaction.

To further localize the binding site for Myef-2 within the MB1 element and determine the specificity of DNA-protein complex, two partially overlapping synthetic oligonucleotides representing the 5`- (MB1) and the 3`- (MB1) non-coding sequence of the MB1 (shown in Fig. 3D) were used as probes in a Southwestern assay. As shown in Fig. 3B, unlike the MB1 probe, MB1 showed a significant binding affinity to the Myef-2 fusion protein, suggesting that the critical nucleotides for binding of the Myef-2 reside in the 12 base pairs of the MB1 that do not overlap with the MB1 sequences. To further identify the important nucleotides for binding of Myef-2 to MB1, we performed Southwestern blot analysis using MB1dl probe, which contains alterations in the pentanucleotides (5`-TGTCCT-3` 5`-atcag-3`) located in the center of the MB1. As shown in Fig. 3D, MB1dl showed insignificant, if any, binding activity corresponding to Myef-2. The mutant oligonucleotide showed binding activity to an unrelated bacterial protein produced in uninduced and induced cells (shown by asterisk in Fig. 3C, lanes1 and 2). These observations corroborate our previous competition bandshift assay data and indicate that the pentanucleotide sequence 5`-TGTCC-3` (as underlined in Fig. 3D) is the important component of the Myef-2 binding site within the MB1(48) . From these data, we conclude that Myef-2 binds efficiently to specific sequences located in the non-coding strand of the MB1 regulatory element of the MBP promoter.

Myef-2 Represses Transcription of the MBP Gene

We employed transient cotransfection assay to investigate the possible role of Myef-2 in modulating transcription of the MBP gene (Fig. 4). Consistent with the restricted tissue specificity of the MBP promoter, the reporter plasmid pMB402, containing a 423-bp segment of the MBP promoter, was expressed more strongly in glial cells (U-87MG, 2.81%; RN22, 2.65%) than in non-glial 3T3 fibroblasts (1.10%) in the absence of exogenous Myef-2. In fact, even this low level of basal expression in non-glial cells is unusual, and no CAT activity was detectable in non-glial Hela cells (data not shown).


Figure 4: Transcriptional activity of Myef-2. Transient cotransfection analysis of Myef-2 transcriptional activity in 3T3 mouse fibroblasts (A), U-87MG human glioblastoma cells (B), and RN22 rat Schwannoma cells (C). Reporter plasmid pMB402 containing mouse MBP promoter sequences from nucleotides -402 to +21 relative to the transcription start site was cotransfected with Myef-2 expression vector pCMV-Myef-2 and/or control plasmid pCMV. CAT activity is expressed as percent conversion (bargraphs), and errorbars represent S.E. of three independent experiments performed with different pCMV-Myef-2 plasmid preparations. Representative CAT assay autoradiograms are shown (insets).



In cotransfection assays, Myef-2 suppressed expression of the MBP promoter 6.5-fold in 3T3 mouse fibroblasts (Fig. 4A). A modest (less than 2-fold) yet detectable decrease was also observed in U-87MG human glioblastoma cells (Fig. 4B) and RN22 rat Schwannoma cells (Fig. 4C). Apparently, the decrease seen in glial cells (U-87MG and RN22) is not greater than the statistical variability of the experiment. Thus, Myef-2 appears to repress transcription of the myelin basic protein gene, primarily in non-glial cells.

To examine the site-dependent repression of MBP gene transcription by Myef-2, mutations were introduced at the Myef-2 binding site shown in Fig. 3D (altering 5`-TTGTCC-3` to 5`-GAGTCC-3`) of the full-length MBP promoter. The mutant plasmid, pMB was introduced either alone or together with the pCMV-Myef-2 into U-87MG and 3T3 cells. As shown in Fig. 5, expression of Myef-2 in the transfected cells had no negative effect on the transcriptional activity of the mutated MBP promoter in U-87MG. Thus, binding of Myef-2 to its target DNA sequence is important for this protein to exert its regulatory function. Fig. 5also illustrates that in cotransfection assay, Myef-2 exhibits no negative activity on transcription of a heterologous simian virus 40 promoter in the transfected cells. These observations strongly suggest that Myef-2 is a sequence-specific DNA binding protein that reduces its transcriptional activity in transfected cells by interacting with its target sequence on MBP promoter.


Figure 5: Transcriptional activity of Myef-2 on the promoters with no Myef-2 binding site. Human glioblastoma cells, U-87MG (A), and mouse fibroblast cells, 3T3 (B), were transfected with the reporter plasmid pMB and pSV-CAT alone or together with Myef-2 expression vector. Cells were harvested 48 h post-transfection, and CAT assay was performed (59). The percent conversion of chloramphenicol to its acetylated forms was determined by liquid scintillation counting of substrate and acetylated forms. Each experiment was repeated multiple times, and results for a representative experiment are shown.



Myef-2 mRNA Is Most Abundant in Mouse Brain and Is Developmentally Regulated

To obtain some clues to the possible functional role of Myef-2 in transcriptional regulation of the MBP gene, we compared the expression profile of Myef-2 and MBP during brain development. Northern blot analysis of total RNA isolated from a panel of mouse tissues and from mouse brain tissue at different stages of development indicated that Myef-2 mRNA was expressed at highest levels in mouse brain (Fig. 6A). Non-neural tissues expressed 16% (i.e. heart or kidney) to 54% (i.e. spleen) of the levels of Myef-2 mRNA in brain as determined by laser densitometry of the autoradiograph. In contrast, MBP mRNA was expressed only in brain tissue (Fig. 6B). Ethidium bromide staining showed that approximately equal amounts of RNA were loaded on both gels (Fig. 6C).


Figure 6: Tissue distribution and developmental accumulation of Myef-2 mRNA. Northern blot analysis of total RNA (20 µg/lane) derived from brain (lane1), heart (lane2), kidney (lane3), liver (lane4), lung (lane5), and spleen (lane6) of young adult mice and from brain tissue of mice aged 3 (lane7), 7 (lane8), 14 (lane9), 18 (lane10), 21 (lane11), 30 (lane12), and 60 days (lane13) and from brain tissue of adult lactating females (lane14). A, tissue distribution and developmental accumulation of Myef-2, showing a major band at 3.7 kb. B, Identical blots show tissue distribution and developmental accumulation of MBP mRNA (2.1 kb). C, ethidium bromide (EtBr) staining of the gels corresponding to the blots.



During development, Myef-2 mRNA levels were maximal in the brain of 7-day-old mice, decreasing more than 3-fold in 14-day-old mice, and remaining relatively constant thereafter throughout maturation of the animal (Fig. 6A). In contrast, MBP mRNA was first detected at postnatal day 7, increasing dramatically between 7 and 14 days, peaking at 18 days, and decreasing to a lower level in the adult mouse (Fig. 6B), in excellent agreement with previous studies (2, 3) . Note that the developmental pattern of Myef-2 mRNA accumulation is very similar to that of SCIP (49, 50) , also called Oct 6 or Tst-1, a member of the POU domain family of transcription factors and a repressor of MBP transcription.

The myef-2 Gene Maps to Mouse Chromosome 2

The Myef2 genetic locus, encoding the Myef-2 gene product, was localized to mouse chromosome 2 by RFLP and simple sequence length polymorphism analysis of a panel of murine genomic DNAs derived from the progeny of an interspecific backcross (Fig. 7). The Myef-2 probe detected a polymorphic 13-kb HindIII band, which was scored for mapping of the locus (see ``Materials and Methods''). Gene order was resolved by minimizing the number of recombinants between loci along the length of the chromosome. The order of the loci and the ratio of the number of recombinants to the total number of N offspring examined are as follows: B2m - 7/190 - Mpmv28 - 1/184 - (Myef2, Avp, Il1a) - 3/92 - D2Mit19). The genetic distances between loci in centiMorgans ± S.E. are: B2m - 3.7 ± 1.4 - Mpmv28 - 0.5 ± 0.5 - Myef2, Avp, Il1a) - 3.3 ± 1.7 - D2Mit19. Fig. 5shows the placement of these genes on the map of mouse chromosome 2. This region of the mouse chromosome contains genes whose human homologs reside on human chromosomes 2, 15, and 20. Therefore, the human homolog of the myef-2 gene likely resides on one of these three chromosomes.


Figure 7: Genetic linkage map showing the chromosomal location of the MYEF2 locus. The chromosome on the left shows the loci typed in the interspecific backcross, with distances between loci given in centimorgan. The chromosome on the right shows a partial version of the consensus linkage map of mouse chromosome 2 (56). MYEF2 is shown in boldfacetype. The chromosomes are aligned at the Il-1a locus. Loci mapped in humans are underlined; locations of these genes in humans are shown between the chromosomes.




DISCUSSION

The Myef-2 gene product appears to be a repressor of transcription from the mouse MBP promoter. It is not surprising that this repressor functions most efficiently in non-glial cells, since those cells do not normally express the endogenous MBP gene and must, therefore, contain all the necessary cofactors required for suppression of MBP expression. Transcriptional repression has been postulated to occur by a number of mechanisms, including direct competition with basal transcription factors, inhibition of the binding of activator proteins to their cognate sites, and ``silencer'' function analogous to activation by enhancers (Ref. 16, also reviewed in Refs. 51 and 52). Identification of the cofactors present in non-glial cells, which allow Myef-2 to efficiently suppress MBP expression, will increase our understanding of transcriptional repression mechanisms in general.

We cannot exclude the possibility that the full-length Myef-2 protein has a transcriptional effect different from the one observed here. In other systems, positive-acting transcription factors have been shown to suppress transcription when truncated, either by competing with the wild-type protein for binding sites on DNA (trans-dominant effect) or by competing for accessory factors by protein-protein interactions (squelching). However, similarities between the developmental profile of Myef-2 expression in mouse brain and that of another MBP transcriptional repressor, SCIP (49) , also called Oct-6 or Tst-1, suggest that this protein may indeed repress MBP gene expression.

Further hypotheses can be formulated by comparison with SCIP. SCIP is a POU domain transcription factor that represses expression of several myelin-specific genes, notably MBP and P0 glycoprotein (50) . SCIP evidently participates in maintenance of the phenotype of the oligodendrocyte progenitor cells, since it is down-regulated when these cells differentiate into oligodendrocytes (53) . The developmental accumulation of SCIP mimics that of Myef-2, with the level of both proteins peaking prior to detectable MBP expression in mouse brain tissue (49) . Whether or not coordinate regulation of these two repressors of myelin gene expression plays a role in some common developmental event during oligodendrocyte differentiation remains to be investigated. Examination of the molecular mechanism of Myef-2 action may help to decipher this cellular differentiation pathway. Also of interest is the finding that SCIP has recently been shown to stimulate transcription of the human neurotropic JC virus promoter (54) in direct contrast to its repressor function on cellular myelin-specific genes. Experiments to assess the effect of Myef-2 on the JC virus promoter are underway in our laboratory.

Myef-2 contains the RRM-type nucleic acid binding motif common to a growing family of proteins that bind single-stranded DNA and RNA in a sequence-specific manner (24, 46) . Some proteins in this class appear to participate in cell type-specific transcriptional regulation, e.g. CBF, a transcriptional repressor of the human smooth muscle -actin gene (20) , and the polypyrimidine tract binding protein, which binds specifically to DNA present in a liver-specific enhancer (16, 55) . Most other members of this family are RNA binding proteins that are involved in RNA metabolism (reviewed in Ref. 24). Although HIV-1 Tat protein provides a model for RNA binding proteins participating in transcriptional regulation, it is unlikely that Myef-2 represses transcription through RNA binding since its cognate site is not transcribed. Probably, Myef-2 interacts with its cognate site in the MBP promoter DNA sequence to exert its transcriptional effect. We hypothesize that the MB1 element may adopt some unusual non-B DNA structure, which facilitates this interaction.

The mouse mutations anx (anorexia), lm (lethal milk), mg (mahogany), pa (pallid), ro (rough), Tsk (tight skin), and we (wellhaarig) map near the Myef-2 gene (56) . The gene product of only the pa mutation (Ebp4.2) has been identified. Two of the remaining mutations (anx and lm) display a neurological phenotype with behavioral abnormalities (summarized in Ref. 57), thus making Myef2 a potential candidate gene. However, Myef2 mRNA is expressed in many tissues and may play a role in biological processes other than myelination. Therefore, a role for Myef-2 in any of these mutations cannot be excluded. Mutation of the Myef-2 gene is apparently not responsible for any known dysmyelinating mouse mutant, since no previously characterized mutant involving dysmyelination maps to this region of mouse chromosome 2 (57, 58) . Interestingly, the epilepsy (EL) mouse is a neurologic mouse strain with unusual susceptibility to seizures, and one of the loci participating in this phenotype, denoted El-2, maps near Mpmv28 on mouse chromosome 2 (40).

Experiments presently in progress in our laboratory include cloning of a full-length Myef-2 cDNA and characterization of the effect of Myef-2 on other oligodendrocyte-specific promoters of both cellular and viral origin. The murine model system allows us to examine the distribution of Myef-2 in mouse brain at the cellular level. By mutating the endogenous Myef-2 gene in transgenic mice, we may determine whether this protein plays a role in myelinogenesis in vivo. Identifying the nature of the biochemical differences between glial and non-glial cells, which allow Myef-2 to function more efficiently in some cell lines than others, will help us to understand the complex regulatory mechanisms involved in expression of myelin basic protein and other myelin-specific gene products.


FOOTNOTES

*
This work was supported by grants awarded by the National Multiple Sclerosis Society and National Institutes of Health (to K. K., S. A., and L. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U13262.

§
A predoctoral fellow of the March of Dimes Birth Defects Foundation.

The recipient of an American Cancer Society Junior Faculty Research Award.

**
To whom all correspondence should be addressed. Tel.: 215-955-5534; Fax: 215-923-8021.

The abbreviations used are: MBP, myelin basic protein; MyEF-2, myelin gene expression factor-2; kb, kilobase(s); CMV, cytomegalovirus; RFLP, restriction fragment length polymorphism; bp, base pair(s); CAT, chloramphenicol acetyltransferase; RRM, RNA recognition motif; hnRNP M4, heterogeneous ribonucleoprotein M4; IPTG, isopropyl-1-thio--D-galactopyranoside.


ACKNOWLEDGEMENTS

We are very grateful to Dr. A. T. Campagnoni for kindly providing cDNA expression library and for valuable comments and helpful discussions. We thank members of the Molecular Neurovirology Section of the Jefferson Institute of Molecular Medicine for sharing reagents and for support and helpful suggestions during the course of this study. We thank Craig Goldstein and Phillip Liaw for technical assistance. We also thank Drs. Wayne N. Frankel and Jonathan P. Stoye for providing the Mpmv-28 flanking probe. We are grateful to Dr. Devjani Chatterjee for assistance with sequence analysis. We thank Marina Hoffman and Jennifer Gordon for critical reading of this manuscript and Cynthia Schriver for preparation of this manuscript.


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