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. 1997 Sep 1;17(17):6657-68.
doi: 10.1523/JNEUROSCI.17-17-06657.1997.

Evidence that the homeodomain protein Gtx is involved in the regulation of oligodendrocyte myelination

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Evidence that the homeodomain protein Gtx is involved in the regulation of oligodendrocyte myelination

R Awatramani et al. J Neurosci. .

Abstract

We have investigated the patterns of postnatal brain expression and DNA binding of Gtx, a homeodomain transcription factor. Gtx mRNA accumulates in parallel with the RNAs encoding the major structural proteins of myelin, myelin basic protein (MBP), and proteolipid protein (PLP) during postnatal brain development; Gtx mRNA decreases in parallel with MBP and PLP mRNAs in the brains of myelin-deficient rats, which have a point mutation in the PLP gene. Gtx mRNA is expressed in differentiated, postmitotic oligodendrocytes but is not found in oligodendrocyte precursors or astrocytes. These data thus demonstrate that Gtx is expressed uniquely in differentiated oligodendrocytes in postnatal rodent brain and that its expression is regulated in parallel with the major myelin protein mRNAs, encoding MBP and PLP, under a variety of physiologically relevant circumstances. Using a Gtx fusion protein produced in bacteria, we have confirmed that Gtx is a sequence-specific DNA-binding protein, which binds DNA sequences containing a core AT-rich homeodomain binding site. Immunoprecipitation of labeled DNA fragments encoding either the MBP or PLP promoter regions with this fusion protein has identified several Gtx-binding fragments, and we have confirmed these data using an electrophoretic mobility shift assay. In this way we have identified four Gtx binding sites within the first 750 bp of the MBP promoter and four Gtx binding sites within the first 1. 3 kb of the PLP promoter. In addition, inspection of the PLP promoter sequence demonstrates the presence of six additional Gtx binding sites. These data, taken together, strongly suggest that Gtx is important for the function of differentiated oligodendrocytes and may be involved in the regulation of myelin-specific gene expression.

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Figures

Fig. 7.
Fig. 7.
Gtx binds to multiple sites on the MBP and PLP promoters. A, Immunoprecipitation analysis of the MBP and PLP promoters. The 750 bp proximal fragment of the human MBP (hMBP) promoter and the 1.3 kb proximal fragment of the rat PLP (rPLP) promoter were digested with various restriction enzymes, and the fragments were end-labeled and incubated with the mbp–Gtx fusion protein, mbp alone, or no protein. Protein–DNA complexes were then immunopreciptated with a polyclonal rabbit anti-mbp antiserum and Staphylococcus protein A-Sepharose beads and separated on a 4%/7 m urea DNA-sequencing gel. Labeled fragments not subjected to immunoprecipitation analysis were run as a control for each promoter (lane C). Sizes (in base pairs) of the labeled fragments from each promoter are indicated on the left andright. B, Electrophoretic mobility shift assay (EMSA) analysis of human MBP promoter–Gtx interactions. Four double-stranded oligonucleotide probes, corresponding to nucleotides −331 to −304 (MBP1), −621 to −598 (MBP2), −636 to −614 (MBP3), and −656 to −630 (MBP4) of the human MBP promoter were each incubated with 1 nm (lanes 1, 5, 9, 13), 10 nm (lanes 2, 6, 10, 14), and 100 nm (lanes 3, 7, 11, 15) Gtx and 1000 nm maltose-binding protein (lanes 4, 8, 12, 16). Protein–DNA complexes were resolved on a nondenaturing polyacrylamide gel. The position of the Gtx–DNA complex is indicated at the left.C, EMSA analysis of rPLP promoter–Gtx interactions. Four double-stranded oligonucleotide probes, corresponding to nucleotides −286 to −259 (PLP1), −434 to −409 (PLP2), −671 to −644 (PLP3), and −1214 to −1188 (PLP4) of the rat PLP promoter were each incubated with 1 nm (lanes 1, 5, 9, 13), 10 nm (lanes 2, 6, 10, 14), and 100 nm (lanes 3, 7, 11, 15) Gtx or 1000 nm maltose-binding protein (lanes 4, 8, 12, 16). Protein–DNA complexes were resolved on a nondenaturing polyacrylamide gel. The position of the Gtx–DNA complex is indicated at the left.D, Summary of human MBP and rat PLP promoter–Gtx interactions. The solid boxed area in each promoter represents the mRNA sequence, beginning at the transcription start site; the thin line represents the upstream promoter sequence. The numbering of the nucleotide sequence for each promoter fragment begins (+1) at the known transcription start site.Positive numbers represent coding sequence;negative numbers represent promoter sequence. The sizes of the labeled fragments from the immunoprecipitation experiment inA are shown below each diagram. Immunoprecipitated fragments are indicated by stars.Arrowheads show the positions of the AT-rich regions corresponding to the probes used in the EMSA in B,C. The arrowheads most proximal to the transcription start sites mark the MBP1 and PLP1 sequences, respectively. Restriction enzymes used were HinfI (H), StyI (S), EcoRI (E), PstI (P), StuI (St), andBamHI (B).
Fig. 1.
Fig. 1.
Analysis of Gtx mRNA accumulation in developing mouse cerebrum. A, Twenty micrograms of total mouse cerebrum RNA from P1 to adult were electrophoresed, blotted, and successively hybridized with radiolabeled cDNA probes encoding Gtx, CNP, PLP, MBP, and GAPDH. B, Twenty micrograms of the same total mouse cerebrum RNA as in A were hybridized to a uniformly labeled, singled-stranded cRNA probe encoding mouse Gtx. The resulting hybrids were digested with RNase A and T1, and the protected fragments were separated on a 4% acrylamide/7 murea DNA-sequencing gel. A schematic diagram of the Gtx cDNA is shownbelow the autoradiogram; the cRNA probe used, encoding nucleotides 672–1052, is underlined. The locations of the undigested probe of 447 bp (lane C) and the protected fragments of 377 bp are indicated at theright.
Fig. 2.
Fig. 2.
RNase protection analysis of Gtx mRNA expression in neural and non-neural adult mouse tissues. Twenty micrograms of total RNA isolated from various adult mouse tissues were hybridized to a uniformly labeled, singled-stranded cRNA probe encoding mouse Gtx and digested with RNase A and T1, and the protected fragments were separated on a 4% acrylamide/7 m urea DNA-sequencing gel. The locations of the undigested probe of 447 bp (lane C) and the protected fragments of 377 bp are indicated at theright.
Fig. 3.
Fig. 3.
Gtx mRNA is expressed in oligodendrocytes.A, Ten micrograms of total RNA, prepared from rat cerebral white matter (CWM) cultures treated with PDGF for various times, were hybridized simultaneously with uniformly labeled cRNA probes encoding Gtx, MBP, and GAPDH. The resulting hybrids were digested with RNase A and T1, and the protected fragments were separated on a 4% acrylamide/7 m urea DNA sequencing gel. RNA from P34 rat brain was used as a control. Astrocyte RNA was prepared from CWM cultures, which had been depleted of oligodendrocytes by complement-mediated cell lysis (Astrocytes lane). The appropriately sized protected fragments of 377 bp (Gtx), 101 bp (MBP), and 324 bp (GAPDH) were observed for each probe. B, Mixed glial cultures, composed of both oligodendrocytes and astrocytes, were prepared from neonatal mouse brain, and the cells fixed onto coverslips and hybridized with a digoxigenin-labeled cDNA full-length probe encoding mouse Gtx. After hybridization the coverslips were washed and developed with nitroblue tretrazolium 5-bromo-4-chloro-3-indoyl phosphate. Note that the reaction product, representing hybridization to the labeled Gtx probe, is concentrated around the oligodendrocyte nucleus (large arrows) and in the oligodendrocyte perinuclear cytoplasm (arrowhead) but is not found in the underlying bed layer of astrocytes (*).
Fig. 4.
Fig. 4.
Northern blot analysis of Gtx mRNA expression in growth factor-stimulated cultures of developing oligodendrocytes. Total RNA was prepared from purified oligodendrocyte precursors cultured in the presence of bFGF and PDGF and at 24, 48, and 72 hr after growth factor withdrawal. Ten micrograms of RNA were electrophoresed per lane, blotted, and sequentially probed with radiolabeled cDNAs encoding Gtx, MBP, and GAPDH.
Fig. 5.
Fig. 5.
RNase protection analysis of Gtx mRNA inmd rat brain. Ten micrograms of total brain RNA frommd and normal rats of various ages were hybridized simultaneously with uniformly labeled cRNA probes encoding Gtx, MBP, and GAPDH. The appropriately sized protected fragments of 377 bp (Gtx), 101 bp (MBP), and 324 bp (GAPDH) were observed for each probe.
Fig. 6.
Fig. 6.
Gtx is a sequence-specific DNA-binding protein. Various amounts of mbp–Gtx (0, 1, 10, 50, and 100 nm) cleaved with factor Xa protease to remove the mbp moiety were incubated with an end-labeled, double-stranded oligonucleotide encoding a Hox A5/A6 binding site (H, lanes 1–5); 0, 1, 10, or 100 nm Gtx was also incubated with an end-labeled, double-stranded oligonucleotide encoding brain CK MEF-2 site (lanes 11–15). A 1000 nm concentration of mbp alone was incubated with probe H (lane 6) and probe CK (lane 15); 100 nm Gtx was incubated with a mutant Hox A5/A6 site (Hm; lane 7). A 50 nm concentration of Gtx was incubated with probe H (lane 8), to which 200-fold excess unlabeled probe H (lane 9) or 200-fold excess unlabeled probe Hm (lane 10) was added. Protein–DNA complexes were resolved on a nondenaturing polyacrylamide gel and are indicated at the left.

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