Interplay of SOX and POU factors in regulation of the Nestin gene in neural primordial cells

Abstract

Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed as markers for neural primordium, suggesting their regulatory link. We have identified adjacent and essential SOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer including these sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimeric forms in transfection assays and was utilized in assessing the specificity of the synergism; combinations of either group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mouse spinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3 expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subventricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) was expressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderate expression was observed in the intermediate zone, distributed in the cytoplasm. These data support the model that synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestin expression. Evidence also suggests that such interactions are involved in the regulation of neural primordial cells.

FIG.1.

Involvement of SOX and POU binding sites in activation of the Nestin neural enhancer. (A) Potential SOX2 binding site found adjacent to the POU factor binding site of the Nestin neural enhancer. The 258-bp-long enhancer sequence in the second intron of the mouse Nestin gene, which is similar to rat and human enhancers, is schematically shown. The blue and red boxes represent the potential SOX binding site and the POU factor binding site, respectively. (B) Nucleotide sequence of a portion of the mouse Nestin enhancer in the gray box in panel A, which is aligned with the corresponding sequences of rat and human. Gray shading indicates conserved nucleotide sequences. The M1 mutation disrupts SOX binding motifs (24, 36, 53) and abolishes SOX2 binding (Fig. 4), while mutation M2 has been shown to eliminate Nestin neural enhancer activity (21). The mutated sequences are shaded in purple. The Nes30 core sequence used in the following analyses is also indicated. (C) Activity of the mouse Nestin neural enhancer (Nes258) and its M1-M2 mutant forms in the electroporated spinal cord of a chicken embryo. Expression vectors for enhancer-dependent EGFP (Reporter) and HcRed1 (Reference) are indicated on top. Electroporation was performed at stage 10, and observations were made after 48 h at stage 21. The HcRed1 reference vector was also included in electroporation. (a, c, and e) EGFP fluorescence in the spinal cord representing activity of Nestin enhancer. (a) Wild-type (WT) enhancer was strongly active in the spinal cord. (c and e) The mutation-containing enhancer, either M1 (c) or M2 (e), was totally inactive. (b, d, and f) HcRed1 fluorescence superimposed on bright-field images. (D) Activity of Nes258 enhancer and its dependence on the SOX and POU binding sites in the neural stem cell line MNS70 and effect of exogenous SOX2 and/or Brn2 expression on the activity. (a) Structure of Nes258 enhancer-bearing luciferase vector and expression vector for SOX2 or Brn2 effectors. CMV, cytomegalovirus. (b) Nes258 enhancer-bearing luciferase vector was transfected into MNS70 neural stem cells, displaying 75-fold activation compared to the enhancer-free vector. Exogenous expression of SOX2 or Brn2 singly augmented the level of activation to roughly 85-fold, and that of SOX2 and Brn2 together activated luciferase expression nearly 100-fold. Using mutant enhancers with defective SOX (M1) or POU (M2) binding sites, activation of the luciferase reporter fell in the range of 30- to 40-fold, and this was not augmented by exogenous SOX2 and/or Brn2 any further. The luciferase activity level generated by the reporter in the absence of enhancer was taken as 1. (b) Expression of Nestin, Sox, and Pou factor genes in the MNS70 line demonstrated by RT-PCR, in comparison with the embryonic spinal cord.

FIG. 2.

Activities of the 30-bp core enhancer (Nes30) in several cultured cells. (A) Structures of the reporter plasmid (left) and wild-type (WT), M1 (SOX2 site mutant), and M2 (POU site mutant) Nes30 sequences. Altered nucleotides are shaded. Lowercase letters indicate linker sequences. An octamerized Nes30 sequence was inserted into the pδ51LucII reporter plasmid (22) in both normal (N) and reversed (R) orientations relative to the direction of transcription. (B) Activities of Nes30-WT, M1, and M2 in chicken primary brain cells and MNS70 neural stem cells. C. Activities of Nes30-WT in chicken primary liver cells, COS7 (kidney) cells, and 10T1/2 fibroblasts. The luciferase activity level generated by the reporter in the absence of enhancer was taken as 1.

FIG. 3.

Synergistic activation of the Nes30 enhancer by exogenous SOX2 and Brn2. (A) Schematic structure of the reporter and SOX2/Brn2-expressing effecter plasmids. The effecter plasmids had full-length cDNAs of SOX or POU factors, which are transcribed from the cytomegalovirus (CMV) promoter. (B) Activation of the Nes30 enhancer by SOX2 and Brn2. Various amounts of effecter plasmids designed to express SOX2 or Brn2, and the reporter plasmids, were transfected into liver cells. (N) and (R) indicate the orientation of the Nes30 enhancer in the reporter plasmid (normal and reversed, respectively). (C) Effect of mutations in the SOX binding site or POU factor binding site on Nes30 enhancer activity. The enhancer was in N orientation. The error bars indicate standard errors.

FIG. 4.

Binding of SOX2 and Brn2 to Nes30 DNA and their protein-protein interaction. (A) Recombinant SOX2(3-202) and Brn2(215-445) were synthesized as fusion proteins with GST and MBP, respectively. (B) Binding of SOX2(3-202) and MBP-Brn2(215-445) with GST removed to wild-type (WT) or mutant (M1 and M2) forms (Fig. 1B) of the Nes30 sequence in vitro, as assessed by EMSA. The recombinant proteins bind to the Nes30-WT sequence efficiently, either alone (lanes 1 and 2) or in combination (lane 3). In lane 3, a slowly migrating band representing a SOX2(3-202)/MBP-Brn2(215-445)/probe ternary complex is observed. A larger amount of Brn2(215-445) increased band intensity, and the formation of the ternary complex was stoichiometric (lanes 4 to 6). Mutation M1 attenuated binding of SOX2(3-202) (lanes 7 and 9), while mutation M2 disrupted the binding of Brn2(215-445) (lanes 11 and 12). (C) Protein interaction of SOX2 and Brn2 without Nes30 DNA. MBP-tagged proteins, LacZα, Brn2(215-445), and Pax6(1-169), were incubated with GST-SOX2(3-202) or GST and coprecipitated with glutathione-Sepharose, and the precipitated MBP fusion proteins were detected by immunoblotting using anti-MBP antibodies.

FIG. 5.

Effects of the arrangement of SOX and POU factor binding sites on the enhancer activity of Nes30. (A) Nucleotide sequences and arrangements of SOX2 and Oct3/4 binding sites found in the enhancers of Fgf4 (3), UTF1 (33), Sox2 (48), and HoxB1 (13) and comparison of those with SOX2 and POU binding sites in the Nes30 sequence. SOX2 and POU binding sequences are indicated by blue and red arrows, respectively. The orientations of both SOX2 and POU sites in the Nes30 sequence are in reverse, compared with the four enhancers indicated above. Two mutant sequences of Nes30 for the arrangement of SOX2 and POU binding sites were prepared. The Nes30-Swap mutant enhancer had the binding sequences in inverted orientations, where the inverted SOX binding sequence is underlined. The Nes30-Ins mutant enhancer had 10 bp inserted between SOX and POU factorbinding sites. (B) Binding of SOX2(3-202) and Brn2(215-445) to the Swap and Ins mutant sequences of Nes30. SOX2(3-202) (lanes 1, 4, and 7), MBP-Brn2(215-445) (lanes 2, 5, and 8), and the combination of SOX2(3-202) and MBP-Brn2(215-445) (lanes 3, 6, and 9) were mixed with the wild-type (WT) (lanes 1 to 3), Swap mutant (lanes 4 to 6), and Ins mutant (lanes 7 to 9). Nes30 probes and their binding were analyzed by EMSA. (C) Effects of Swap and Ins mutations on enhancer activity of Nes30 in transfected liver cells. Various amounts of plasmids to express SOX2 or Brn2 were transfected to liver cells, together with a luciferase-encoding reporter plasmid carrying an octamerized enhancer sequence. (N) and (R) indicate the orientation of the Nes30 enhancer in the reporter plasmid (normal and reversed, respectively). The Swap and Ins mutations both inactivated enhancer activity. The error bars indicate standard errors.

FIG. 6.

Selective combination of SOX and POU factors in activation of the Nes30 enhancer. Various amounts of plasmids to express SOX or POU factors were transfected into liver cells, together with a luciferase-encoding reporter plasmid carrying the octamerized enhancer sequence. (N) indicates the normal orientation of the Nes30 enhancer in the reporter plasmid. (A) Combinations of Brn2 with different SOX proteins. SOX1, SOX2, and SOX3 (group B1) strongly activated the enhancer, and SOX11 (group C) moderately activated the enhancer, while SOX9 (group E) and SOX14 and -21 (group B2) showed no activation. WT, wild type. The error bars indicate standard errors. (B) Combination of SOX2 with different POU factors. All class III POU factors (although Brn1 was relatively inactive) and class V Oct3/4, when combined with SOX2, activated the Nes30 enhancer by nearly 100-fold, whereas class II Oct2 did so moderately. In contrast, Brn5 did not activate the enhancer.

FIG. 7.

Expression of Nestin, group B1 SOX, SOX11, and Brn2 in E10.5 spinal cord. (A) Cross sections of E10.5 spinal cord at trunk level immunostained for Nestin (a), SOX2 (b), SOX1/(2)/3 (c), SOX11 (d), and Brn2 (e) and stained with DAPI (4′,6′-diamidino-2-phenylindole) (f). The scale bar indicates 100 μm. (B) High-magnification confocal images of median regions of the spinal cord (boxed in panel A) immunostained for SOX2 (a), SOX11 (b), SOX2 (green) and SOX11 (red) (c), SOX1/(2)/3 (d), Brn2 (e), and Brn2 (green) and Nestin (red) (f). The ventricle is toward the left. The scale bar indicates 50 μm.

FIG. 8.

Expression of Nestin, group B1 SOX, SOX11, and Brn2 in E13.5 spinal cord. (A) Cross sections of E13.5 spinal cord at trunk level immunostained for Nestin (a), SOX2 (b), SOX1/(2)/3 (c), SOX11 (d), Brn2 (e), and DAPI (4′,6′-diamidino-2-phenylindole) (f). The scale bar indicates 100 μm. (B) High-magnification confocal images of median regions of the spinal cord roughly corresponding to the rectangles in panel A, which were immunostained for SOX2 (a), SOX11 (b), SOX1/(2)/3 (c), SOX2 (green) and SOX11 (red) (d), Brn2 (e), and Brn2 (green) and Nestin (red) (f). The scale bar indicates 50 μm. The ventricle is toward the left. The ventricular zone (VZ), subventricular zone (SVZ), and intermediate zone (IZ) are indicated. Note that subcellular localization of Brn2 is clearly different in the VZ-SVZ and IZ. (C) Further enlargement of Brn2-immunostained specimens of the areas indicated in panel A, image e, which are also stained with DAPI (blue). In the ventricular zone (a), Brn2 signals are largely identified within the nucleus, although significant signals are also observed in the cytoplasm. In the nucleus, the heterochromatic regions strongly stained with DAPI are devoid of Brn2 signal. In the intermediate-zone areas (b and c), Brn2 signals are largely within the cytoplasm. The scale bar indicates 10 μm.

FIG. 9.

Model of Nestin gene regulation in the embryonic spinal cord. Nestin is expressed in the cells with their cell bodies in the ventricular zone (VZ) and subventricular zone (SVZ). In the ventricular zone, SOX1/2/3 (group B SOX) interact with Brn2 (and other class III POU factors) on the Nes30 sequence (Fig. 1 and 2) within the nucleus. In the subventricular zone, SOX11 (and possibly other group C SOX, i.e., SOX4 and SOX12) interacts with class III POU factors. In the cells with cell body localization in the intermediate zone (IZ), moderate expression of SOX11 and Brn2 also takes place, but sequestering the majority of Brn2 activity in the cytoplasm results in the absence of Nestin expression in these cells.