Interplay of SOX and POU factors in regulation of the Nestin gene in neural primordial cells.
Journal: 2004/December - Molecular and Cellular Biology
ISSN: 0270-7306
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.
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Mol Cell Biol 24(20): 8834-8846

Interplay of SOX and POU Factors in Regulation of the <em>Nestin</em> Gene in Neural Primordial Cells

Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan, Laboratory of Molecular Cell Biology and Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People’s Republic of China2
Corresponding author. Mailing address: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-7963. Fax: 81-6-6877-1738. E-mail: pj.ca.u-akaso.cmc.cph@65016j.
Received 2004 Jan 28; Revised 2004 Mar 12; Accepted 2004 Jul 23.

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.

Abstract

Specific gene regulation for the determination of neural primordial cells is of high interest for its possible contribution to stem cell biology, where neural tissues are of primary importance. The majority of the neural primordial cells present in the central nervous system (CNS) from embryonic to adult stages (2, 47) have been documented to express the intermediate filament protein Nestin (11, 28, 41). Given that transcriptional regulation determining a cell type is largely reflected by the regulation of genes specific to the cell type, we focused on the regulation of the Nestin gene to better understand gene regulation that is determinative for the neural primordial state.

Previous studies of rat and human Nestin genes indicated that Nestin expression in the CNS, at least during embryonic stages, is largely regulated by a nervous system-specific enhancer located in the 3′ half of the second intron (21, 29, 58, 61). After an analysis of a 257-bp-long Nestin enhancer in the rat, Josephson et al. (21) identified putative binding sites for POU, AP2, and a hormone-responsive element; further assessment of their involvement in Nestin enhancer activity was performed using transgenic mouse embryos. Mutational alterations of the downstream POU factor sites, bound by class III POU factors in vitro, largely eliminated the activity of the enhancer; mutations of other sites altered the domain of the CNS in which the enhancer showed activity (21). These previous results indicated that the activation of the Nestin neural enhancer is fundamentally dependent on the binding of a POU factor to the downstream site and that other nuclear factors may cooperate in establishing this enhancer activity.

POU family transcription factors contain the DNA-binding POU domain, consisting of the POU-specific domain and homeodomain, and are grouped into six classes (4, 19, 27). The class III POU factors, Brn1, Brn2, Brn4, and Oct6 (also called Tst1/SCIP), are widely expressed in the developing CNS, with extensive regional overlap (1, 18), and presumably share redundant functions. Corroborating this notion, single-knockout mice lacking Brn1, Brn2, Brn4, or Oct6 do not have significant neural phenotypes, and only the simultaneous loss of Brn1 and Brn2 in double-knockout mice causes a mild CNS phenotype (4, 30, 46).

Transcription factors expressed in tight association with the neural primordia are group B1 SOX proteins, represented by SOX2 and including SOX1 and SOX3 (8, 37, 40, 45, 51, 57). SOX proteins have similar DNA-binding HMG domains (24, 36, 53) and are classified into groups A through J according to the characteristics of the HMG domain and extra-HMG domain sequences (6).

Early neurogenesis in a variety of vertebrate systems is associated with expression of group B1 Sox genes (31, 37, 45, 49). In the chicken, an enhancer for Sox2 that directly responds to neural induction signals has been identified and has been shown to be conserved among amniotes (49). In Xenopus, forced expression of Sox2, in combination with basic fibroblast growth factor, initiates the neural development of the ectoderm (31). It has also been demonstrated that group B1 SOX activity is involved in maintaining the neural primordia in the embryonic neural tube (7, 15). Taken together, these observations indicate that the expression of group B1 SOX proteins is directly involved in establishing and maintaining neural primordial cell populations.

SOX and POU transcription factors often regulate genes interdependently, forming a complex on the DNA regulatory site (12, 24). In this study, we demonstrate that group B1 and group C SOX proteins interact with POU factors and activate the Nestin neural enhancer. In the embryonic spinal cord, Nestin expression occurs in the cells expressing group B1 SOX and Brn2 in their nuclei (ventricular zone) and in those expressing group C SOX and Brn2 in the nucleus (subventricular zone). In the intermediate zone, the cells coexpress SOX11 and Brn2; however, Brn2 is largely sequestered in the cytoplasm. Thus, the participation of the POU factors in transcriptional regulation is determined by their subcellular localization. This regulated interaction between transcription factors presumably underlies the mechanisms that the determine the neural primordial cell state.

Acknowledgments

We thank members of the Kondoh laboratory for stimulating discussions and Masato Nakafuku for the provision of MNS70 cells.

This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12CE2007) and from the Human Frontier Science Program (RGP0040/2001-M) to H.K. and from the National Key Basic Research and Developmental Program (G1999054000 and 2002CB713802) and the National Natural Science Foundation of China (39870283 and 39930090) to N.J.

Acknowledgments

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