Inhibition of DNA binding of Sox2 by the SUMO conjugation

doi:10.1016/j.bbrc.2006.10.130

Biochemical and Biophysical Research Communications

Volume 351, Issue 4, 29 December 2006, Pages 920-926

https://doi.org/10.1016/j.bbrc.2006.10.130 Get rights and content

Abstract

Sox2 is a member of the high mobility group (HMG) domain DNA-binding proteins for transcriptional control and chromatin architecture. The HMG domain of Sox2 binds the DNA to facilitate transactivation by the cooperative transcription factors such as Oct3/4. We report that mouse Sox2 is modified by SUMO at lysine 247. Substitution of the target lysine to arginine lost the sumoylation but little affected transcriptional potential or nuclear localization of Sox2. By contrast with the unmodified form, Sox2 fused to SUMO-1 did not augment transcription via the Fgf4 enhancer in the presence of Oct3/4. Further, SUMO-1-conjugated Sox2 at the lysine 247 or at the carboxyl terminus reduced the binding to the Fgf4 enhancer. These indicate that Sox2 sumoylation negatively regulates its transcriptional role through impairing the DNA binding.

Section snippets

Materials and methods

Antibodies and cell lines. Rabbit anti-Sox2 [14] and goat anti-GST (Amersham Pharmacia Biotech) polyclonal antibodies, and mouse anti-Oct3/4 (C-10) (Santa Cruz Biotechnology), anti-GMP-1 (SUMO-1, Zymed Laboratories), and anti-FLAG M5 (Sigma) monoclonal antibodies were used. HeLa, AZ-521, AGS cells, and mouse embryonic stem cells were used in this study.

Construction of expression plasmids. As described in Supplementary Materials and Methods, pcDNA3-FLAG (pcDNA3-FLAG-Sox2 WT, pcDNA3-FLAG-Oct3/4

Sumoylation of Sox2 at lysine 247

We examined whether Sox2 possesses the consensus sequences for SUMO conjugation. Because amino acids 246–249 of mouse Sox2 coexist with the minimal consensus sequence, Lys²⁴⁷ seemed to be a putative SUMO acceptor site (Fig. 1A). The lysine residue of Sox2 is perfectly conserved among species, including human, mouse, and rat. To test whether Sox2 can be conjugated with SUMO at the target site, we generated two plasmids to express Sox2 mutant in which Lys²⁴⁷ was substituted with arginine or

Discussion

In the present study, we report that Sox2 is a substrate for SUMO modification at the lysine 247 in vivo and in vitro. As was the case of other transcription factors, a relatively small proportion of Sox2 was found as the conjugated form. It is of great interest that sumoylated Sox2 either at lysine 247 or at the carboxyl terminus markedly reduced the binding to the Fgf4 enhancer in the presence of Oct3/4 (Fig. 4), indicating that SUMO modification of Sox2 inhibits the DNA-binding activity. The

Acknowledgments

This work was supported by a Grant-in-Aid for 21st Century Center of Excellence (COE) Research, and by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (M.N.). S.T. is a COE Junior Research Associate.

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HMG proteins: dynamic players in gene regulation and differentiation
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SOX2 is a transcription factor belonging to the SOX gene family, whose activity has been associated with the maintenance of the stemness and self-renewal of embryonic stem cells (ESCs), as well as the induction of differentiated cells into induced pluripotent stem cells (iPSCs). Moreover, accumulating studies have shown that SOX2 is amplified in various cancers, notably in esophageal squamous cell carcinoma (ESCC). In addition, SOX2 expression is linked to multiple malignant processes, including proliferation, migration, invasion, and drug resistance. Taken together, targeting SOX2 might shed light on novel approaches for cancer therapy. In this review, we aim to summarize the current knowledge regarding SOX2 in the development of esophagus and ESCC. We also highlight several therapeutic strategies for targeting SOX2 in different cancer types, which can provide new tools to treat cancers possessing abnormal levels of SOX2 protein.
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Citation Excerpt :
This article contains supporting information (14, 49, 50, 56, 59–61, 68, 72, 74, 75, 81, 84, 94–110).
Protein O-GlcNAcylation is a dynamic posttranslational modification that is catalyzed by the enzyme O-GlcNAc transferase (OGT) and is essential for neurodevelopment and postnatal neuronal function. Missense mutations in OGT segregate with a novel X-linked intellectual disability syndrome, the OGT congenital disorder of glycosylation (OGT-CDG). One hypothesis for the etiology of OGT-CDG is that loss of OGT activity leads to hypo-O-GlcNAcylation of as yet unidentified, specific neuronal proteins, affecting essential embryonic, and postnatal neurodevelopmental processes; however, the identity of these O-GlcNAcylated proteins is not known. Here, we used bioinformatic techniques to integrate sequence conservation, structural data, clinical data, and the available literature to identify 22 candidate proteins that convey OGT-CDG. We found using gene ontology and PANTHER database data that these candidate proteins are involved in diverse processes including Ras/MAPK signaling, translational repression, cytoskeletal dynamics, and chromatin remodeling. We also identify pathogenic missense variants at O-GlcNAcylation sites that segregate with intellectual disability. This work establishes a preliminary platform for the mechanistic dissection of the links between protein O-GlcNAcylation and neurodevelopment in OGT-CDG.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Sumoylation of Sox2 at lysine 247

Discussion

Acknowledgments

References (25)

HMG proteins: dynamic players in gene regulation and differentiation

Curr. Opin. Genet. Dev.

Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators

Curr. Opin. Genet. Dev.

Pairing SOX off: with partners in the regulation of embryonic development

Trends Genet.

Modulation of the activity of multiple transcriptional activation domains by the DNA binding domains mediates the synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4 enhancer

J. Biol. Chem.

SUMO promotes HDAC-mediated transcriptional repression

Mol. Cell

Genetic players in esophageal atresia and tracheoesophageal fistula

Curr. Opin. Genet. Dev.

Repression of SOX6 transcriptional activity by SUMO modification

FEBS Lett.

Sumoylation of the SOX10 transcription factor regulates its transcriptional activity

FEBS Lett.

The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system

J. Biol. Chem.

Overproduction of eukaryotic SUMO-1- and SUMO-2-conjugated proteins in Escherichia coli

Anal. Biochem.

SUMO-1 modification represses Sp3 transcriptional activation and modulates its subnuclear localization

Mol. Cell

Multipotent cell lineages in early mouse development depend on SOX2 function

Genes Dev.

Cited by (100)

SUMOylation effects on neural stem cells self-renewal, differentiation, and survival

MYB regulates the SUMO protease SENP1 and its novel interaction partner UXT, modulating MYB target genes and the SUMO landscape

The roles of the SOX2 protein in the development of esophagus and esophageal squamous cell carcinoma, and pharmacological target for therapy

SUMO control of nervous system development

p38 MAPK-dependent phosphorylation of transcription factor SOX2 promotes an adaptive response to BRAF inhibitors in melanoma cells

Bioinformatic prediction of putative conveyers of O-GlcNAc transferase intellectual disability