High Mobility Group Domain Research Articles

Nuclear Expression of the Sox 11 Transcription Factor in Mantle Cell Lymphoma, and Cytoplasmic Expression in Follicular Lymphoma and Multiple Myeloma: Pathogenetic Implications.

Abstract 2925Poster Board II-901Mantle cell lymphoma (MCL) is characterized by nuclear cyclin D1 expression resulting from the t(11;14)(q13;q32). As cyclin D1 overexpression alone is insufficient for B-cell transformation, we have investigated other potentially contributing mutations. Sox11 is a member of a large family of transcription factors containing a DNA-binding high-mobility group (HMG) domain and shares homology with Sox4, which is involved in lymphopoiesis. Recent reports have identified Sox11 expression in the majority of MCL, suggesting it may contribute to pathogenesis (Ek et al, Blood 2008;111:800; Wang et al, Br J Haematol 2008;143:248). Methods:Patients with MCL diagnosed at the Univ. of Virginia from 1997-2008 and for whom nodal, marrow or spleen tissue blocks were available were identified from the Pathology database. Follicular lymphoma (FL), marginal zone lymphoma (MZL), small lymphocytic lymphoma (SLL), hairy cell leukemia (HCL) and multiple myeloma (MM) were also analyzed. Formalin-fixed, paraffin-embedded samples were stained by immunohistochemistry for cyclinD1, Sox11 (rabbit a/Sox11, 1:100, Sigma) and Ki-67. Nuclear and/or cytoplasmic expression was determined by two of us (YT, JC). Results:All MCL samples revealed nuclear cyclin D1 expression. Nuclear but not cytoplasmic Sox11 expression was identified in all 24 non-blastoid and in 5/6 blastoid samples; 1 blastoid sample showed only cytoplasmic Sox11 staining (Table). Four of 5 HCL expressed cyclin D1 but were negative for Sox11; 4/5 MM also were cyclin D1-positive, with 3 positive for cytoplasmic Sox11 including 2 of the cyclin D1-positive cases. All 5 FL showed cytoplasmic Sox11 positivity, whereas all MZL and SLL were negative for cytoplasmic or nuclear staining.TypePatientsSamplesNuclear Cyclin D1+Nuclear Sox11+Cytoplasmic Sox11+MCL, non –blastoid222424240MCL, blastoid56651FL55N.D.105MZL66000SLL66000HCL255400MM3554031N.D. = Not Done23 spleen, 2 bone marrow samples3Bone marrow samples Conclusions:Nuclear Sox11 expression was uniformly identified in MCL, with the exception of cytoplasmic expression in a single blastoid case. No nuclear Sox11 was present in HCL or MM despite the expression of cyclin D1, although 3 MM showed cytoplasmic Sox11 staining. These findings suggest that nuclear Sox11 overexpression is not a direct result of dysregulated cyclin D1 signaling but instead occurs by alternative mechanisms. The significance of cytoplasmic staining remains uncertain. A potential pathogenetic role for Sox11 and possibly other Sox family transcription factors warrants further investigation in MCL and other lymphoproliferative neoplasms for diagnostic use and therapeutic targeting. Disclosures:No relevant conflicts of interest to declare.

Novel SOX2 mutations and genotype–phenotype correlation in anophthalmia and microphthalmia

SOX2 represents a High Mobility Group domain containing transcription factor that is essential for normal development in vertebrates. Mutations in SOX2 are known to result in a spectrum of severe ocular phenotypes in humans, also typically associated with other systemic defects. Ocular phenotypes include anophthalmia/microphthalmia (A/M), optic nerve hypoplasia, ocular coloboma and other eye anomalies. We screened 51 unrelated individuals with A/M and identified SOX2 mutations in the coding region of the gene in 10 individuals. Seven of the identified mutations are novel alterations, while the remaining three individuals carry the previously reported recurrent 20-nucleotide deletion in SOX2, c.70del20. Among the SOX2-positive cases, seven patients had bilateral A/M and mutations resulting in premature termination of the normal protein sequence (7/38; 18% of all bilateral cases), one patient had bilateral A/M associated with a single amino acid insertion (1/38; 3% of bilateral cases), and the final two patients demonstrated unilateral A/M associated with missense mutations (2/13; 15% of all unilateral cases). These findings and review of previously reported cases suggest a potential genotype/phenotype correlation for SOX2 mutations with missense changes generally leading to less severe ocular defects. In addition, we report a new familial case of affected siblings with maternal mosaicism for the identified SOX2 mutation, which further underscores the importance of parental testing to provide accurate genetic counseling to families.

Familial Frameshift SRY Mutation Inherited from a Mosaic Father with Testicular Dysgenesis Syndrome

The SRY gene encodes a transcription factor responsible for initiating testis differentiation. Mutations in SRY almost always result in XY sex reversal with pure gonadal dysgenesis and an increased risk of gonadal tumor. Most of these mutations are de novo, affecting only one individual in a family. Only a small subset of mutations is shared between a phenotypically normal father and one or more of his affected children. Incomplete penetrance and somatic mosaicism are two hypotheses that may explain a normal phenotype in a father carrying a SRY mutation. We describe a family with two sisters with XY sex reversal and pure gonadal dysgenesis and a phenotypically normal brother. A novel constitutional frameshift SRY mutation was identified in both sisters and was absent in the brother. The single base pair deletion (c.71delA) led to a premature stop codon in position 60 of the protein, removing entirely the high-mobility group domain and the DNA-binding domain of SRY. The father of the three children presented with hypospadias; cryptorchidism; testicular seminoma and oligoasthenozoospermia, an association termed testicular dysgenesis syndrome (TDS); and the SRY mutation in a mosaic state in the peripheral blood and the tumor. This observation of somatic and germinal mosaicism for a SRY mutation may explain the variable penetrance in some familial gonadal dysgenesis. Importantly, the present report is the first one describing the association of SRY mutation in a male with TDS. This suggests that mutations in a sex-determining gene may contribute to the pathogenesis of TDS.

A new mechanism of SOX9 action to regulate PKCα expression in the intestine epithelium

Variations of protein kinase C (PKC) expression greatly influence the proliferation-to-differentiation transition (PDT) of intestinal epithelial cells and might have an important impact on intestinal tumorigenesis. We demonstrate here that the expression of PKCalpha in proliferating intestinal epithelial cells is repressed both in vitro and in vivo by the SOX9 transcription factor. This repression does not require DNA binding of the SOX9 high-mobility group (HMG) domain but is mediated through a new mechanism of SOX9 action requiring the central and highly conserved region of SOXE members. Because SOX9 expression is itself upregulated by Wnt-APC signaling in intestinal epithelial cells, the present study points out this transcription factor as a molecular link between the Wnt-APC pathway and PKCalpha. These results provide a potential explanation for the decrease of PKCalpha expression in colorectal cancers with constitutive activation of the Wnt-APC pathway.

The Armadillo Repeat-containing Protein, ARMCX3, Physically and Functionally Interacts with the Developmental Regulatory Factor Sox10

Sox10 is a member of the group E Sox transcription factor family and plays key roles in neural crest development and subsequent cellular differentiation. Sox10 binds to regulatory sequences in target genes via its conserved high mobility group domain. In most cases, Sox10 exerts its transcriptional effects in concert with other DNA-binding factors, adaptor proteins, and nuclear import proteins. These interactions can lead to synergistic gene activation and can be cell type-specific. In earlier work, we demonstrated that Sox10 transactivates the nicotinic acetylcholine receptor alpha3 and beta4 subunit genes and does so only in neuronal-like cell lines, raising the possibility that Sox10 mediates its effects via interactions with co-regulatory factors. Here we describe the identification of the armadillo repeat-containing protein, ARMCX3, as a Sox10-interacting protein. Biochemical analyses indicate that ARMCX3 is an integral membrane protein of the mitochondrial outer membrane. Others have shown that Sox10 is a nucleocytoplasmic shuttling protein. We extend this observation and demonstrate that, in the cytoplasm, Sox10 is peripherally associated with the mitochondrial outer membrane. Both Sox10 and ARMCX3 are expressed in mouse brain and spinal cord as well as several cell lines. Overexpression of ARMCX3 increased the amount of mitochondrially associated Sox10. In addition, although ARMCX3 does not possess intrinsic transcriptional activity, it does enhance transactivation of the nicotinic acetylcholine receptor alpha3 and beta4 subunit gene promoters by Sox10. These results suggest that Sox10 is a membrane-associated factor whose transcriptional function is increased by direct interactions with ARMCX3 and raise the possibility of a signal transduction cascade between the nucleus and mitochondria through Sox10/ARMCX3 interactions.

The Structure of Sox17 Bound to DNA Reveals a Conserved Bending Topology but Selective Protein Interaction Platforms

Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 Å resolution. Sox17 targets the minor groove and bends the DNA by approximately 80°. The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness.

Aplasia of cochlear nerves and olfactory bulbs in association with SOX10 mutation

A 17-month-old boy was referred with profound sensorineural hearing loss (SNHL), severe visual impairment and developmental delay. Neuroimaging identified hypomyelination and cochlear nerve aplasia. He was noted to have fair skin and hair and multiple areas of cutaneous hyperpigmentation. Previous investigations including karyotype, array comparative genomic hybridization (aCGH) and a full metabolic screen were normal. A novel missense mutation of the highly conserved high mobility group (HMG) domain of SOX10 was identified (Q174P:c.521A>C). This case represents the first description of aplasia of the cochlear nerve due to a SOX10 mutation.

The histone methylase KMTox interacts with the redox‐sensor peroxiredoxin‐1 and targets genes involved in Toxoplasma gondii antioxidant defences

The ability of living cells to alter their gene expression patterns in response to environmental changes is essential for viability. Oxidative stress represents a common threat for all aerobic life. In normally growing cells, in which hydrogen peroxide generation is transient or pulsed, the antioxidant systems efficiently control its concentration. Intracellular parasites must also protect themselves against the oxidative burst imposed by the host. In this work, we have investigated the role of KMTox, a new histone lysine methyltransferase, in the obligate intracellular parasite Toxoplasma gondii. KMTox is a nuclear protein that holds a High Mobility Group domain, which is thought to recognize bent DNA. The enzyme methylates both histones H4 and H2A in vitro with a great preference for the substrate in reduced conditions. Importantly, KMTox interacts specifically with the typical 2-cys peroxiredoxin-1 and the binding is to some extent enhanced upon oxidation. It appears that the cellular functions that are primarily regulated by the KMTox are antioxidant defences and maintenance of cellular homeostasis. KMTox may regulate gene expression in T. gondii by providing the rapid re-arrangement of chromatin domains and by interacting with the redox-sensor TgPrx1 contribute to establish the antioxidant 'firewall' in T. gondii.

Activation of Wingless Targets Requires Bipartite Recognition of DNA by TCF

Specific recognition of DNA by transcription factors is essential for precise gene regulation. In Wingless (Wg) signaling in Drosophila, target gene regulation is controlled by T cell factor (TCF), which binds to specific DNA sequences through a high mobility group (HMG) domain. However, there is considerable variability in TCF binding sites, raising the possibility that they are not sufficient for target location. Some isoforms of human TCF contain a domain, termed the C-clamp, that mediates binding to an extended sequence in vitro. However, the significance of this extended sequence for the function of Wnt response elements (WREs) is unclear. In this report, we identify a cis-regulatory element that, to our knowledge, was previously unpublished. The element, named the TCF Helper site (Helper site), is essential for the activation of several WREs. This motif greatly augments the ability of TCF binding sites to respond to Wg signaling. Drosophila TCF contains a C-clamp that enhances in vitro binding to TCF-Helper site pairs and is required for transcriptional activation of WREs containing Helper sites. A genome-wide search for clusters of TCF and Helper sites identified two new WREs. Our data suggest that DNA recognition by fly TCF occurs through a bipartite mechanism, involving both the HMG domain and the C-clamp, which enables TCF to locate and activate WREs in the nucleus.

Purification, crystallization and preliminary X-ray diffraction analysis of the HMG domain of Sox17 in complex with DNA.

Sox17 is a member of the SRY-related high-mobility group (HMG) of transcription factors that have been shown to direct endodermal differentiation in early mammalian development. The LAMA1 gene encoding the alpha-chain of laminin-1 has been reported to be directly bound and regulated by Sox17. This paper describes the details of initial crystallization attempts with the HMG domain of mouse Sox17 (mSox17-HMG) with a 16-mer DNA element derived from the LAMA1 enhancer and optimization strategies to obtain a better diffracting crystal. The best diffracting crystal was obtained in a condition containing 0.1 M Tris-HCl pH 7.4, 0.2 M MgCl(2), 30% PEG 3350 using the hanging-drop vapour-diffusion method. A highly redundant in-house data set was collected to 2.75 A resolution with 99% completeness. The presence of the mSox17-HMG-DNA complex within the crystals was confirmed and Matthews analysis indicated the presence of one complex per asymmetric unit.

Microsporidia Evolved from Ancestral Sexual Fungi

Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested microsporidia share a common ancestor with fungi [2-7]. However, the exact nature of this phylogenetic relationship is unclear because of unusual features of microsporidial genomes, which are compact with fewer and highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, or whether microsporidia are a sister group to all fungi. Here, we present evidence addressing this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster governing sexual reproduction in which a high mobility group (HMG) transcription-factor gene is flanked by triose-phosphate transporter (TPT) and RNA helicase genes [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order. Genome-wide synteny analysis reveals multiple other loci conserved between microsporidia and zygomycetes to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor and suggest microsporidia may have an extant sexual cycle.

Analysis of the MAT1-2-1 gene of Colletotrichum lindemuthianum

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.

Analysis of the <i>MAT1-2-1</i> gene of <i>Colletotrichum lindemuthianum</i>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.

Mating Type and the Genetic Basis of Self-Fertility in the Model Fungus Aspergillus nidulans

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.

A Dominant-negative Form of Mouse SOX2 Induces Trophectoderm Differentiation and Progressive Polyploidy in Mouse Embryonic Stem Cells

SOX2 plays an important role in early embryogenesis by cooperating with OCT4 in regulating gene expression in fertilized eggs, yet the precise mechanism through which SOX2 accomplishes this important function remains poorly understood. Here, we describe the identification of two nuclear localization signals (NLS) in SOX2 and the generation of a dominant-negative mutant (Dmu-mSox2) by mutating these two NLS in its high mobility group domain. Characterization of this mutant demonstrated that SOX2 shuttles between the cytoplasm and nucleus using these two NLS. The mutant has lost its ability to interact with OCT4, but remains competent to interact with wild-type SOX2. Functionally, Dmu-mSox2 is inactive and unable to cooperate with OCT4 in transactivating target promoters bearing its binding sites. However, Dmu-mSox2 is able to inhibit the activity of wild-type SOX2 and subsequently suppress the activity of downstream genes such as Oct4 and Nanog. When stably expressed in embryonic stem (ES) cells, Dmu-mSox2 triggered progressive doublings of cell ploidy (>8N), leading to differentiation into the trophectoderm lineage. Knockdown of Sox2 by small interfering RNA also induced trophectoderm differentiation and polyploid formation in mouse ES cells. These results suggest that SOX2 maintains stem cell pluripotency by shuttling between the nucleus and cytoplasm in cooperation with OCT4 to prevent trophectoderm differentiation and polyploid formation in ES cells.

SOX6 Suppresses Cyclin D1 Promoter Activity by Interacting with β-Catenin and Histone Deacetylase 1, and Its Down-regulation Induces Pancreatic β-Cell Proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from beta-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with beta-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of beta-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withbeta-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Sox15 and Fhl3 transcriptionally coactivate Foxk1 and regulate myogenic progenitor cells

The regulation of myogenic progenitor cells during muscle regeneration is not clearly understood. We have previously shown that the Foxk1 gene, a member of the forkhead/winged helix family of transcription factors, is expressed in myogenic progenitor cells in adult skeletal muscle. In the present study, we utilize transgenic technology and demonstrate that the 4.6 kb upstream fragment of the Foxk1 gene directs beta-galactosidase expression to the myogenic progenitor cell population. We further establish that Sox15 directs Foxk1 expression to the myogenic progenitor cell population, as it binds to an evolutionarily conserved site and recruits Fhl3 to transcriptionally coactivate Foxk1 gene expression. Knockdown of endogenous Sox15 results in perturbed cell cycle kinetics and decreased Foxk1 expression. Furthermore, Sox15 mutant mice display perturbed skeletal muscle regeneration, due in part to decreased numbers of satellite cells and decreased Foxk1 expression. These studies demonstrate that Sox15, Fhl3 and Foxk1 function to coordinately regulate the myogenic progenitor cell population and skeletal muscle regeneration.

Human SSRP1 Has Spt16-dependent and -independent Roles in Gene Transcription

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were approximately 170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 ( approximately 1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells.

Direct Interactions of Runx2 and Canonical Wnt Signaling Induce FGF18

Canonical Wnt signaling is clearly required for skeletal development and bone formation. However, the targets of Wnt signaling that convert this signal into bone are unclear. Identification of these targets will yield insight into normal bone physiology and suggest new therapeutics for treatment of bone disease. Here we show that an essential regulator of bone development, FGF18, is a direct target of canonical Wnt signaling. A single DNA binding site for the Wnt-dependent transcription factors TCF/Lef accounted for the stimulation of the fgf18 promoter in response to Wnt signaling. Additionally, targeted disruption of betacat blocked fgf18 expression in vivo. Partially overlapping the TCF/Lef binding site is a Runx2 binding site and experiments showed that Runx2 and TCF/Lef work cooperatively to induce fgf18 expression. RNA interference knockdown of Runx2 inhibited and Runx2 forced expression augmented the induction of fgf18 by canonical Wnt signaling. Significantly, Runx2 formed a complex with Lef1 or TCF4 and this complex bound the composite binding site in the fgf18 promoter. These results demonstrate that two transcription pathways that are essential for bone, physically and functionally converge at the fgf18 promoter.

Inhibition of DNA binding of Sox2 by the SUMO conjugation

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.