Structural basis for nuclear import selectivity of pioneer transcription factor SOX2

Jagga, Bikshapathi; Edwards, Megan R.; Pagin, Miriam; Wagstaff, Kylie M.; Aragão, D.; Roman, Noelia; Nanson, J.D.; Raidal, Shane R.; Dominado, Nicole; Stewart, Murray; Jans, David A.; Hime, Gary R.; Nicolis, Silvia K.; Basler, Christopher F.; Forwood, Jade K.

doi:10.1038/s41467-020-20194-0

Cited by 32 publications

(35 citation statements)

References 67 publications

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“…These observations made us ask if re‐expression of Sox2 into Sox2‐deleted cells 5 (Supplementary Figure 4) would also reincrease levels of Fos and Socs3 expression. As expected, Sox2 re‐expression in Sox2‐deleted NSC fully recovered normal long‐term self‐renewal (Supplementary Figure 4); by contrast, empty vector transduction is unable to rescue long‐term self‐renewal of Sox2‐deleted NSC (Reference 31 and data not shown). A selective advantage of Sox2‐transduced cells was again observed by FACS analysis through passaging, until almost 100% of the cells were represented by transduced cells (Supplementary Figure 4).…”

Section: Resultssupporting

confidence: 70%

Sox2 Controls Neural Stem Cell Self-Renewal Through a Fos-Centered Gene Regulatory Network

et al. 2021

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The Sox2 transcription factor is necessary for the long-term self-renewal of neural stem cells (NSCs). Its mechanism of action is still poorly defined. To identify molecules regulated by Sox2, and acting in mouse NSC maintenance, we transduced, into Sox2-deleted NSC, genes whose expression is strongly downregulated following Sox2 loss (Fos, Jun, Egr2), individually or in combination. Fos alone rescued long-term proliferation, as shown by in vitro cell growth and clonal analysis. Furthermore, pharmacological inhibition by T-5224 of FOS/JUN AP1 complex binding to its targets decreased cell proliferation and expression of the putative target Suppressor of cytokine signaling 3 (Socs3). Additionally, Fos requirement for efficient long-term proliferation was demonstrated by the reduction of NSC clones capable of long-term expansion following CRISPR/Cas9-mediated Fos inactivation. Previous work showed that the Socs3 gene is strongly downregulated following Sox2 deletion, and its re-expression by lentiviral transduction rescues long-term NSC proliferation. Fos appears to be an upstream regulator of Socs3, possibly together with Jun and Egr2; indeed, Sox2 re-expression in Sox2-deleted NSC progressively activates both Fos and Socs3 expression; in turn, Fos transduction activates Socs3 expression. Based on available SOX2 ChIPseq and ChIA-PET data, we propose a model whereby Sox2 is a direct activator of both Socs3 and Fos, as well as possibly Jun and Egr2; furthermore, we provide direct evidence for FOS and JUN binding on Socs3 promoter, suggesting direct transcriptional regulation. These results provide the basis for developing a model of a network of interactions, regulating critical effectors of NSC proliferation and long-term maintenance.

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Section: Resultssupporting

confidence: 70%

Sox2 Controls Neural Stem Cell Self-Renewal Through a Fos-Centered Gene Regulatory Network

et al. 2021

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“…Sox2-del NSC transduction with lentiviral constructs, expressing Sox2 , Fos , or Socs3 , was as in [ 8 , 9 , 14 ]. After 6–8 passages in NSC proliferation medium [ 8 , 9 ], during which essentially all cells had become positive for the virally transduced Sox2 , or Fos , or Socs3 expression construct [ 8 , 9 ], cells were induced to differentiate.…”

Section: Methodsmentioning

confidence: 99%

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes

Pagin

Pernebrink

Pitasi

et al. 2021

Cells

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The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC’s long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a “mixed” neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the “neuronal” genes are bound by both SOX2 and AP1.

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“…This suggests that in the absence of the rest of the sequence in the HMG box, the presumed helical segment of the iPep is no longer stabilized in its helical conformation due to the loss of the above interactions with the neighboring helix and coil. A recent structure of SOX2 NLS [35] from the N-and C-terminal (similar in sequence to SOX2 iPep) bound to importin-α in the minor groove and major groove also showed disordered conformation in alignment with the CD data. In contrast, when bound to nucleosomes [36,37], these NLS regions are in close proximity, and in a closed conformation in particular the region 57 KRLRALHMKEH 67 is helical.…”

Section: Secondary Structure Of Sox2 Ipepmentioning

confidence: 57%

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor

Gandhi

Wang

Sorolla

et al. 2021

IJMS

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SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

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Structural basis for nuclear import selectivity of pioneer transcription factor SOX2

Cited by 32 publications

References 67 publications

Sox2 Controls Neural Stem Cell Self-Renewal Through a Fos-Centered Gene Regulatory Network

Sox2 Controls Neural Stem Cell Self-Renewal Through a Fos-Centered Gene Regulatory Network

FOS Rescues Neuronal Differentiation of Sox2-Deleted Neural Stem Cells by Genome-Wide Regulation of Common SOX2 and AP1(FOS-JUN) Target Genes

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor

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