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Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS

View Article: PubMed Central - PubMed

ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

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Functional interactions between Grg4 and HDAC/Tcf4 are required to repress Gli2A induction of Nkx2.2 in vivo.(A) Co-transfection of Tcf4R with Gli2A (both at (at 2.0 μg/μl) suppresses induction of Nkx2.2. (B) Predicted repressor complex for Tcf4R-mediated repression. (C, D) Co-transfection of a full-length Grg4 construct with Gli2A+TcfR also suppresses Nkx2.2 induction. (E-H) Co-transfection of Grg4 deletion constructs with Gli2A+TcfR. Both Grg4-ΔQ and Grg4-Q domain proteins prevent Tcf4R from blocking Gli2A-mediated induction of Nkx2.2 (seen in A). Antibody staining for the Myc-epitope tag was used to detect Grg4 constructs in all figures; GFP expression marks cells transfected with Tcf4R, while Gli activity was monitored by assaying Nkx2.2 expression. (I) Quantification of induced Nkx2.2 cells in each experiment, (J) Model for the regulation of Nkx2.2 by Tcf repressors involving HDAC activity and chromatin remodeling. *p<0.001, **p>0.05.
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pone.0163267.g004: Functional interactions between Grg4 and HDAC/Tcf4 are required to repress Gli2A induction of Nkx2.2 in vivo.(A) Co-transfection of Tcf4R with Gli2A (both at (at 2.0 μg/μl) suppresses induction of Nkx2.2. (B) Predicted repressor complex for Tcf4R-mediated repression. (C, D) Co-transfection of a full-length Grg4 construct with Gli2A+TcfR also suppresses Nkx2.2 induction. (E-H) Co-transfection of Grg4 deletion constructs with Gli2A+TcfR. Both Grg4-ΔQ and Grg4-Q domain proteins prevent Tcf4R from blocking Gli2A-mediated induction of Nkx2.2 (seen in A). Antibody staining for the Myc-epitope tag was used to detect Grg4 constructs in all figures; GFP expression marks cells transfected with Tcf4R, while Gli activity was monitored by assaying Nkx2.2 expression. (I) Quantification of induced Nkx2.2 cells in each experiment, (J) Model for the regulation of Nkx2.2 by Tcf repressors involving HDAC activity and chromatin remodeling. *p<0.001, **p>0.05.

Mentions: We next sought to establish whether Gro/TLE/Grg proteins act by bridging Tcf4 repressor and HDAC1 functions to regulate progenitor gene patterning in the ventral spinal cord. Gro/TLE/Grg factors are multi-domain proteins that do not bind directly to DNA but rather function to bring together transcription factors and other proteins in a repressor complex [8]. To do this, we performed transfection assays in chick embryos using full-length and truncated forms of the Grg4 protein, which was chosen on the basis of prior studies in mouse and chick that showed it is one of two Gro/TLE/Grg family genes expressed in ventral neural progenitors during neurogenesis in chicken and mouse embryos [5]. Co-transfection of a full-length Grg4 protein with Gli2A+Tcf4R did not block inhibition of Nkx2.2 (Fig 4C, 4D and 4I). We next co-transfected truncated forms of Grg4 that were missing key protein-protein interaction domains. We reasoned that mis-expression of a Grg4-ΔQ protein lacking the Tcf-binding and tetramerization “Q-domain” but retaining the “GP-domain” that binds to HDAC [6,8] along with Gli2A+Tcf4R should block the ability of Tcf4R to repress the induction of Nkx2.2 by dominantly interfering with the ability of endogenous Grg proteins to form a Tcf4-Grg-HDAC repressor complex at the endogenous Nkx2.2 locus (see Fig 4F). Consistent with this, there was a significant increase in the number of Nkx2.2+ cells in triple-transfected embryos (Fig 4E and 4I) compared to controls transfected with Gli2A+Tcf4R alone (Fig 4A). Similarly, co-transfection of a Grg4 protein containing only the Q domain but not the GP domain (“Grg4-Q”) should also block complex formation by inhibiting the association of endogenous Grg proteins with Tcf4 (Fig 4H). As in the experiments above, a significant increase in the number of Nkx2.2+ cells were detected in triple-transfected embryos, compared to controls (Fig 4G and 4I). We also examined Olig2 expression as a control, and found no changes in any experiment (S4 Fig). Taken together, these results indicate that Tcf4 repressor activity requires functional association with both Grg and HDAC proteins, and suggest a model for how Tcf repressor activity selectively regulates the domain-restricted expression of Nkx2.2 via an HDAC-dependent mechanism, possibly involving chromatin modification (Fig 4J).


Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS
Functional interactions between Grg4 and HDAC/Tcf4 are required to repress Gli2A induction of Nkx2.2 in vivo.(A) Co-transfection of Tcf4R with Gli2A (both at (at 2.0 μg/μl) suppresses induction of Nkx2.2. (B) Predicted repressor complex for Tcf4R-mediated repression. (C, D) Co-transfection of a full-length Grg4 construct with Gli2A+TcfR also suppresses Nkx2.2 induction. (E-H) Co-transfection of Grg4 deletion constructs with Gli2A+TcfR. Both Grg4-ΔQ and Grg4-Q domain proteins prevent Tcf4R from blocking Gli2A-mediated induction of Nkx2.2 (seen in A). Antibody staining for the Myc-epitope tag was used to detect Grg4 constructs in all figures; GFP expression marks cells transfected with Tcf4R, while Gli activity was monitored by assaying Nkx2.2 expression. (I) Quantification of induced Nkx2.2 cells in each experiment, (J) Model for the regulation of Nkx2.2 by Tcf repressors involving HDAC activity and chromatin remodeling. *p<0.001, **p>0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036887&req=5

pone.0163267.g004: Functional interactions between Grg4 and HDAC/Tcf4 are required to repress Gli2A induction of Nkx2.2 in vivo.(A) Co-transfection of Tcf4R with Gli2A (both at (at 2.0 μg/μl) suppresses induction of Nkx2.2. (B) Predicted repressor complex for Tcf4R-mediated repression. (C, D) Co-transfection of a full-length Grg4 construct with Gli2A+TcfR also suppresses Nkx2.2 induction. (E-H) Co-transfection of Grg4 deletion constructs with Gli2A+TcfR. Both Grg4-ΔQ and Grg4-Q domain proteins prevent Tcf4R from blocking Gli2A-mediated induction of Nkx2.2 (seen in A). Antibody staining for the Myc-epitope tag was used to detect Grg4 constructs in all figures; GFP expression marks cells transfected with Tcf4R, while Gli activity was monitored by assaying Nkx2.2 expression. (I) Quantification of induced Nkx2.2 cells in each experiment, (J) Model for the regulation of Nkx2.2 by Tcf repressors involving HDAC activity and chromatin remodeling. *p<0.001, **p>0.05.
Mentions: We next sought to establish whether Gro/TLE/Grg proteins act by bridging Tcf4 repressor and HDAC1 functions to regulate progenitor gene patterning in the ventral spinal cord. Gro/TLE/Grg factors are multi-domain proteins that do not bind directly to DNA but rather function to bring together transcription factors and other proteins in a repressor complex [8]. To do this, we performed transfection assays in chick embryos using full-length and truncated forms of the Grg4 protein, which was chosen on the basis of prior studies in mouse and chick that showed it is one of two Gro/TLE/Grg family genes expressed in ventral neural progenitors during neurogenesis in chicken and mouse embryos [5]. Co-transfection of a full-length Grg4 protein with Gli2A+Tcf4R did not block inhibition of Nkx2.2 (Fig 4C, 4D and 4I). We next co-transfected truncated forms of Grg4 that were missing key protein-protein interaction domains. We reasoned that mis-expression of a Grg4-ΔQ protein lacking the Tcf-binding and tetramerization “Q-domain” but retaining the “GP-domain” that binds to HDAC [6,8] along with Gli2A+Tcf4R should block the ability of Tcf4R to repress the induction of Nkx2.2 by dominantly interfering with the ability of endogenous Grg proteins to form a Tcf4-Grg-HDAC repressor complex at the endogenous Nkx2.2 locus (see Fig 4F). Consistent with this, there was a significant increase in the number of Nkx2.2+ cells in triple-transfected embryos (Fig 4E and 4I) compared to controls transfected with Gli2A+Tcf4R alone (Fig 4A). Similarly, co-transfection of a Grg4 protein containing only the Q domain but not the GP domain (“Grg4-Q”) should also block complex formation by inhibiting the association of endogenous Grg proteins with Tcf4 (Fig 4H). As in the experiments above, a significant increase in the number of Nkx2.2+ cells were detected in triple-transfected embryos, compared to controls (Fig 4G and 4I). We also examined Olig2 expression as a control, and found no changes in any experiment (S4 Fig). Taken together, these results indicate that Tcf4 repressor activity requires functional association with both Grg and HDAC proteins, and suggest a model for how Tcf repressor activity selectively regulates the domain-restricted expression of Nkx2.2 via an HDAC-dependent mechanism, possibly involving chromatin modification (Fig 4J).

View Article: PubMed Central - PubMed

ABSTRACT

The generation of functionally distinct neuronal subtypes within the vertebrate central nervous system (CNS) requires the precise regulation of progenitor gene expression in specific neuronal territories during early embryogenesis. Accumulating evidence has implicated histone deacetylase (HDAC) proteins in cell specification, proliferation, and differentiation in diverse embryonic and adult tissues. However, although HDAC proteins have shown to be expressed in the developing vertebrate neural tube, their specific role in CNS neural progenitor fate specification remains unclear. Prior work from our lab showed that the Tcf7l2/Tcf4 transcription factor plays a key role in ventral progenitor lineage segregation by differential repression of two key specification factors, Nkx2.2 and Olig2. In this study, we found that administration of HDAC inhibitors (Valproic Acid (VPA), Trichostatin-A (TSA), or sodium butyrate) in chick embryos in ovo disrupted normal progenitor gene segregation in the developing neural tube, indicating that HDAC activity is required for this process. Further, using functional and pharmacological approaches in vivo, we found that HDAC activity is required for the differential repression of Nkx2.2 and Olig2 by Tcf7l2/Tcf4. Finally, using dominant-negative functional assays, we provide evidence that Tcf7l2/Tcf4 repression also requires Gro/TLE/Grg co-repressor factors. Together, our data support a model where the transcriptional repressor activity of Tcf7l2/Tcf4 involves functional interactions with both HDAC and Gro/TLE/Grg co-factors at specific target gene regulatory elements in the developing neural tube, and that this activity is required for the proper segregation of the Nkx2.2 (p3) and Olig2 (pMN) expressing cells from a common progenitor pool.

No MeSH data available.


Related in: MedlinePlus