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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.

No MeSH data available.


Tcf and HDAC1 proteins occupy Nkx2.2 but not Olig2 regulatory sequences.ChIP-reChIP experiments showing interaction of Tcf4 and HDAC1 with Nkx2.2 but not Olig2 regulatory regions. (A) Left column: Primary IP assays with HDAC1, IgG (negative control), or AcHistone3 (AcHis3) (positive control) antibodies from chromatin prepared from E10.5 mouse embryos. Right column: re-ChIP assays with Tcf3/4, IgG (negative control), or AcHistone3 (positive control) antibodies from primary IP elution. (B) Sequential chromatin immunoprecipitation assay schema. (C) Densitometry analysis of primary IP (left), and re-IP (right) results. The data are expressed as mean± SEM from three independent experiments.
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pone.0163267.g003: Tcf and HDAC1 proteins occupy Nkx2.2 but not Olig2 regulatory sequences.ChIP-reChIP experiments showing interaction of Tcf4 and HDAC1 with Nkx2.2 but not Olig2 regulatory regions. (A) Left column: Primary IP assays with HDAC1, IgG (negative control), or AcHistone3 (AcHis3) (positive control) antibodies from chromatin prepared from E10.5 mouse embryos. Right column: re-ChIP assays with Tcf3/4, IgG (negative control), or AcHistone3 (positive control) antibodies from primary IP elution. (B) Sequential chromatin immunoprecipitation assay schema. (C) Densitometry analysis of primary IP (left), and re-IP (right) results. The data are expressed as mean± SEM from three independent experiments.

Mentions: These results raise the possibility that Tcf4 protein functionally interacts with HDAC1 at the Nkx2.2, but not Olig2, locus. To address this, we performed ChIP-reChIP experiments using the previously identified cis-regulatory sequences for these genes (Nkx2.2p3-CRM, Olig2pMN-CRM) [11,12,19] to first pull-down HDAC1 and then probe for the presence of Tcf4 protein (Fig 3). As predicted, both HDAC1 and Tcf4 could only be detected at the Nkx2.2p3-CRM (Fig 3). These findings are consistent with our data that indicate a selective functional interaction of Tcf4 with HDAC1 at the Nkx2.2 but not Olig2 locus.


Tcf7l2/Tcf4 Transcriptional Repressor Function Requires HDAC Activity in the Developing Vertebrate CNS
Tcf and HDAC1 proteins occupy Nkx2.2 but not Olig2 regulatory sequences.ChIP-reChIP experiments showing interaction of Tcf4 and HDAC1 with Nkx2.2 but not Olig2 regulatory regions. (A) Left column: Primary IP assays with HDAC1, IgG (negative control), or AcHistone3 (AcHis3) (positive control) antibodies from chromatin prepared from E10.5 mouse embryos. Right column: re-ChIP assays with Tcf3/4, IgG (negative control), or AcHistone3 (positive control) antibodies from primary IP elution. (B) Sequential chromatin immunoprecipitation assay schema. (C) Densitometry analysis of primary IP (left), and re-IP (right) results. The data are expressed as mean± SEM from three independent experiments.
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Related In: Results  -  Collection

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pone.0163267.g003: Tcf and HDAC1 proteins occupy Nkx2.2 but not Olig2 regulatory sequences.ChIP-reChIP experiments showing interaction of Tcf4 and HDAC1 with Nkx2.2 but not Olig2 regulatory regions. (A) Left column: Primary IP assays with HDAC1, IgG (negative control), or AcHistone3 (AcHis3) (positive control) antibodies from chromatin prepared from E10.5 mouse embryos. Right column: re-ChIP assays with Tcf3/4, IgG (negative control), or AcHistone3 (positive control) antibodies from primary IP elution. (B) Sequential chromatin immunoprecipitation assay schema. (C) Densitometry analysis of primary IP (left), and re-IP (right) results. The data are expressed as mean± SEM from three independent experiments.
Mentions: These results raise the possibility that Tcf4 protein functionally interacts with HDAC1 at the Nkx2.2, but not Olig2, locus. To address this, we performed ChIP-reChIP experiments using the previously identified cis-regulatory sequences for these genes (Nkx2.2p3-CRM, Olig2pMN-CRM) [11,12,19] to first pull-down HDAC1 and then probe for the presence of Tcf4 protein (Fig 3). As predicted, both HDAC1 and Tcf4 could only be detected at the Nkx2.2p3-CRM (Fig 3). These findings are consistent with our data that indicate a selective functional interaction of Tcf4 with HDAC1 at the Nkx2.2 but not Olig2 locus.

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.