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Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival.

Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, Luo EJ, Neve RL, Reichardt LF, Huang EJ - J. Cell Biol. (2004)

Bottom Line: Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

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HIPK2 interacts with Brn3a and promotes Brn3a binding to DNA elements. (A) Isolation of HIPK2 by yeast two-hybrid screen. The domain between amino acids 752 and 897 of HIPK2 interacts with full-length Brn3a, Brn3b, and Brn3c. Interaction between Brn3a and HIPK2 is mediated by the POU homeodomain (Brn3a POUHD), not the POU specific domain (POUS) or non-POU domain (Brn3a NP). Data represent mean ± SEM (n = 3). A schematic diagram of Brn3a and HIPK2 interaction. (B) Brn3a and HIPK2 are present in a protein complex that can be coimmunoprecipitated with Brn3a antibody, but not preimmune sera (PI). Interaction between Brn3a and HIPK2 is reduced by increasing wash stringency. Whereas detergents NP-40 (1%; wash 1) and sodium deoxycholate (0.5%; wash 2) preserve Brn3a–HIPK2 interaction, the presence of ionic detergent SDS (0.1%; wash 3) significantly reduces such interaction. (C) Electrophoretic mobility shift assays for Brn3a and HIPK2. HIPK2 enhances Brn3a binding to consensus DNA element (b3s1). Routinely, 2 μl of in vitro translated Brn3a protein is added (lanes 4–7) and increasing amount of HIPK2 (1, 2, and 5 μl) is added to the reaction (lanes 5–7). Although Brn3a antibody produces a supershift, the addition of HIPK2 antibody does not (lanes 10–14).
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fig1: HIPK2 interacts with Brn3a and promotes Brn3a binding to DNA elements. (A) Isolation of HIPK2 by yeast two-hybrid screen. The domain between amino acids 752 and 897 of HIPK2 interacts with full-length Brn3a, Brn3b, and Brn3c. Interaction between Brn3a and HIPK2 is mediated by the POU homeodomain (Brn3a POUHD), not the POU specific domain (POUS) or non-POU domain (Brn3a NP). Data represent mean ± SEM (n = 3). A schematic diagram of Brn3a and HIPK2 interaction. (B) Brn3a and HIPK2 are present in a protein complex that can be coimmunoprecipitated with Brn3a antibody, but not preimmune sera (PI). Interaction between Brn3a and HIPK2 is reduced by increasing wash stringency. Whereas detergents NP-40 (1%; wash 1) and sodium deoxycholate (0.5%; wash 2) preserve Brn3a–HIPK2 interaction, the presence of ionic detergent SDS (0.1%; wash 3) significantly reduces such interaction. (C) Electrophoretic mobility shift assays for Brn3a and HIPK2. HIPK2 enhances Brn3a binding to consensus DNA element (b3s1). Routinely, 2 μl of in vitro translated Brn3a protein is added (lanes 4–7) and increasing amount of HIPK2 (1, 2, and 5 μl) is added to the reaction (lanes 5–7). Although Brn3a antibody produces a supershift, the addition of HIPK2 antibody does not (lanes 10–14).

Mentions: The important roles of Brn3a in regulating its own expression and the expression of Trk receptors and members of the Bcl-2 family motivated us to investigate additional components that regulate Brn3a-mediated gene expression. Using the yeast two-hybrid screen and a cDNA library prepared from mouse E10-11 embryos (Hollenberg et al., 1995), we isolated several independent clones that interacted with full-length Brn3a, one of which encoded protein sequence between amino acids 752 and 897 of HIPK2, a Ser/Thr kinase that interacts with homeodomain transcription factors of the Nkx family (Fig. 1; Kim et al., 1998). Further characterizations showed that full-length HIPK2 also interacted with Brn3a (not depicted) and that HIPK2 interacted with the POU homeodomain (POUHD) of Brn3a, but not POU specific (POUS) or non-POU domain (Fig. 1, A and B). Not surprisingly, given the high sequence homology in the POU domain among members of the Brn3 family (>95%; Ryan and Rosenfeld, 1997), HIPK2 also interacted with Brn3b and Brn3c (Fig. 1 A).


Interaction of Brn3a and HIPK2 mediates transcriptional repression of sensory neuron survival.

Wiggins AK, Wei G, Doxakis E, Wong C, Tang AA, Zang K, Luo EJ, Neve RL, Reichardt LF, Huang EJ - J. Cell Biol. (2004)

HIPK2 interacts with Brn3a and promotes Brn3a binding to DNA elements. (A) Isolation of HIPK2 by yeast two-hybrid screen. The domain between amino acids 752 and 897 of HIPK2 interacts with full-length Brn3a, Brn3b, and Brn3c. Interaction between Brn3a and HIPK2 is mediated by the POU homeodomain (Brn3a POUHD), not the POU specific domain (POUS) or non-POU domain (Brn3a NP). Data represent mean ± SEM (n = 3). A schematic diagram of Brn3a and HIPK2 interaction. (B) Brn3a and HIPK2 are present in a protein complex that can be coimmunoprecipitated with Brn3a antibody, but not preimmune sera (PI). Interaction between Brn3a and HIPK2 is reduced by increasing wash stringency. Whereas detergents NP-40 (1%; wash 1) and sodium deoxycholate (0.5%; wash 2) preserve Brn3a–HIPK2 interaction, the presence of ionic detergent SDS (0.1%; wash 3) significantly reduces such interaction. (C) Electrophoretic mobility shift assays for Brn3a and HIPK2. HIPK2 enhances Brn3a binding to consensus DNA element (b3s1). Routinely, 2 μl of in vitro translated Brn3a protein is added (lanes 4–7) and increasing amount of HIPK2 (1, 2, and 5 μl) is added to the reaction (lanes 5–7). Although Brn3a antibody produces a supershift, the addition of HIPK2 antibody does not (lanes 10–14).
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getmorefigures.php?uid=PMC2172556&req=5

fig1: HIPK2 interacts with Brn3a and promotes Brn3a binding to DNA elements. (A) Isolation of HIPK2 by yeast two-hybrid screen. The domain between amino acids 752 and 897 of HIPK2 interacts with full-length Brn3a, Brn3b, and Brn3c. Interaction between Brn3a and HIPK2 is mediated by the POU homeodomain (Brn3a POUHD), not the POU specific domain (POUS) or non-POU domain (Brn3a NP). Data represent mean ± SEM (n = 3). A schematic diagram of Brn3a and HIPK2 interaction. (B) Brn3a and HIPK2 are present in a protein complex that can be coimmunoprecipitated with Brn3a antibody, but not preimmune sera (PI). Interaction between Brn3a and HIPK2 is reduced by increasing wash stringency. Whereas detergents NP-40 (1%; wash 1) and sodium deoxycholate (0.5%; wash 2) preserve Brn3a–HIPK2 interaction, the presence of ionic detergent SDS (0.1%; wash 3) significantly reduces such interaction. (C) Electrophoretic mobility shift assays for Brn3a and HIPK2. HIPK2 enhances Brn3a binding to consensus DNA element (b3s1). Routinely, 2 μl of in vitro translated Brn3a protein is added (lanes 4–7) and increasing amount of HIPK2 (1, 2, and 5 μl) is added to the reaction (lanes 5–7). Although Brn3a antibody produces a supershift, the addition of HIPK2 antibody does not (lanes 10–14).
Mentions: The important roles of Brn3a in regulating its own expression and the expression of Trk receptors and members of the Bcl-2 family motivated us to investigate additional components that regulate Brn3a-mediated gene expression. Using the yeast two-hybrid screen and a cDNA library prepared from mouse E10-11 embryos (Hollenberg et al., 1995), we isolated several independent clones that interacted with full-length Brn3a, one of which encoded protein sequence between amino acids 752 and 897 of HIPK2, a Ser/Thr kinase that interacts with homeodomain transcription factors of the Nkx family (Fig. 1; Kim et al., 1998). Further characterizations showed that full-length HIPK2 also interacted with Brn3a (not depicted) and that HIPK2 interacted with the POU homeodomain (POUHD) of Brn3a, but not POU specific (POUS) or non-POU domain (Fig. 1, A and B). Not surprisingly, given the high sequence homology in the POU domain among members of the Brn3 family (>95%; Ryan and Rosenfeld, 1997), HIPK2 also interacted with Brn3b and Brn3c (Fig. 1 A).

Bottom Line: Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Show MeSH
Related in: MedlinePlus