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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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Stage-specific up-regulation of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants. (A) The mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in the trigeminal ganglion of HIPK2−/− mutants and wild-type littermates are determined using qRT-PCR assays. No alteration in the mRNA level is detected at E13.5. In contrast, significant increases in the mRNA levels of Brn3a, TrkA, and Bcl-xL are present at E14.5 and E15.5. The level of Bax mRNA level remains unchanged from E13.5 to E15.5. The numbers of samples examined for each age group are: E13.5, n = 3; E14.5, n = 3; and E15.5, n = 5. All data represent mean ± SEM. ns indicates no significant difference and * indicates P < 0.01 (t test). (B) Western blot and quantitative analyses of protein expression in trigeminal ganglion of HIPK2−/− mutants and wild-type littermates. At E13.5, only Brn3a protein shows a modest increase in the trigeminal ganglion of HIPK2−/− mutants, whereas the protein levels of TrkA, Bcl-xL, Bcl-2, and Bax remain unchanged. In contrast, the protein levels of Brn3a, TrkA, and Bcl-xL increase by 2.6-, 1.6-, and 1.7-fold in HIPK2−/− mutant trigeminal ganglion at E15.5, whereas the levels of Bax and Bcl-2 show no change.
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fig6: Stage-specific up-regulation of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants. (A) The mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in the trigeminal ganglion of HIPK2−/− mutants and wild-type littermates are determined using qRT-PCR assays. No alteration in the mRNA level is detected at E13.5. In contrast, significant increases in the mRNA levels of Brn3a, TrkA, and Bcl-xL are present at E14.5 and E15.5. The level of Bax mRNA level remains unchanged from E13.5 to E15.5. The numbers of samples examined for each age group are: E13.5, n = 3; E14.5, n = 3; and E15.5, n = 5. All data represent mean ± SEM. ns indicates no significant difference and * indicates P < 0.01 (t test). (B) Western blot and quantitative analyses of protein expression in trigeminal ganglion of HIPK2−/− mutants and wild-type littermates. At E13.5, only Brn3a protein shows a modest increase in the trigeminal ganglion of HIPK2−/− mutants, whereas the protein levels of TrkA, Bcl-xL, Bcl-2, and Bax remain unchanged. In contrast, the protein levels of Brn3a, TrkA, and Bcl-xL increase by 2.6-, 1.6-, and 1.7-fold in HIPK2−/− mutant trigeminal ganglion at E15.5, whereas the levels of Bax and Bcl-2 show no change.

Mentions: The coexpression of Brn3a and HIPK2 in sensory ganglion and the suppressive effects of HIPK2 on Brn3a-mediated gene expression suggest that loss of HIPK2 could lead to increases in the expression of Brn3a and its downstream targets. To test this hypothesis, we used qRT-PCR assays to determine the mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in trigeminal ganglion from individual wild-type and HIPK2−/− embryos collected at E13.5, 14.5, and 15.5. Loss of HIPK2 resulted in a significant increase in Brn3a, TrkA, and Bcl-xL mRNA at E14.5 and E15.5, with no significant change at E13.5. Specifically, the level of Brn3a mRNA increased by 2.7-fold at E14.5 and twofold at E15.5 (Fig. 6 A; P < 0.01, t test). The levels of TrkA and Bcl-xL mRNA also showed significant, albeit smaller increases at E14.5 and E15.5 (Fig. 6 A). In contrast, the level of Bax mRNA remained unchanged from E13.5 to E15.5. Although Brn3a mRNA showed no significant increase at E13.5, Brn3a protein was modestly increased in some HIPK2−/− mutants at E13.5 (∼1.2-fold), suggesting that HIPK2 may also regulate Brn3a protein stability at this stage (Fig. 6 B). At E15.5, the protein levels of Brn3a, TrkA, and Bcl-xL proteins showed consistent increases by 2.6-, 1.6-, and 1.7-fold, respectively (Fig. 6 B). In contrast, levels of Bcl-2 and Bax proteins were not altered at any time point examined. Together, increases in the mRNA and proteins of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants suggest that HIPK2 negatively regulates Brn3a-mediated gene expression in vivo.


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)

Stage-specific up-regulation of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants. (A) The mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in the trigeminal ganglion of HIPK2−/− mutants and wild-type littermates are determined using qRT-PCR assays. No alteration in the mRNA level is detected at E13.5. In contrast, significant increases in the mRNA levels of Brn3a, TrkA, and Bcl-xL are present at E14.5 and E15.5. The level of Bax mRNA level remains unchanged from E13.5 to E15.5. The numbers of samples examined for each age group are: E13.5, n = 3; E14.5, n = 3; and E15.5, n = 5. All data represent mean ± SEM. ns indicates no significant difference and * indicates P < 0.01 (t test). (B) Western blot and quantitative analyses of protein expression in trigeminal ganglion of HIPK2−/− mutants and wild-type littermates. At E13.5, only Brn3a protein shows a modest increase in the trigeminal ganglion of HIPK2−/− mutants, whereas the protein levels of TrkA, Bcl-xL, Bcl-2, and Bax remain unchanged. In contrast, the protein levels of Brn3a, TrkA, and Bcl-xL increase by 2.6-, 1.6-, and 1.7-fold in HIPK2−/− mutant trigeminal ganglion at E15.5, whereas the levels of Bax and Bcl-2 show no change.
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fig6: Stage-specific up-regulation of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants. (A) The mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in the trigeminal ganglion of HIPK2−/− mutants and wild-type littermates are determined using qRT-PCR assays. No alteration in the mRNA level is detected at E13.5. In contrast, significant increases in the mRNA levels of Brn3a, TrkA, and Bcl-xL are present at E14.5 and E15.5. The level of Bax mRNA level remains unchanged from E13.5 to E15.5. The numbers of samples examined for each age group are: E13.5, n = 3; E14.5, n = 3; and E15.5, n = 5. All data represent mean ± SEM. ns indicates no significant difference and * indicates P < 0.01 (t test). (B) Western blot and quantitative analyses of protein expression in trigeminal ganglion of HIPK2−/− mutants and wild-type littermates. At E13.5, only Brn3a protein shows a modest increase in the trigeminal ganglion of HIPK2−/− mutants, whereas the protein levels of TrkA, Bcl-xL, Bcl-2, and Bax remain unchanged. In contrast, the protein levels of Brn3a, TrkA, and Bcl-xL increase by 2.6-, 1.6-, and 1.7-fold in HIPK2−/− mutant trigeminal ganglion at E15.5, whereas the levels of Bax and Bcl-2 show no change.
Mentions: The coexpression of Brn3a and HIPK2 in sensory ganglion and the suppressive effects of HIPK2 on Brn3a-mediated gene expression suggest that loss of HIPK2 could lead to increases in the expression of Brn3a and its downstream targets. To test this hypothesis, we used qRT-PCR assays to determine the mRNA levels of Brn3a, TrkA, Bcl-xL, and Bax in trigeminal ganglion from individual wild-type and HIPK2−/− embryos collected at E13.5, 14.5, and 15.5. Loss of HIPK2 resulted in a significant increase in Brn3a, TrkA, and Bcl-xL mRNA at E14.5 and E15.5, with no significant change at E13.5. Specifically, the level of Brn3a mRNA increased by 2.7-fold at E14.5 and twofold at E15.5 (Fig. 6 A; P < 0.01, t test). The levels of TrkA and Bcl-xL mRNA also showed significant, albeit smaller increases at E14.5 and E15.5 (Fig. 6 A). In contrast, the level of Bax mRNA remained unchanged from E13.5 to E15.5. Although Brn3a mRNA showed no significant increase at E13.5, Brn3a protein was modestly increased in some HIPK2−/− mutants at E13.5 (∼1.2-fold), suggesting that HIPK2 may also regulate Brn3a protein stability at this stage (Fig. 6 B). At E15.5, the protein levels of Brn3a, TrkA, and Bcl-xL proteins showed consistent increases by 2.6-, 1.6-, and 1.7-fold, respectively (Fig. 6 B). In contrast, levels of Bcl-2 and Bax proteins were not altered at any time point examined. Together, increases in the mRNA and proteins of Brn3a, TrkA, and Bcl-xL in the trigeminal ganglion of HIPK2−/− mutants suggest that HIPK2 negatively regulates Brn3a-mediated gene expression in vivo.

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