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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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Targeted deletion of HIPK2 and generation of HIPK2  mice. (A) Homologous recombination with the targeting construct deletes exon 3 (1st coding exon), and simultaneously inserts LacZ and a selection marker neomycin (PGK-neo) gene in-frame with the first ATG of HIPK2. Arrows indicate the orientation of PGK-neo, thymidine kinase (hsv-tk) and LacZ genes. Primers 1–3 are used for genotyping. (B) Genomic DNA from G418-resistant ES colonies was screened by Southern blots using 5′ and 3′ probes indicated in A. (C) Southern blot genotyping of wild type (wt), HIPK2+/− (+/−), and HIPK2−/− (−/−) progeny using the same probes. (D) Total RNA from MEFs derived from wild type, HIPK2+/−, and HIPK2−/− mutants was probed with a cDNA fragment containing 5′ UTR and first coding exon of HIPK2. Level of HIPK2 expression is reduced in HIPK2+/− mutants and is completely absent in HIPK2−/− mutants. (E) mRNA from trigeminal ganglia was amplified by RT-PCR using primers for the first coding exon (exon 3) of HIPK2 and the last two exons (exon 15–16). Consistent with results from the Northern blot, HIPK2 expression is not detected in the trigeminal ganglion of HIPK2−/− mutants. (F and G) The presence of LacZ in HIPK2 locus provides a convenient tool to detect HIPK2 expression in the developing trigeminal ganglion (F) and dorsal root ganglion (G) at E13.5. Bars: (F) 250 μm; (G) 100 μm.
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fig5: Targeted deletion of HIPK2 and generation of HIPK2 mice. (A) Homologous recombination with the targeting construct deletes exon 3 (1st coding exon), and simultaneously inserts LacZ and a selection marker neomycin (PGK-neo) gene in-frame with the first ATG of HIPK2. Arrows indicate the orientation of PGK-neo, thymidine kinase (hsv-tk) and LacZ genes. Primers 1–3 are used for genotyping. (B) Genomic DNA from G418-resistant ES colonies was screened by Southern blots using 5′ and 3′ probes indicated in A. (C) Southern blot genotyping of wild type (wt), HIPK2+/− (+/−), and HIPK2−/− (−/−) progeny using the same probes. (D) Total RNA from MEFs derived from wild type, HIPK2+/−, and HIPK2−/− mutants was probed with a cDNA fragment containing 5′ UTR and first coding exon of HIPK2. Level of HIPK2 expression is reduced in HIPK2+/− mutants and is completely absent in HIPK2−/− mutants. (E) mRNA from trigeminal ganglia was amplified by RT-PCR using primers for the first coding exon (exon 3) of HIPK2 and the last two exons (exon 15–16). Consistent with results from the Northern blot, HIPK2 expression is not detected in the trigeminal ganglion of HIPK2−/− mutants. (F and G) The presence of LacZ in HIPK2 locus provides a convenient tool to detect HIPK2 expression in the developing trigeminal ganglion (F) and dorsal root ganglion (G) at E13.5. Bars: (F) 250 μm; (G) 100 μm.

Mentions: The suppressive effects of HIPK2 in vitro and the coexpression of Brn3a and HIPK2 in sensory ganglia suggest that both may be important components in the transcriptional mechanism that controls sensory neuron survival and cell death by regulating a common set of prosurvival genes. To test this hypothesis, we deleted HIPK2 in embryonic stem (ES) cells. The mouse HIPK2 locus encompasses ∼150 kb and contains 16 exons (unpublished data). We targeted exon 3, which encodes the first ATG and the entire HIPK2 kinase domain (from amino acids 192 to 520). The majority of exon 3 was replaced with the reporter gene LacZ, followed by a polyadenylation signal sequence and the selection marker neomycin gene. Four positive ES clones were identified by Southern blot analysis using specific 5′ and 3′ probes, three were injected into blastocysts (Fig. 5, A and B). Germline transmission of the targeted allele was confirmed by Southern blot analyses (Fig. 5 C). Northern blot analysis using a probe that hybridized with a sequence in the exons 3 and 4 confirmed that HIPK2 mRNA was undetectable in mouse embryonic fibroblasts (MEFs) derived from homozygous HIPK2 (HIPK2−/−) mutants (Fig. 5 D). The absence of HIPK2 transcripts in the trigeminal ganglion of HIPK2−/− mutants was further confirmed by RT-PCR using primers that detected sequences in exon 3 and in exons 15–16 of HIPK2 (Fig. 5 E). The insertion of LacZ in–frame with the coding sequence of HIPK2 (HIPK2LacZ) provided an excellent lineage tracer for cells expressing HIPK2. Consistent with the in situ hybridization and immunostaining results (Fig. 4), significant HIPK2LacZ staining intensity was detected in the developing sensory ganglia, including the trigeminal and dorsal root ganglia, diencephalon, and spinal cord at E13.5 (Fig. 5, F and G).


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)

Targeted deletion of HIPK2 and generation of HIPK2  mice. (A) Homologous recombination with the targeting construct deletes exon 3 (1st coding exon), and simultaneously inserts LacZ and a selection marker neomycin (PGK-neo) gene in-frame with the first ATG of HIPK2. Arrows indicate the orientation of PGK-neo, thymidine kinase (hsv-tk) and LacZ genes. Primers 1–3 are used for genotyping. (B) Genomic DNA from G418-resistant ES colonies was screened by Southern blots using 5′ and 3′ probes indicated in A. (C) Southern blot genotyping of wild type (wt), HIPK2+/− (+/−), and HIPK2−/− (−/−) progeny using the same probes. (D) Total RNA from MEFs derived from wild type, HIPK2+/−, and HIPK2−/− mutants was probed with a cDNA fragment containing 5′ UTR and first coding exon of HIPK2. Level of HIPK2 expression is reduced in HIPK2+/− mutants and is completely absent in HIPK2−/− mutants. (E) mRNA from trigeminal ganglia was amplified by RT-PCR using primers for the first coding exon (exon 3) of HIPK2 and the last two exons (exon 15–16). Consistent with results from the Northern blot, HIPK2 expression is not detected in the trigeminal ganglion of HIPK2−/− mutants. (F and G) The presence of LacZ in HIPK2 locus provides a convenient tool to detect HIPK2 expression in the developing trigeminal ganglion (F) and dorsal root ganglion (G) at E13.5. Bars: (F) 250 μm; (G) 100 μm.
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fig5: Targeted deletion of HIPK2 and generation of HIPK2 mice. (A) Homologous recombination with the targeting construct deletes exon 3 (1st coding exon), and simultaneously inserts LacZ and a selection marker neomycin (PGK-neo) gene in-frame with the first ATG of HIPK2. Arrows indicate the orientation of PGK-neo, thymidine kinase (hsv-tk) and LacZ genes. Primers 1–3 are used for genotyping. (B) Genomic DNA from G418-resistant ES colonies was screened by Southern blots using 5′ and 3′ probes indicated in A. (C) Southern blot genotyping of wild type (wt), HIPK2+/− (+/−), and HIPK2−/− (−/−) progeny using the same probes. (D) Total RNA from MEFs derived from wild type, HIPK2+/−, and HIPK2−/− mutants was probed with a cDNA fragment containing 5′ UTR and first coding exon of HIPK2. Level of HIPK2 expression is reduced in HIPK2+/− mutants and is completely absent in HIPK2−/− mutants. (E) mRNA from trigeminal ganglia was amplified by RT-PCR using primers for the first coding exon (exon 3) of HIPK2 and the last two exons (exon 15–16). Consistent with results from the Northern blot, HIPK2 expression is not detected in the trigeminal ganglion of HIPK2−/− mutants. (F and G) The presence of LacZ in HIPK2 locus provides a convenient tool to detect HIPK2 expression in the developing trigeminal ganglion (F) and dorsal root ganglion (G) at E13.5. Bars: (F) 250 μm; (G) 100 μm.
Mentions: The suppressive effects of HIPK2 in vitro and the coexpression of Brn3a and HIPK2 in sensory ganglia suggest that both may be important components in the transcriptional mechanism that controls sensory neuron survival and cell death by regulating a common set of prosurvival genes. To test this hypothesis, we deleted HIPK2 in embryonic stem (ES) cells. The mouse HIPK2 locus encompasses ∼150 kb and contains 16 exons (unpublished data). We targeted exon 3, which encodes the first ATG and the entire HIPK2 kinase domain (from amino acids 192 to 520). The majority of exon 3 was replaced with the reporter gene LacZ, followed by a polyadenylation signal sequence and the selection marker neomycin gene. Four positive ES clones were identified by Southern blot analysis using specific 5′ and 3′ probes, three were injected into blastocysts (Fig. 5, A and B). Germline transmission of the targeted allele was confirmed by Southern blot analyses (Fig. 5 C). Northern blot analysis using a probe that hybridized with a sequence in the exons 3 and 4 confirmed that HIPK2 mRNA was undetectable in mouse embryonic fibroblasts (MEFs) derived from homozygous HIPK2 (HIPK2−/−) mutants (Fig. 5 D). The absence of HIPK2 transcripts in the trigeminal ganglion of HIPK2−/− mutants was further confirmed by RT-PCR using primers that detected sequences in exon 3 and in exons 15–16 of HIPK2 (Fig. 5 E). The insertion of LacZ in–frame with the coding sequence of HIPK2 (HIPK2LacZ) provided an excellent lineage tracer for cells expressing HIPK2. Consistent with the in situ hybridization and immunostaining results (Fig. 4), significant HIPK2LacZ staining intensity was detected in the developing sensory ganglia, including the trigeminal and dorsal root ganglia, diencephalon, and spinal cord at E13.5 (Fig. 5, F and G).

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