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Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

Yamanaka T, Tosaki A, Miyazaki H, Kurosawa M, Furukawa Y, Yamada M, Nukina N - Hum. Mol. Genet. (2010)

Bottom Line: We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons.We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides.Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

ABSTRACT
In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

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Reduced DNA binding of Brn-2 in R6/2 mouse brain cortex. (A–D) Cortical lysates prepared using high-salt buffer from12-week-old R6/2 (TG; n = 3) or control (WT; n = 3) mice were subjected to EMSA using probe 20I (left) or 2M (right). (A) Nucleotide sequences of sense oligonucleotides of the probes. The binding consensuses of POU domain factors are underlined. (B) Three shifted bands corresponding to the probe-complex containing Oct-1, Brn-1 and Brn-2 are indicated. (C) Super-shift assay using anti-Brn-1, anti-Brn-2 or IgG for probe 20I (left) or 2M (right). (D) Quantification of the amount of protein complexes with probe 20I (left) and 2M (right). Values are means ± SD (**P < 0.01). (E) The cortical lysates of R6/2 or control mice were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP) and β-actin. (F) Quantification of the amount of Brn-2, Brn-1 and β-actin. Values are means ± SD (*P < 0.05).
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DDQ087F1: Reduced DNA binding of Brn-2 in R6/2 mouse brain cortex. (A–D) Cortical lysates prepared using high-salt buffer from12-week-old R6/2 (TG; n = 3) or control (WT; n = 3) mice were subjected to EMSA using probe 20I (left) or 2M (right). (A) Nucleotide sequences of sense oligonucleotides of the probes. The binding consensuses of POU domain factors are underlined. (B) Three shifted bands corresponding to the probe-complex containing Oct-1, Brn-1 and Brn-2 are indicated. (C) Super-shift assay using anti-Brn-1, anti-Brn-2 or IgG for probe 20I (left) or 2M (right). (D) Quantification of the amount of protein complexes with probe 20I (left) and 2M (right). Values are means ± SD (**P < 0.01). (E) The cortical lysates of R6/2 or control mice were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP) and β-actin. (F) Quantification of the amount of Brn-2, Brn-1 and β-actin. Values are means ± SD (*P < 0.05).

Mentions: Interestingly, two other probes, 20I (NF-A3) and 2M (OCT), contain ATGCAAA sequences, which is the binding site for POU domain transcription factors (Fig. 1A) (18). Using these probes, we observed three shifted bands by EMSA (Fig. 1B). The upper, middle and lower bands correspond to the probe complex containing Oct-1 (Pou2f1), Brn-1 (Pou3f3) and Brn-2 (Pou3f2, N-Oct-3), respectively (19). Indeed, the middle and lower bands were further shifted by the addition of antibodies specific to Brn-1 and Brn-2, respectively (Fig. 1C, Supplementary Material, Fig. S2A). Importantly, the Brn-2-probe complex was specifically reduced in R6/2 samples, whereas probe complexes containing Oct-1 and Brn-1 were not affected (Fig. 1B). Quantified data showed that the binding of Brn-2 was reduced by 10–20% of control levels in R6/2 cortical lysates (Fig. 1D). The difference in degree of the reduction between these probes may be due to the difference in the number of binding sites (Fig. 1A). These data indicate that Brn-2 is specifically affected by mutant Nhtt among the POU domain factors active in the cortical lysates.


Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction.

Yamanaka T, Tosaki A, Miyazaki H, Kurosawa M, Furukawa Y, Yamada M, Nukina N - Hum. Mol. Genet. (2010)

Reduced DNA binding of Brn-2 in R6/2 mouse brain cortex. (A–D) Cortical lysates prepared using high-salt buffer from12-week-old R6/2 (TG; n = 3) or control (WT; n = 3) mice were subjected to EMSA using probe 20I (left) or 2M (right). (A) Nucleotide sequences of sense oligonucleotides of the probes. The binding consensuses of POU domain factors are underlined. (B) Three shifted bands corresponding to the probe-complex containing Oct-1, Brn-1 and Brn-2 are indicated. (C) Super-shift assay using anti-Brn-1, anti-Brn-2 or IgG for probe 20I (left) or 2M (right). (D) Quantification of the amount of protein complexes with probe 20I (left) and 2M (right). Values are means ± SD (**P < 0.01). (E) The cortical lysates of R6/2 or control mice were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP) and β-actin. (F) Quantification of the amount of Brn-2, Brn-1 and β-actin. Values are means ± SD (*P < 0.05).
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DDQ087F1: Reduced DNA binding of Brn-2 in R6/2 mouse brain cortex. (A–D) Cortical lysates prepared using high-salt buffer from12-week-old R6/2 (TG; n = 3) or control (WT; n = 3) mice were subjected to EMSA using probe 20I (left) or 2M (right). (A) Nucleotide sequences of sense oligonucleotides of the probes. The binding consensuses of POU domain factors are underlined. (B) Three shifted bands corresponding to the probe-complex containing Oct-1, Brn-1 and Brn-2 are indicated. (C) Super-shift assay using anti-Brn-1, anti-Brn-2 or IgG for probe 20I (left) or 2M (right). (D) Quantification of the amount of protein complexes with probe 20I (left) and 2M (right). Values are means ± SD (**P < 0.01). (E) The cortical lysates of R6/2 or control mice were subjected to western blot analysis using antibodies against Brn-2 (C-2AP), Brn-1 (C-2AP) and β-actin. (F) Quantification of the amount of Brn-2, Brn-1 and β-actin. Values are means ± SD (*P < 0.05).
Mentions: Interestingly, two other probes, 20I (NF-A3) and 2M (OCT), contain ATGCAAA sequences, which is the binding site for POU domain transcription factors (Fig. 1A) (18). Using these probes, we observed three shifted bands by EMSA (Fig. 1B). The upper, middle and lower bands correspond to the probe complex containing Oct-1 (Pou2f1), Brn-1 (Pou3f3) and Brn-2 (Pou3f2, N-Oct-3), respectively (19). Indeed, the middle and lower bands were further shifted by the addition of antibodies specific to Brn-1 and Brn-2, respectively (Fig. 1C, Supplementary Material, Fig. S2A). Importantly, the Brn-2-probe complex was specifically reduced in R6/2 samples, whereas probe complexes containing Oct-1 and Brn-1 were not affected (Fig. 1B). Quantified data showed that the binding of Brn-2 was reduced by 10–20% of control levels in R6/2 cortical lysates (Fig. 1D). The difference in degree of the reduction between these probes may be due to the difference in the number of binding sites (Fig. 1A). These data indicate that Brn-2 is specifically affected by mutant Nhtt among the POU domain factors active in the cortical lysates.

Bottom Line: We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons.We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides.Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Structural Neuropathology, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

ABSTRACT
In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

Show MeSH
Related in: MedlinePlus