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The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

Prentice LM, d'Anglemont de Tassigny X, McKinney S, Ruiz de Algara T, Yap D, Turashvili G, Poon S, Sutcliffe M, Allard P, Burleigh A, Fee J, Huntsman DG, Colledge WH, Aparicio SA - BMC Genomics (2011)

Bottom Line: Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone.The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus.Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada.

ABSTRACT

Background: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods.

Results: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.

Conclusions: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

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Classification of 198 differentially expressed genes in the discovery set. The figure shows the number of significant genes identified at each step of the microarray analysis procedure with subsequent filtering steps for statistical significance and fold change expression differences. WT vs KO represents a joint comparison of all data, whereas WT vs GKO and KKO represent separated analyses. The numbers of genes falling through the analysis are shown separately for gene level and exon level summarization. The pie chart shows the grouping of molecular functions for the 198 gene set.
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Figure 1: Classification of 198 differentially expressed genes in the discovery set. The figure shows the number of significant genes identified at each step of the microarray analysis procedure with subsequent filtering steps for statistical significance and fold change expression differences. WT vs KO represents a joint comparison of all data, whereas WT vs GKO and KKO represent separated analyses. The numbers of genes falling through the analysis are shown separately for gene level and exon level summarization. The pie chart shows the grouping of molecular functions for the 198 gene set.

Mentions: We first isolated RNA from micro-dissected hypothalamic tissue of knockout and wild-type mice and hybridized this to Exon 1.0 arrays (methods). We compared quantile normalized probe intensity values from Affymetrix whole mouse exon array chips where hybridization was performed with wild-type (WT), Gpr54 knockout (GKO) or Kiss1 knockout (KKO) hypothalamic RNA. Affymetrix results were compared between genotypes, specifically gene expression of all wild-type mice were grouped together and compared with all knockout mice grouped together (WT vs KO) or with Kiss1 knockout mice alone (WT vs KKO) or Gpr54 knockout mice alone (WT vs GKO). Additionally, Gpr54 knockout mice were compared with Kiss1 knockout mice (GKO vs KKO) to give a total of four groups (Figure 1). Gene level and exon level summarization was used in the comparisons and from each of these we selected genes showing a p-value < 0.05 and an expression fold difference of 1.5 or greater, as candidates for further analysis (yellow highlighted regions in the volcano plots shown in Additional file 1).


The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

Prentice LM, d'Anglemont de Tassigny X, McKinney S, Ruiz de Algara T, Yap D, Turashvili G, Poon S, Sutcliffe M, Allard P, Burleigh A, Fee J, Huntsman DG, Colledge WH, Aparicio SA - BMC Genomics (2011)

Classification of 198 differentially expressed genes in the discovery set. The figure shows the number of significant genes identified at each step of the microarray analysis procedure with subsequent filtering steps for statistical significance and fold change expression differences. WT vs KO represents a joint comparison of all data, whereas WT vs GKO and KKO represent separated analyses. The numbers of genes falling through the analysis are shown separately for gene level and exon level summarization. The pie chart shows the grouping of molecular functions for the 198 gene set.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111392&req=5

Figure 1: Classification of 198 differentially expressed genes in the discovery set. The figure shows the number of significant genes identified at each step of the microarray analysis procedure with subsequent filtering steps for statistical significance and fold change expression differences. WT vs KO represents a joint comparison of all data, whereas WT vs GKO and KKO represent separated analyses. The numbers of genes falling through the analysis are shown separately for gene level and exon level summarization. The pie chart shows the grouping of molecular functions for the 198 gene set.
Mentions: We first isolated RNA from micro-dissected hypothalamic tissue of knockout and wild-type mice and hybridized this to Exon 1.0 arrays (methods). We compared quantile normalized probe intensity values from Affymetrix whole mouse exon array chips where hybridization was performed with wild-type (WT), Gpr54 knockout (GKO) or Kiss1 knockout (KKO) hypothalamic RNA. Affymetrix results were compared between genotypes, specifically gene expression of all wild-type mice were grouped together and compared with all knockout mice grouped together (WT vs KO) or with Kiss1 knockout mice alone (WT vs KKO) or Gpr54 knockout mice alone (WT vs GKO). Additionally, Gpr54 knockout mice were compared with Kiss1 knockout mice (GKO vs KKO) to give a total of four groups (Figure 1). Gene level and exon level summarization was used in the comparisons and from each of these we selected genes showing a p-value < 0.05 and an expression fold difference of 1.5 or greater, as candidates for further analysis (yellow highlighted regions in the volcano plots shown in Additional file 1).

Bottom Line: Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone.The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus.Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Oncology and Breast Cancer Program, British Columbia Cancer Research Centre, 675 West 10th Avenue, Vancouver, BC, V5Z 1L3, Canada.

ABSTRACT

Background: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods.

Results: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot.

Conclusions: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.

Show MeSH