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The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.

Qiu J, Kleineidam A, Gouraud S, Yao ST, Greenwood M, Hoe SZ, Hindmarch C, Murphy D - Endocrinology (2014)

Bottom Line: In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out.Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration.These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences (J.Q., A.K., S.G., S.T.Y., M.G., C.H., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; and Department of Physiology (S.Z.H., C.H., D.M.), Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.

ABSTRACT
The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

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Stat1/Stat3 EMSA. Biotin-labeled double-stranded oligonucleotide probes were incubated with nuclear proteins extracted from control or dehydrated SON. After electrophoresis, complexes were transferred to a membrane for ECL detection. A, Lane 1, probe only; lane 2, probe with control SON nuclear extract; lane 3, probe with dehydrated nuclear extract; lane 4, probe, control SON nuclear extract, and an excess of unlabeled oligonucleotide probe; lane 5, probe, dehydrated SON nuclear extract, and an excess of unlabeled oligonucleotide probe. The arrow indicates the position of a specific complex. B, Three further independent EMSA experiments (control [C], 1–3; and dehydrated [D], 1–3) confirm an increase in Stat1/Stat3 complexes after dehydration. C, Supershift analysis with specific Stat1 or Stat3 antibodies reveals that the Stat1/Sta3 complex in the dehydrated SON contains Stat3 but not Stat1. Lane 1, EMSA with control extract; lane 2, EMSA with dehydrated extract; lane 3, supershift EMSA with dehydrated extract and Stat1 antibody; lane 4, supershift EMSA with dehydrated extract and Stat3 antibody. The specific complex is disrupted by the Stat3 antibody, not the Stat1 antibody.
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Figure 2: Stat1/Stat3 EMSA. Biotin-labeled double-stranded oligonucleotide probes were incubated with nuclear proteins extracted from control or dehydrated SON. After electrophoresis, complexes were transferred to a membrane for ECL detection. A, Lane 1, probe only; lane 2, probe with control SON nuclear extract; lane 3, probe with dehydrated nuclear extract; lane 4, probe, control SON nuclear extract, and an excess of unlabeled oligonucleotide probe; lane 5, probe, dehydrated SON nuclear extract, and an excess of unlabeled oligonucleotide probe. The arrow indicates the position of a specific complex. B, Three further independent EMSA experiments (control [C], 1–3; and dehydrated [D], 1–3) confirm an increase in Stat1/Stat3 complexes after dehydration. C, Supershift analysis with specific Stat1 or Stat3 antibodies reveals that the Stat1/Sta3 complex in the dehydrated SON contains Stat3 but not Stat1. Lane 1, EMSA with control extract; lane 2, EMSA with dehydrated extract; lane 3, supershift EMSA with dehydrated extract and Stat1 antibody; lane 4, supershift EMSA with dehydrated extract and Stat3 antibody. The specific complex is disrupted by the Stat3 antibody, not the Stat1 antibody.

Mentions: EMSA was used to confirm increased binding to the Stat1/Stat3 (3.7-fold increase; P = .0027, n = 3) (Figure 2), c-Myc-Max (1.7-fold increase; P = .0413, n = 4) (Figure 3), and Pbx1 (1.3-fold increase; P = .0445, n = 3) (Figure 4) probes after dehydration. In all cases, probe binding was reduced by the addition of an excess of unlabeled probe (Figures 2A, 3A, and 4A). The presence of Stat3, but not Stat1, in the protein-Stat1/Stat3 probe complex was demonstrated by antibody supershift (Figure 2C). An increase in the activity of c-Myc in the dehydrated SON was confirmed by the use of the TransAM c-Myc activity ELISA assay (1.4-fold increase, ±0.31; P = .026, n = 7).


The use of protein-DNA, chromatin immunoprecipitation, and transcriptome arrays to describe transcriptional circuits in the dehydrated male rat hypothalamus.

Qiu J, Kleineidam A, Gouraud S, Yao ST, Greenwood M, Hoe SZ, Hindmarch C, Murphy D - Endocrinology (2014)

Stat1/Stat3 EMSA. Biotin-labeled double-stranded oligonucleotide probes were incubated with nuclear proteins extracted from control or dehydrated SON. After electrophoresis, complexes were transferred to a membrane for ECL detection. A, Lane 1, probe only; lane 2, probe with control SON nuclear extract; lane 3, probe with dehydrated nuclear extract; lane 4, probe, control SON nuclear extract, and an excess of unlabeled oligonucleotide probe; lane 5, probe, dehydrated SON nuclear extract, and an excess of unlabeled oligonucleotide probe. The arrow indicates the position of a specific complex. B, Three further independent EMSA experiments (control [C], 1–3; and dehydrated [D], 1–3) confirm an increase in Stat1/Stat3 complexes after dehydration. C, Supershift analysis with specific Stat1 or Stat3 antibodies reveals that the Stat1/Sta3 complex in the dehydrated SON contains Stat3 but not Stat1. Lane 1, EMSA with control extract; lane 2, EMSA with dehydrated extract; lane 3, supershift EMSA with dehydrated extract and Stat1 antibody; lane 4, supershift EMSA with dehydrated extract and Stat3 antibody. The specific complex is disrupted by the Stat3 antibody, not the Stat1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256826&req=5

Figure 2: Stat1/Stat3 EMSA. Biotin-labeled double-stranded oligonucleotide probes were incubated with nuclear proteins extracted from control or dehydrated SON. After electrophoresis, complexes were transferred to a membrane for ECL detection. A, Lane 1, probe only; lane 2, probe with control SON nuclear extract; lane 3, probe with dehydrated nuclear extract; lane 4, probe, control SON nuclear extract, and an excess of unlabeled oligonucleotide probe; lane 5, probe, dehydrated SON nuclear extract, and an excess of unlabeled oligonucleotide probe. The arrow indicates the position of a specific complex. B, Three further independent EMSA experiments (control [C], 1–3; and dehydrated [D], 1–3) confirm an increase in Stat1/Stat3 complexes after dehydration. C, Supershift analysis with specific Stat1 or Stat3 antibodies reveals that the Stat1/Sta3 complex in the dehydrated SON contains Stat3 but not Stat1. Lane 1, EMSA with control extract; lane 2, EMSA with dehydrated extract; lane 3, supershift EMSA with dehydrated extract and Stat1 antibody; lane 4, supershift EMSA with dehydrated extract and Stat3 antibody. The specific complex is disrupted by the Stat3 antibody, not the Stat1 antibody.
Mentions: EMSA was used to confirm increased binding to the Stat1/Stat3 (3.7-fold increase; P = .0027, n = 3) (Figure 2), c-Myc-Max (1.7-fold increase; P = .0413, n = 4) (Figure 3), and Pbx1 (1.3-fold increase; P = .0445, n = 3) (Figure 4) probes after dehydration. In all cases, probe binding was reduced by the addition of an excess of unlabeled probe (Figures 2A, 3A, and 4A). The presence of Stat3, but not Stat1, in the protein-Stat1/Stat3 probe complex was demonstrated by antibody supershift (Figure 2C). An increase in the activity of c-Myc in the dehydrated SON was confirmed by the use of the TransAM c-Myc activity ELISA assay (1.4-fold increase, ±0.31; P = .026, n = 7).

Bottom Line: In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out.Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration.These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences (J.Q., A.K., S.G., S.T.Y., M.G., C.H., D.M.), University of Bristol, Bristol BS1 3NY, United Kingdom; and Department of Physiology (S.Z.H., C.H., D.M.), Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia.

ABSTRACT
The supraoptic nucleus (SON) of the hypothalamus is responsible for maintaining osmotic stability in mammals through its elaboration of the antidiuretic hormone arginine vasopressin. Upon dehydration, the SON undergoes a function-related plasticity, which includes remodeling of morphology, electrical properties, and biosynthetic activity. This process occurs alongside alterations in steady state transcript levels, which might be mediated by changes in the activity of transcription factors. In order to identify which transcription factors might be involved in changing patterns of gene expression, an Affymetrix protein-DNA array analysis was carried out. Nuclear extracts of SON from dehydrated and control male rats were analyzed for binding to the 345 consensus DNA transcription factor binding sequences of the array. Statistical analysis revealed significant changes in binding to 26 consensus elements, of which EMSA confirmed increased binding to signal transducer and activator of transcription (Stat) 1/Stat3, cellular Myelocytomatosis virus-like cellular proto-oncogene (c-Myc)-Myc-associated factor X (Max), and pre-B cell leukemia transcription factor 1 sequences after dehydration. Focusing on c-Myc and Max, we used quantitative PCR to confirm previous transcriptomic analysis that had suggested an increase in c-Myc, but not Max, mRNA levels in the SON after dehydration, and we demonstrated c-Myc- and Max-like immunoreactivities in SON arginine vasopressin-expressing cells. Finally, by comparing new data obtained from Roche-NimbleGen chromatin immunoprecipitation arrays with previously published transcriptomic data, we have identified putative c-Myc target genes whose expression changes in the SON after dehydration. These include known c-Myc targets, such as the Slc7a5 gene, which encodes the L-type amino acid transporter 1, ribosomal protein L24, histone deactylase 2, and the Rat sarcoma proto-oncogene (Ras)-related nuclear GTPase.

Show MeSH
Related in: MedlinePlus