Limits...
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.

Show MeSH

Related in: MedlinePlus

c-Myc- and Max mRNAs in the SON. Relative mRNA expression levels of (A) Myc and (B) Max in SON of 3 days of dehydrated compared with control rats (n = 6 each group). Error bar is ±SEM. **, P < .01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256826&req=5

Figure 5: c-Myc- and Max mRNAs in the SON. Relative mRNA expression levels of (A) Myc and (B) Max in SON of 3 days of dehydrated compared with control rats (n = 6 each group). Error bar is ±SEM. **, P < .01.

Mentions: We have previously used Affymetrix GeneChip analysis to identify changes in the expression of mRNAs encoding transcription factors in the rat SON as a consequence of 3 days of dehydration (25). Examination of these datasets suggested a significant (∼1.64-fold) increase in c-Myc mRNAs but no change in the expression of Max transcripts. This has been confirmed by qPCR. c-Myc mRNA levels increased with dehydration (1.287 ± 0.058; P < .01, n = 6) (Figure 5), but Max transcript levels did not significantly change (1.085 ± 0.036; not significant, n = 6) (Figure 5). Both c-Myc- and Max-like immunoreactivities were found in the SON of both control and dehydrated rats (Figure 6A). c-Myc staining is both nuclear and cytosolic, whereas Max is mainly nuclear. Because the SON contains 2 distinct MCN populations, those that express AVP and those that express OT, we sought to identify which of these cell types express c-Myc or Max. c-Myc- and Max-like immunoreactivities are found in both AVP and OT expressing MCNs.


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)

c-Myc- and Max mRNAs in the SON. Relative mRNA expression levels of (A) Myc and (B) Max in SON of 3 days of dehydrated compared with control rats (n = 6 each group). Error bar is ±SEM. **, P < .01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: c-Myc- and Max mRNAs in the SON. Relative mRNA expression levels of (A) Myc and (B) Max in SON of 3 days of dehydrated compared with control rats (n = 6 each group). Error bar is ±SEM. **, P < .01.
Mentions: We have previously used Affymetrix GeneChip analysis to identify changes in the expression of mRNAs encoding transcription factors in the rat SON as a consequence of 3 days of dehydration (25). Examination of these datasets suggested a significant (∼1.64-fold) increase in c-Myc mRNAs but no change in the expression of Max transcripts. This has been confirmed by qPCR. c-Myc mRNA levels increased with dehydration (1.287 ± 0.058; P < .01, n = 6) (Figure 5), but Max transcript levels did not significantly change (1.085 ± 0.036; not significant, n = 6) (Figure 5). Both c-Myc- and Max-like immunoreactivities were found in the SON of both control and dehydrated rats (Figure 6A). c-Myc staining is both nuclear and cytosolic, whereas Max is mainly nuclear. Because the SON contains 2 distinct MCN populations, those that express AVP and those that express OT, we sought to identify which of these cell types express c-Myc or Max. c-Myc- and Max-like immunoreactivities are found in both AVP and OT expressing MCNs.

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