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Identification of regulatory elements that control PPARγ expression in adipocyte progenitors.

Chou WL, Galmozzi A, Partida D, Kwan K, Yeung H, Su AI, Saez E - PLoS ONE (2013)

Bottom Line: Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression.Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes.Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.

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PPARγ CS1-5_LacZ positive cells express markers of adipose progenitors.Paraffin-embedded serial sections of X-gal stained subcutaneous WAT derived from PPARγ CS1-5_LacZ line 1 transgenic mice were analyzed by immunohistochemistry. Note that LacZ positive cells in transgenic fat pads express mural/endothelial/adipose progenitor cell markers (CD29, SMA), but not perilipin (mature adipocytes). Arrows point to several examples of the same LacZ positive cells in all serial sections, so that the overlap of markers can be evaluated.
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pone-0072511-g005: PPARγ CS1-5_LacZ positive cells express markers of adipose progenitors.Paraffin-embedded serial sections of X-gal stained subcutaneous WAT derived from PPARγ CS1-5_LacZ line 1 transgenic mice were analyzed by immunohistochemistry. Note that LacZ positive cells in transgenic fat pads express mural/endothelial/adipose progenitor cell markers (CD29, SMA), but not perilipin (mature adipocytes). Arrows point to several examples of the same LacZ positive cells in all serial sections, so that the overlap of markers can be evaluated.

Mentions: To evaluate the extent to which these sequences control PPARγ expression in vivo (i.e. behave as enhancer elements that dictate tissue-specific PPARγ expression), we cloned all 5 elements together into an Hsp68-LacZ reporter vector to generate PPARγ CS1-5_Hsp68-LacZ transgenic mice (referred hereafter as PPARγ CS1-5_LacZ; Supplemental Fig. 1). The Hsp68 minimal promoter was chosen because this is a widely used basal promoter for in vivo enhancer analysis [30]. To establish if these 5 conserved elements are sufficient to drive expression of the LacZ reporter in a pattern similar to that of endogenous PPARγ, we analyzed LacZ expression by X-gal staining in tissues of 5 independently-derived PPARγ CS1-5_LacZ transgenic lines. One line (line 1) showed very strong X-gal staining in brown fat and in all white adipose depots (Fig. 1B). To check the specificity of reporter expression, we analyzed LacZ expression in skeletal muscle, liver, spleen, and pancreas and found no X-gal staining in these organs (Fig. 1B and Supplemental Fig. 5). The pattern of X-gal staining in this PPARγ CS1-5_LacZ transgenic line mirrored that seen in PPARγ (+/−) heterozygous mice in which an allele of PPARγ was targeted by an in-frame insertion of a neomycin-LacZ construct (β-geo) into exon 2 of PPARγ [24]. Analysis of LacZ expression across tissues by RT-qPCR and Western Blot indicated that the PPARγ CS1-5_LacZ transgene was expressed in a similar pattern to that of endogenous PPARγ (Fig. 2), with greatest expression of mRNA and protein in fat depots, and lower levels in selected other organs. This adipose-enriched pattern of expression of the transgene suggested that these 5 conserved sequences contain most of the regulatory elements necessary for tissue-specific PPARγ expression. Two additional PPARγ CS1-5_LacZ transgenic lines (lines 6 and 7) showed an identical, but weaker, pattern of X-gal staining and LacZ mRNA expression, indicating that the pattern of transgene expression we observed is not the consequence of integration effects.


Identification of regulatory elements that control PPARγ expression in adipocyte progenitors.

Chou WL, Galmozzi A, Partida D, Kwan K, Yeung H, Su AI, Saez E - PLoS ONE (2013)

PPARγ CS1-5_LacZ positive cells express markers of adipose progenitors.Paraffin-embedded serial sections of X-gal stained subcutaneous WAT derived from PPARγ CS1-5_LacZ line 1 transgenic mice were analyzed by immunohistochemistry. Note that LacZ positive cells in transgenic fat pads express mural/endothelial/adipose progenitor cell markers (CD29, SMA), but not perilipin (mature adipocytes). Arrows point to several examples of the same LacZ positive cells in all serial sections, so that the overlap of markers can be evaluated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3757023&req=5

pone-0072511-g005: PPARγ CS1-5_LacZ positive cells express markers of adipose progenitors.Paraffin-embedded serial sections of X-gal stained subcutaneous WAT derived from PPARγ CS1-5_LacZ line 1 transgenic mice were analyzed by immunohistochemistry. Note that LacZ positive cells in transgenic fat pads express mural/endothelial/adipose progenitor cell markers (CD29, SMA), but not perilipin (mature adipocytes). Arrows point to several examples of the same LacZ positive cells in all serial sections, so that the overlap of markers can be evaluated.
Mentions: To evaluate the extent to which these sequences control PPARγ expression in vivo (i.e. behave as enhancer elements that dictate tissue-specific PPARγ expression), we cloned all 5 elements together into an Hsp68-LacZ reporter vector to generate PPARγ CS1-5_Hsp68-LacZ transgenic mice (referred hereafter as PPARγ CS1-5_LacZ; Supplemental Fig. 1). The Hsp68 minimal promoter was chosen because this is a widely used basal promoter for in vivo enhancer analysis [30]. To establish if these 5 conserved elements are sufficient to drive expression of the LacZ reporter in a pattern similar to that of endogenous PPARγ, we analyzed LacZ expression by X-gal staining in tissues of 5 independently-derived PPARγ CS1-5_LacZ transgenic lines. One line (line 1) showed very strong X-gal staining in brown fat and in all white adipose depots (Fig. 1B). To check the specificity of reporter expression, we analyzed LacZ expression in skeletal muscle, liver, spleen, and pancreas and found no X-gal staining in these organs (Fig. 1B and Supplemental Fig. 5). The pattern of X-gal staining in this PPARγ CS1-5_LacZ transgenic line mirrored that seen in PPARγ (+/−) heterozygous mice in which an allele of PPARγ was targeted by an in-frame insertion of a neomycin-LacZ construct (β-geo) into exon 2 of PPARγ [24]. Analysis of LacZ expression across tissues by RT-qPCR and Western Blot indicated that the PPARγ CS1-5_LacZ transgene was expressed in a similar pattern to that of endogenous PPARγ (Fig. 2), with greatest expression of mRNA and protein in fat depots, and lower levels in selected other organs. This adipose-enriched pattern of expression of the transgene suggested that these 5 conserved sequences contain most of the regulatory elements necessary for tissue-specific PPARγ expression. Two additional PPARγ CS1-5_LacZ transgenic lines (lines 6 and 7) showed an identical, but weaker, pattern of X-gal staining and LacZ mRNA expression, indicating that the pattern of transgene expression we observed is not the consequence of integration effects.

Bottom Line: Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression.Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes.Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Physiology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states.

Show MeSH
Related in: MedlinePlus