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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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Conserved PPARγ sequences 1 to 5 are transcriptionally active in adipocyte precursors, but not in mature fat cells.Real-time qPCR analysis of LacZ (A), PPARγ (B), and adiponectin (C) expression in the stroma-vascular (SVF) and adipocyte fractions of fat pads derived from wild type, PPARγ (+/−), and PPARγ CS1-5_LacZ line 1 mice (6–7 weeks, n = 3 per group). Error bars denote mean ± S.D. (D) Conditionally immortalized SV cells from subcutaneous WAT of line 1 transgenic mice at day 8 post-induction of adipocyte differentiation. Nile-red stains adipocyte neutral lipids. Levels of LacZ (E) and PPARγ (F) mRNA expression during the course of adipocyte differentiation in these cells. Error bars denote mean ± S.D.
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pone-0072511-g003: Conserved PPARγ sequences 1 to 5 are transcriptionally active in adipocyte precursors, but not in mature fat cells.Real-time qPCR analysis of LacZ (A), PPARγ (B), and adiponectin (C) expression in the stroma-vascular (SVF) and adipocyte fractions of fat pads derived from wild type, PPARγ (+/−), and PPARγ CS1-5_LacZ line 1 mice (6–7 weeks, n = 3 per group). Error bars denote mean ± S.D. (D) Conditionally immortalized SV cells from subcutaneous WAT of line 1 transgenic mice at day 8 post-induction of adipocyte differentiation. Nile-red stains adipocyte neutral lipids. Levels of LacZ (E) and PPARγ (F) mRNA expression during the course of adipocyte differentiation in these cells. Error bars denote mean ± S.D.

Mentions: Adipocytes develop in coordination with the vasculature, which supplies oxygen, nutrients, and endocrine factors, and provides a niche for pericyte-derived adipocyte progenitors [16], [32]. To explore the compartment(s) within adipose depots where CS1 to 5 PPARγ sequences are transcriptionally active, we measured LacZ and PPARγ mRNA expression after separation of the stromal-vascular (SV) and adipocyte fractions of WAT and BAT depots of wild type, PPARγ (+/−), and transgenic PPARγ CS1-5_ LacZ mice. As expected, LacZ expression driven by the entire endogenous PPARγ locus (as in PPARγ [+/−] mice) was predominantly associated with the differentiated adipocyte compartment, particularly in WAT depots (Fig. 3A). In contrast, we found that the CS1-5 PPARγ sequences activated LacZ mRNA expression almost exclusively in the SV compartment, and not in the adipocyte fraction of either WAT or BAT. This pattern of LacZ expression observed in transgenic line 1 was confirmed in two other transgenic lines (lines 6 and 7; see Supplementary Fig. 3 for line 7 data). Endogenous PPARγ mRNA was detected in the SV fraction, but was significantly enriched in the adipocyte compartment, with no differences among mice of different genotypes (Fig. 3B). The quality of our fractions was verified by measuring expression of adiponectin, a mature adipocyte marker that could only be detected in the adipocyte fraction (Fig. 3C). These results indicate that PPARγ CS 1 to 5 are transcriptionally active only in the SV fraction that contains adipocyte progenitors and preadipocytes, as well as other cells that do not contribute to the adipose lineage. Interestingly, expression of transgenic LacZ, but not that derived from the endogenous locus (PPARγ [+/−] mice), was consistently higher in BAT compared to WAT (Fig. 3A), perhaps a reflection of the larger vascular network that is present in BAT.


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)

Conserved PPARγ sequences 1 to 5 are transcriptionally active in adipocyte precursors, but not in mature fat cells.Real-time qPCR analysis of LacZ (A), PPARγ (B), and adiponectin (C) expression in the stroma-vascular (SVF) and adipocyte fractions of fat pads derived from wild type, PPARγ (+/−), and PPARγ CS1-5_LacZ line 1 mice (6–7 weeks, n = 3 per group). Error bars denote mean ± S.D. (D) Conditionally immortalized SV cells from subcutaneous WAT of line 1 transgenic mice at day 8 post-induction of adipocyte differentiation. Nile-red stains adipocyte neutral lipids. Levels of LacZ (E) and PPARγ (F) mRNA expression during the course of adipocyte differentiation in these cells. Error bars denote mean ± S.D.
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Related In: Results  -  Collection

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

pone-0072511-g003: Conserved PPARγ sequences 1 to 5 are transcriptionally active in adipocyte precursors, but not in mature fat cells.Real-time qPCR analysis of LacZ (A), PPARγ (B), and adiponectin (C) expression in the stroma-vascular (SVF) and adipocyte fractions of fat pads derived from wild type, PPARγ (+/−), and PPARγ CS1-5_LacZ line 1 mice (6–7 weeks, n = 3 per group). Error bars denote mean ± S.D. (D) Conditionally immortalized SV cells from subcutaneous WAT of line 1 transgenic mice at day 8 post-induction of adipocyte differentiation. Nile-red stains adipocyte neutral lipids. Levels of LacZ (E) and PPARγ (F) mRNA expression during the course of adipocyte differentiation in these cells. Error bars denote mean ± S.D.
Mentions: Adipocytes develop in coordination with the vasculature, which supplies oxygen, nutrients, and endocrine factors, and provides a niche for pericyte-derived adipocyte progenitors [16], [32]. To explore the compartment(s) within adipose depots where CS1 to 5 PPARγ sequences are transcriptionally active, we measured LacZ and PPARγ mRNA expression after separation of the stromal-vascular (SV) and adipocyte fractions of WAT and BAT depots of wild type, PPARγ (+/−), and transgenic PPARγ CS1-5_ LacZ mice. As expected, LacZ expression driven by the entire endogenous PPARγ locus (as in PPARγ [+/−] mice) was predominantly associated with the differentiated adipocyte compartment, particularly in WAT depots (Fig. 3A). In contrast, we found that the CS1-5 PPARγ sequences activated LacZ mRNA expression almost exclusively in the SV compartment, and not in the adipocyte fraction of either WAT or BAT. This pattern of LacZ expression observed in transgenic line 1 was confirmed in two other transgenic lines (lines 6 and 7; see Supplementary Fig. 3 for line 7 data). Endogenous PPARγ mRNA was detected in the SV fraction, but was significantly enriched in the adipocyte compartment, with no differences among mice of different genotypes (Fig. 3B). The quality of our fractions was verified by measuring expression of adiponectin, a mature adipocyte marker that could only be detected in the adipocyte fraction (Fig. 3C). These results indicate that PPARγ CS 1 to 5 are transcriptionally active only in the SV fraction that contains adipocyte progenitors and preadipocytes, as well as other cells that do not contribute to the adipose lineage. Interestingly, expression of transgenic LacZ, but not that derived from the endogenous locus (PPARγ [+/−] mice), was consistently higher in BAT compared to WAT (Fig. 3A), perhaps a reflection of the larger vascular network that is present in BAT.

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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Related in: MedlinePlus