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Dual functions of TAF7L in adipocyte differentiation.

Zhou H, Kaplan T, Li Y, Grubisic I, Zhang Z, Wang PJ, Eisen MB, Tjian R - Elife (2013)

Bottom Line: Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well.Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters.In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology , Howard Hughes Medical Institute, University of California, Berkeley , Berkeley , United States ; Li Ka Shing Center For Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley , Berkeley , United States.

ABSTRACT
The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

No MeSH data available.


Related in: MedlinePlus

Gene expression analysis of TAF7L-expressing C2C12 cells.(A)–(F) Time course of gene expression by RT-qPCR analysis of Taf7l (A), Pparγ (B), C/ebpα (C), Myf5 (D), Adipoq (E), and Fabp4 (F) in C2C12.CNTL and C2C12.TAF7L cells at 0D, 1D, 2D, 3D, 4D and 5D post adipogenic induction. D, days; CNTL, C2C12.CNTL; TAF7L, C2C12.TAF7L. mRNA levels in C2C12.CNTL cells at 0D were assigned to 1, mRNA levels of each gene at 0D, 1D, 2D, 3D, 4D, and 5D in both C2C12.CNTL and C2C12.TAF7L cells during adipogenesis were compared to mRNA levels in C2C12.CNTL cells at 0D respectively, data is mean from triplicates.DOI:http://dx.doi.org/10.7554/eLife.00170.011
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fig5s1: Gene expression analysis of TAF7L-expressing C2C12 cells.(A)–(F) Time course of gene expression by RT-qPCR analysis of Taf7l (A), Pparγ (B), C/ebpα (C), Myf5 (D), Adipoq (E), and Fabp4 (F) in C2C12.CNTL and C2C12.TAF7L cells at 0D, 1D, 2D, 3D, 4D and 5D post adipogenic induction. D, days; CNTL, C2C12.CNTL; TAF7L, C2C12.TAF7L. mRNA levels in C2C12.CNTL cells at 0D were assigned to 1, mRNA levels of each gene at 0D, 1D, 2D, 3D, 4D, and 5D in both C2C12.CNTL and C2C12.TAF7L cells during adipogenesis were compared to mRNA levels in C2C12.CNTL cells at 0D respectively, data is mean from triplicates.DOI:http://dx.doi.org/10.7554/eLife.00170.011

Mentions: A complementary strategy to probe the capacity of transcription factors to influence specific differentiation pathways involves the ‘reprogramming of cell fate’. For instance, transdifferentiation of C2C12 myoblasts into adipocytes by ectopic expression of PPARγ and/or C/EBPa under adipogenic permissive conditions helped establish these sequence-specific enhancer binding factors as key regulators of adipogenesis (Tontonoz et al., 1994a, 1994b; Hu et al., 1995; Kajimura et al., 2009). Therefore, we tested the adipogenic function of TAF7L by an analogous ‘gain of function’ approach with forced introduction of TAF7L or control vector into C2C12 myoblasts. First, we generated TAF7L-expressing (C2C12.TAF7L) or control C2C12 (C2C12.CNTL) stable cell lines by transfecting either FLAG-TAF7L or empty vector followed by neomycin selection. As detected by FLAG antibody through Western blot analysis, C2C12.TAF7L stable cells achieved modestly elevated expression levels of FLAG-TAF7L protein (Figure 5B). Similarly, C2C12.TAF7L cells express roughly eightfold higher Taf7l mRNA levels than C2C12.CNTL cells (Figure 5—figure supplement 1A). Next, we treated both C2C12.TAF7L and C2C12.CNTL stable cell lines with the four standard adipogenic inducers for 5 days and then applied Oil red O staining. A large proportion of C2C12.TAF7L cells developed into lipid-laden cells while no detectable C2C12.CNTL cells produced lipid droplets (Figure 5A). Gene expression analysis by RT-qPCR confirmed that C2C12.TAF7L cells have markedly increased mRNA levels of a subset of adipocyte-specific genes including Adipsin, Resistin, Pparγ, C/EBPα, Adipoq and Fabp4 relative to C2C12.CNTL cells post differentiation (Figure 5C and Figure 5—figure supplement 1B,C,E,F). By contrast, myoblast-gene Myf5 is downregulated in C2C12.TAF7L cells prior to and post adipogenic induction (Figure 5—figure supplement 1D). Furthermore, we performed mRNA-seq on differentiated C2C12.TAF7L and C2C12.CNTL cells and these genome-wide expression studies revealed that indeed, a number of adipocyte-specific genes become highly upregulated in C2C12.TAF7L cells compared to C2C12.CNTL cells post adipogenesis (Figure 5D). Gene ontology analysis of genes upregulated fivefold or more in differentiated C2C12.TAF7L cells vs C2C12.CNTL cells indicated that ∼32% of these genes are involved in either adipocyte differentiation or function. Notably, in accordance with the role of Taf7l in spermatogenesis that was reported previously, ∼10% of these up-regulated genes are involved in spermatogenesis and sexual reproduction (Figure 5E). Taken together, these results indicate that ectopic expression of TAF7L in C2C12 myoblasts, even at modestly elevated levels, is capable of inducing upregulation of Taf7l itself and other important adipogenic transcription factors including Pparγ and C/ebpα (Figure 5—figure supplement 1A–C) thereby reprogramming a significant portion of C2C12 cells into adipocytes upon induction (Figure 5—figure supplement 1E,F), providing further evidence for TAF7L as a pro-adipogenic regulator.10.7554/eLife.00170.010Figure 5.Ectopic expression of TAF7L transdifferentiates C2C12 myoblasts into adipocytes under adipogenic induction.


Dual functions of TAF7L in adipocyte differentiation.

Zhou H, Kaplan T, Li Y, Grubisic I, Zhang Z, Wang PJ, Eisen MB, Tjian R - Elife (2013)

Gene expression analysis of TAF7L-expressing C2C12 cells.(A)–(F) Time course of gene expression by RT-qPCR analysis of Taf7l (A), Pparγ (B), C/ebpα (C), Myf5 (D), Adipoq (E), and Fabp4 (F) in C2C12.CNTL and C2C12.TAF7L cells at 0D, 1D, 2D, 3D, 4D and 5D post adipogenic induction. D, days; CNTL, C2C12.CNTL; TAF7L, C2C12.TAF7L. mRNA levels in C2C12.CNTL cells at 0D were assigned to 1, mRNA levels of each gene at 0D, 1D, 2D, 3D, 4D, and 5D in both C2C12.CNTL and C2C12.TAF7L cells during adipogenesis were compared to mRNA levels in C2C12.CNTL cells at 0D respectively, data is mean from triplicates.DOI:http://dx.doi.org/10.7554/eLife.00170.011
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fig5s1: Gene expression analysis of TAF7L-expressing C2C12 cells.(A)–(F) Time course of gene expression by RT-qPCR analysis of Taf7l (A), Pparγ (B), C/ebpα (C), Myf5 (D), Adipoq (E), and Fabp4 (F) in C2C12.CNTL and C2C12.TAF7L cells at 0D, 1D, 2D, 3D, 4D and 5D post adipogenic induction. D, days; CNTL, C2C12.CNTL; TAF7L, C2C12.TAF7L. mRNA levels in C2C12.CNTL cells at 0D were assigned to 1, mRNA levels of each gene at 0D, 1D, 2D, 3D, 4D, and 5D in both C2C12.CNTL and C2C12.TAF7L cells during adipogenesis were compared to mRNA levels in C2C12.CNTL cells at 0D respectively, data is mean from triplicates.DOI:http://dx.doi.org/10.7554/eLife.00170.011
Mentions: A complementary strategy to probe the capacity of transcription factors to influence specific differentiation pathways involves the ‘reprogramming of cell fate’. For instance, transdifferentiation of C2C12 myoblasts into adipocytes by ectopic expression of PPARγ and/or C/EBPa under adipogenic permissive conditions helped establish these sequence-specific enhancer binding factors as key regulators of adipogenesis (Tontonoz et al., 1994a, 1994b; Hu et al., 1995; Kajimura et al., 2009). Therefore, we tested the adipogenic function of TAF7L by an analogous ‘gain of function’ approach with forced introduction of TAF7L or control vector into C2C12 myoblasts. First, we generated TAF7L-expressing (C2C12.TAF7L) or control C2C12 (C2C12.CNTL) stable cell lines by transfecting either FLAG-TAF7L or empty vector followed by neomycin selection. As detected by FLAG antibody through Western blot analysis, C2C12.TAF7L stable cells achieved modestly elevated expression levels of FLAG-TAF7L protein (Figure 5B). Similarly, C2C12.TAF7L cells express roughly eightfold higher Taf7l mRNA levels than C2C12.CNTL cells (Figure 5—figure supplement 1A). Next, we treated both C2C12.TAF7L and C2C12.CNTL stable cell lines with the four standard adipogenic inducers for 5 days and then applied Oil red O staining. A large proportion of C2C12.TAF7L cells developed into lipid-laden cells while no detectable C2C12.CNTL cells produced lipid droplets (Figure 5A). Gene expression analysis by RT-qPCR confirmed that C2C12.TAF7L cells have markedly increased mRNA levels of a subset of adipocyte-specific genes including Adipsin, Resistin, Pparγ, C/EBPα, Adipoq and Fabp4 relative to C2C12.CNTL cells post differentiation (Figure 5C and Figure 5—figure supplement 1B,C,E,F). By contrast, myoblast-gene Myf5 is downregulated in C2C12.TAF7L cells prior to and post adipogenic induction (Figure 5—figure supplement 1D). Furthermore, we performed mRNA-seq on differentiated C2C12.TAF7L and C2C12.CNTL cells and these genome-wide expression studies revealed that indeed, a number of adipocyte-specific genes become highly upregulated in C2C12.TAF7L cells compared to C2C12.CNTL cells post adipogenesis (Figure 5D). Gene ontology analysis of genes upregulated fivefold or more in differentiated C2C12.TAF7L cells vs C2C12.CNTL cells indicated that ∼32% of these genes are involved in either adipocyte differentiation or function. Notably, in accordance with the role of Taf7l in spermatogenesis that was reported previously, ∼10% of these up-regulated genes are involved in spermatogenesis and sexual reproduction (Figure 5E). Taken together, these results indicate that ectopic expression of TAF7L in C2C12 myoblasts, even at modestly elevated levels, is capable of inducing upregulation of Taf7l itself and other important adipogenic transcription factors including Pparγ and C/ebpα (Figure 5—figure supplement 1A–C) thereby reprogramming a significant portion of C2C12 cells into adipocytes upon induction (Figure 5—figure supplement 1E,F), providing further evidence for TAF7L as a pro-adipogenic regulator.10.7554/eLife.00170.010Figure 5.Ectopic expression of TAF7L transdifferentiates C2C12 myoblasts into adipocytes under adipogenic induction.

Bottom Line: Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well.Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters.In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology , Howard Hughes Medical Institute, University of California, Berkeley , Berkeley , United States ; Li Ka Shing Center For Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley , Berkeley , United States.

ABSTRACT
The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

No MeSH data available.


Related in: MedlinePlus