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Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Palian BM, Rohira AD, Johnson SA, He L, Zheng N, Dubeau L, Stiles BL, Johnson DL - PLoS Genet. (2014)

Bottom Line: PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes.We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation.Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, and the Norris Comprehensive Cancer Center, Los Angeles, California, United States of America.

ABSTRACT
Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

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Maf1 occupies the FASN promoter to repress lipogenic gene expression.(A) Maf1 negatively regulates cellular FASN and ACC1 protein expression. Protein lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNAs (left) or HepG2 cells engineered to express doxycycline-inducible Maf1-HA as described in Fig. 4 (right) were subjected to immunoblot analysis using antibodies against ACC1, FASN, SREBP, Maf1, HA (right, ectopic Maf1-HA), or β-actin. (B) Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN promoter. Chromatin immunoprecipitations were performed with Huh7 cells stably expressing Maf1 shRNA or HepG2-doxycycline-inducible Maf1-HA cells. ChIP analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS (black and white bars). Bars represent Maf1 (top) or SREBP1c (bottom) occupancy relative to input and IgG. Maf1 occupancy at the TSS displayed statistically significant differences, Maf1 siRNA compared to non-silencing control, p = 0.001, and doxycycline versus no doxycycline treatment, p = 0.0001. (C) Altering Maf1 levels changes Maf1 occupancy at the tRNALeu gene promoter. ChIP analysis was performed as in (B) with primers specific for the tRNALeu promoter. Values shown for all graphs are the means ±S.E. (n = 4); Student t-test, Maf1 shRNA p = 0.0026, Maf1-HA p = 0.0013.
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pgen-1004789-g005: Maf1 occupies the FASN promoter to repress lipogenic gene expression.(A) Maf1 negatively regulates cellular FASN and ACC1 protein expression. Protein lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNAs (left) or HepG2 cells engineered to express doxycycline-inducible Maf1-HA as described in Fig. 4 (right) were subjected to immunoblot analysis using antibodies against ACC1, FASN, SREBP, Maf1, HA (right, ectopic Maf1-HA), or β-actin. (B) Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN promoter. Chromatin immunoprecipitations were performed with Huh7 cells stably expressing Maf1 shRNA or HepG2-doxycycline-inducible Maf1-HA cells. ChIP analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS (black and white bars). Bars represent Maf1 (top) or SREBP1c (bottom) occupancy relative to input and IgG. Maf1 occupancy at the TSS displayed statistically significant differences, Maf1 siRNA compared to non-silencing control, p = 0.001, and doxycycline versus no doxycycline treatment, p = 0.0001. (C) Altering Maf1 levels changes Maf1 occupancy at the tRNALeu gene promoter. ChIP analysis was performed as in (B) with primers specific for the tRNALeu promoter. Values shown for all graphs are the means ±S.E. (n = 4); Student t-test, Maf1 shRNA p = 0.0026, Maf1-HA p = 0.0013.

Mentions: We next determined whether Maf1 repression of FASN transcription was mediated by the direct recruitment of Maf1 to the FASN promoter. Consistent with Maf1-mediated changes in mRNAs above, we found that down regulation of Maf1 in Huh7 cells resulted in an increase in both FASN and ACC1 protein expression, whereas ectopic expression of Maf1 in HepG2 cells produced a decrease in these proteins (Fig. 5A). Using chromatin immunoprecipitation assays we asked whether Maf1 was enriched in the region that conferred Maf1-mediated repression in comparison with Maf1 occupancy at sequences upstream of this region. Maf1 occupancy was enriched in sequences encompassing the transcription start site and SREBP1c binding site of the FASN promoter in both Huh7 and HepG2 cells (Fig. 5B, top). We next manipulated Maf1 expression in these cells. Analysis of Maf1 occupancy revealed that down regulation of Maf1 resulted in diminished recruitment of Maf1 to the FASN promoter (Fig. 5B, top left), whereas induction of Maf1-HA expression resulted in the enhanced recruitment of the HA-tagged Maf1 to the FASN promoter (Fig. 5B, top right). These changes in Maf1 occupancy on the FASN promoter observed when cellular Maf1 levels are altered further validate the ChIP signals and were similar to those observed for the Maf1-targeted tRNALeu gene (Fig. 5C). As expression of SREBP1c protein was unchanged despite altered Maf1 expression (Fig. 5A), we further assessed whether changes in Maf1 expression and promoter occupancy would affect SREBP1c binding. Analysis revealed that changes in Maf1 occupancy did not alter SREBP1c binding to the FASN promoter (Fig. 5B, bottom). These results indicate that at least one mechanism by which Maf1 represses lipogenic gene expression is through the ability of Maf1 to directly target the promoters of these genes. However, given that our previous work revealed that Maf1 represses the expression of the TATA-binding protein, TBP [9], we further assessed whether Maf1-mediated changes in TBP could indirectly regulate lipogenic gene expression. Cellular TBP amounts were increased in Huh7 cells or down-regulated in HepG2 cells (Supplemental Data, Figure S1). In both cases, altered expression of TBP had no affect on the expression of either FASN or ACC1 mRNA. Together, these results support the idea that Maf1 directly targets and negatively regulates genes required for de novo lipogenesis and that this effect does not impair the binding of SREBP1c at the promoter.


Maf1 is a novel target of PTEN and PI3K signaling that negatively regulates oncogenesis and lipid metabolism.

Palian BM, Rohira AD, Johnson SA, He L, Zheng N, Dubeau L, Stiles BL, Johnson DL - PLoS Genet. (2014)

Maf1 occupies the FASN promoter to repress lipogenic gene expression.(A) Maf1 negatively regulates cellular FASN and ACC1 protein expression. Protein lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNAs (left) or HepG2 cells engineered to express doxycycline-inducible Maf1-HA as described in Fig. 4 (right) were subjected to immunoblot analysis using antibodies against ACC1, FASN, SREBP, Maf1, HA (right, ectopic Maf1-HA), or β-actin. (B) Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN promoter. Chromatin immunoprecipitations were performed with Huh7 cells stably expressing Maf1 shRNA or HepG2-doxycycline-inducible Maf1-HA cells. ChIP analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS (black and white bars). Bars represent Maf1 (top) or SREBP1c (bottom) occupancy relative to input and IgG. Maf1 occupancy at the TSS displayed statistically significant differences, Maf1 siRNA compared to non-silencing control, p = 0.001, and doxycycline versus no doxycycline treatment, p = 0.0001. (C) Altering Maf1 levels changes Maf1 occupancy at the tRNALeu gene promoter. ChIP analysis was performed as in (B) with primers specific for the tRNALeu promoter. Values shown for all graphs are the means ±S.E. (n = 4); Student t-test, Maf1 shRNA p = 0.0026, Maf1-HA p = 0.0013.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263377&req=5

pgen-1004789-g005: Maf1 occupies the FASN promoter to repress lipogenic gene expression.(A) Maf1 negatively regulates cellular FASN and ACC1 protein expression. Protein lysates isolated from Huh7 cells stably infected with nsRNA or Maf1 shRNAs (left) or HepG2 cells engineered to express doxycycline-inducible Maf1-HA as described in Fig. 4 (right) were subjected to immunoblot analysis using antibodies against ACC1, FASN, SREBP, Maf1, HA (right, ectopic Maf1-HA), or β-actin. (B) Altering Maf1 levels changes occupancy of Maf1 but not SREBP on the FASN promoter. Chromatin immunoprecipitations were performed with Huh7 cells stably expressing Maf1 shRNA or HepG2-doxycycline-inducible Maf1-HA cells. ChIP analysis was performed with antibodies against Maf1, SREBP1c, and IgG. qPCR was performed with an upstream primers set (gray bars) and a set encompassing the transcription start site TSS (black and white bars). Bars represent Maf1 (top) or SREBP1c (bottom) occupancy relative to input and IgG. Maf1 occupancy at the TSS displayed statistically significant differences, Maf1 siRNA compared to non-silencing control, p = 0.001, and doxycycline versus no doxycycline treatment, p = 0.0001. (C) Altering Maf1 levels changes Maf1 occupancy at the tRNALeu gene promoter. ChIP analysis was performed as in (B) with primers specific for the tRNALeu promoter. Values shown for all graphs are the means ±S.E. (n = 4); Student t-test, Maf1 shRNA p = 0.0026, Maf1-HA p = 0.0013.
Mentions: We next determined whether Maf1 repression of FASN transcription was mediated by the direct recruitment of Maf1 to the FASN promoter. Consistent with Maf1-mediated changes in mRNAs above, we found that down regulation of Maf1 in Huh7 cells resulted in an increase in both FASN and ACC1 protein expression, whereas ectopic expression of Maf1 in HepG2 cells produced a decrease in these proteins (Fig. 5A). Using chromatin immunoprecipitation assays we asked whether Maf1 was enriched in the region that conferred Maf1-mediated repression in comparison with Maf1 occupancy at sequences upstream of this region. Maf1 occupancy was enriched in sequences encompassing the transcription start site and SREBP1c binding site of the FASN promoter in both Huh7 and HepG2 cells (Fig. 5B, top). We next manipulated Maf1 expression in these cells. Analysis of Maf1 occupancy revealed that down regulation of Maf1 resulted in diminished recruitment of Maf1 to the FASN promoter (Fig. 5B, top left), whereas induction of Maf1-HA expression resulted in the enhanced recruitment of the HA-tagged Maf1 to the FASN promoter (Fig. 5B, top right). These changes in Maf1 occupancy on the FASN promoter observed when cellular Maf1 levels are altered further validate the ChIP signals and were similar to those observed for the Maf1-targeted tRNALeu gene (Fig. 5C). As expression of SREBP1c protein was unchanged despite altered Maf1 expression (Fig. 5A), we further assessed whether changes in Maf1 expression and promoter occupancy would affect SREBP1c binding. Analysis revealed that changes in Maf1 occupancy did not alter SREBP1c binding to the FASN promoter (Fig. 5B, bottom). These results indicate that at least one mechanism by which Maf1 represses lipogenic gene expression is through the ability of Maf1 to directly target the promoters of these genes. However, given that our previous work revealed that Maf1 represses the expression of the TATA-binding protein, TBP [9], we further assessed whether Maf1-mediated changes in TBP could indirectly regulate lipogenic gene expression. Cellular TBP amounts were increased in Huh7 cells or down-regulated in HepG2 cells (Supplemental Data, Figure S1). In both cases, altered expression of TBP had no affect on the expression of either FASN or ACC1 mRNA. Together, these results support the idea that Maf1 directly targets and negatively regulates genes required for de novo lipogenesis and that this effect does not impair the binding of SREBP1c at the promoter.

Bottom Line: PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes.We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation.Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, and the Norris Comprehensive Cancer Center, Los Angeles, California, United States of America.

ABSTRACT
Maf1 was initially identified as a transcriptional repressor of RNA pol III-transcribed genes, yet little is known about its other potential target genes or its biological function. Here, we show that Maf1 is a key downstream target of PTEN that drives both its tumor suppressor and metabolic functions. Maf1 expression is diminished with loss of PTEN in both mouse models and human cancers. Consistent with its role as a tumor suppressor, Maf1 reduces anchorage-independent growth and tumor formation in mice. PTEN-mediated changes in Maf1 expression are mediated by PTEN acting on PI3K/AKT/FoxO1 signaling, revealing a new pathway that regulates RNA pol III-dependent genes. This regulatory event is biologically relevant as diet-induced PI3K activation reduces Maf1 expression in mouse liver. We further identify lipogenic enzymes as a new class of Maf1-regulated genes whereby Maf1 occupancy at the FASN promoter opposes SREBP1c-mediated transcription activation. Consistent with these findings, Maf1 inhibits intracellular lipid accumulation and increasing Maf1 expression in mouse liver abrogates diet-mediated induction of lipogenic enzymes and triglycerides. Together, these results establish a new biological role for Maf1 as a downstream effector of PTEN/PI3K signaling and reveal that Maf1 is a key element by which this pathway co-regulates lipid metabolism and oncogenesis.

Show MeSH
Related in: MedlinePlus