Progress 11/01/01 to 10/31/05
Outputs
We have characterized the mechanism by which thyroid hormone activates the hepatic transcription of the acetyl-CoA carboxylase (ACC) gene in chickens. The active form of thyroid hormone, triiodothyronine (T3), interacts with the nuclear T3 receptor (TR) bound to T3 response element (T3RE) on promoter 2 of the ACC gene. This region of the ACC gene is associated with coactivators (CBP, TRAP/ARC) that interact with the basal transcription apparatus or modify chromatin structure through changes in histone acetylation. In addition, the ACC gene binds sterol regulatory element binding protein-1 (SREBP-1). SREBP-1 directly interacts with TR on ACC promoter 2 to stimulate ACC gene transcription. We also showed that agents that inhibit ACC gene transcription (i.e. glucagon and medium chain fatty acids) act by disrupting the positive interaction between TR and SREBP-1 on ACC promoter 2. In addition, we demonstrated that alterations in SREBP-1 expression play a role in mediating
the dietary regulation of ACC gene transcription in intact chickens. Lastly, we demonstrated that the ACC gene is a target of the liver X receptor (LXR) and that artificial agonists of LXR activate ACC gene transcription in chicken liver. The mechanism for this effect involves an interaction between LXR and SREBP-1 on ACC promoter 2.
Impacts
The identification of small molecules that modulate SREBP-1 activity will provide an approach to control lipid levels in poultry. In addition, the SREBP-1 gene provides a loci for genetic selection in poultry.
Publications
- Wang, Y., Yin, L., and Hillgartner, F. B. (2001) The homeodomain proteins PBX and MEIS1 are accessory factors that enhance thyroid hormone regulation of the malic enzyme gene in hepatocytes. J. Biol. Chem. 276, 23838-23848.
- Wang, Y., Zhang, Y., and Hillgartner, F. B. (2002) Chicken ovalbumin upstream promoter transcription factor and E-box binding proteins enhance thyroid hormone responsiveness of the malic enzyme gene in avian hepatocytes. Biochem. J. 361, 391-400.
- Yin, L., Zhang, Y., and Hillgartner, F. B. (2002) Sterol regulatory element-binding protein 1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase transcription in hepatocytes. J. Biol. Chem. 277,19554-19565.
- Zhang, Y., Yin, L., and Hillgartner, F. B. (2003) SREBP 1 integrates the actions of thyroid hormone, insulin, cAMP, and medium-chain fatty acids on ACC transcription in hepatocytes. J. Lipid Res. 44, 356-368.
- Zhang, Y., Yin, L., and Hillgartner, F. B. (2001) Thyroid hormone stimulates acetyl-CoA carboxylase transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element. J. Biol. Chem. 276, 974-983.
- Zhang, Y. and Hillgartner, F. B. (2004) Starvation and feeding a high-carbohydrate, low-fat diet regulate the expression of sterol regulatory element-binding protein 1 in chickens. J. Nutr. 134, 2205-2210
- Yin, L., Wang, Y., Dridi, S., Vinson, C., and Hillgartner, F. B. (2005) Role of CCAAT/enhancer-binding protein, histone acetylation, and coactivator recruitment in the regulation of malic enzyme transcription by thyroid hormone. Mol. Cell. Endocrinol. 245, 43-52
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Progress 10/01/02 to 09/30/03
Outputs
In previous work, we showed that activation of acetyl-CoA carboxylase (ACC) transcription by 3,5,3'-triiodothyronine (T3) is mediated by a cis-acting regulatory unit (-101 to -71 bp) that binds the nuclear T3 receptor (TR) and sterol regulatory element-binding protein-1 (SREBP-1). We also demonstrated that SREBP-1 directly interacted with TR on the ACC gene to enhance T3-induced transcription. During the past year, we further investigated the role of SREBP-1 in mediating the actions of T3, insulin, and cyclic AMP on ACC transcription. We found that treating chick hepatocytes with T3 or insulin stimulated a 4-fold increase in the concentration of the mature, active form of SREBP-1. When T3 and insulin were added together, a 7-fold increase in the mature SREBP-1 concentration was observed. Time course studies indicated that the T3-induced increase in mature SREBP-1 abundance was closely associated with changes in ACC transcription and that the mechanism mediating the
effect of T3 on mature SREBP-1 was distinct from that mediating the effect of insulin. Transfection analyses indicated that inhibition of ACC transcription by cyclic AMP was mediated by ACC sequences between -101 and -71 bp. Treatment with cyclic AMP suppressed the increase in mature SREBP-1 abundance caused by T3 and insulin. These results establish a new interaction between the SREBP-1 and TR signaling pathways and demonstrate that SREBP-1 plays an active role in mediating the effects of T3, insulin, and cyclic AMP on ACC transcription. In other work, we identified and characterized the full-length cDNA for chicken SREBP-1 and investigated the regulation of this protein by alterations in nutritional status. We found that feeding previously starved chicks a high-carbohydrate, low-fat diet stimulated a robust increase (14-fold at 5 h of feeding) in the concentration of mature SREBP-1 in liver. Feeding a high-carbohydrate, low-fat diet also increased the concentration of precursor
SREBP-1 and SREBP-1 mRNA in chick liver; however, the magnitude of this effect was substantially lower than that observed for mature SREBP-1. DNA binding experiments demonstrated that three protein complexes containing SREBP bound the ACC sterol regulatory element (SRE) in chick liver and that the binding activity of two of these complexes was increased by consumption of a high-carbohydrate, low-fat diet. Additional analyses showed that feeding a high-carbohydrate, low-fat diet had no effect on the concentration of mature SREBP-2 and the binding of SREBP-2 to the ACC SRE in chick liver. These results indicate that alterations in the concentration of mature SREBP-1 play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACC transcription in chick liver and that diet-induced changes in mature SREBP-1 concentration in chick liver are mediated primarily by a posttranslational mechanism. Because insulin, T3, and cyclic AMP regulate mature SREBP-1
concentration primarily by a posttranslational mechanism, we postulate that these hormones are involved in mediating diet-induced changes in SREBP-1 concentration in intact chickens.
Impacts
Factors controlling the expression of the mature form of SREBP-1 provide a means to regulate fat accretion in chickens.
Publications
- 1. Zhang, Y., Yin, L., and Hillgartner, F. B. (2003) SREBP-1 integrates the actions of thyroid hormone, insulin, cAMP, and medium-chain fatty acids on ACC transcription in hepatocytes. J. Lipid Res. 44, 356-368.
- 2. Zhang, Y. and Hillgartner, F. B. (2004) Starvation and feeding a high-carbohydrate, low-fat diet regulate the expression of sterol regulatory element-binding protein-1 in chickens. J. Nutr. In press.
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Progress 11/01/01 to 10/30/02
Outputs
In previous work, we characterized a 3,5,3O-triiodothyronine response element (T3RE) in acetyl-CoA carboxylase (ACC) promoter 2 that mediated 3,5,3O-triiodothyronine (T3) regulation of ACC transcription in chick embryo hepatocytes. Sequence comparison analysis revealed the presence of a sterol regulatory element-1 (SRE-1) located 5 bp downstream of the ACC T3RE. In this project, we have set out to investigate the role of this SRE-1 in modulating T3 regulation of ACC transcription. Transfection analyses demonstrated that the SRE-1 enhanced T3-induced ACC transcription by more than 2-fold in hepatocytes. The effect of the SRE-1 on T3 responsiveness required the presence of the T3RE in its native orientation. In pull-down experiments, the mature form of SREBP-1 specifically bound the nuclear T3 receptor (TR), and the presence of T3 enhanced this interaction. A region of TR containing the DNA binding domain plus flanking sequences (amino acids 21-157) was required for
interaction with SREBP-1, and a region of SREBP-1 containing the basic-helix-loop-helix-leucine zipper domain (amino acids 300 to 389) was required for interaction with TR. In gel mobility shift experiments, TR, retinoid X receptor, and mature SREBP-1 formed a tetrameric complex on a DNA probe containing the ACC T3RE and SRE-1, and the presence of T3 enhanced the formation of this complex. Formation of the tetrameric complex stabilized the binding of SREBP-1 to the SRE-1. Thus far, the results indicate that SREBP-1 directly interacts with TR-RXR in an orientation-specific manner to enhance T3-induced ACC transcription in hepatocytes. In other work, we have characterized cis-acting accessory elements that enhance T3 regulation of malic enzyme transcription in chick embryo hepatocytes. One of these elements (designated region E) binds the homeodomain proteins, PBX and Meis1. We have developed evidence that PBX and Meis1 augment T3 responsiveness via direct interactions with TR-RXR
heterodimers. Thus SREBP-1 and PBX-Meis1 appear to act via a similar mechanism to enhance transcription of the ACC and malic enzyme genes, respectively. The orphan nuclear hormone receptor, COUP-TF, binds another T3 accessory element (designated region G) in the malic enzyme gene. The pattern of binding of COUP-TF to region G varies substantially between hepatocytes and non-hepatic cell types suggesting a mechanism by which region G confers cell-type dependent differences in T3 regulation of malic enzyme transcription.
Impacts
The above defines a new target for manipulating fat synthesis. Pharmacological agents that disrupt the interaction between SREBP and the nuclear thyroid hormone receptor and between PBX and the nuclear thyroid hormone receptor should provide a selective means for inhibiting fat accretion in chickens.
Publications
- 1. Zhang, Y., Yin, L., and Hillgartner, F.B. (2001) Thyroid hormone stimulates acetyl-CoA carboxylase transcription in hepatocytes by modulating the composition of nuclear receptor complexes bound to a thyroid hormone response element. J. Biol. Chem. 276, 974-983.
- 2. Wang, Y., Yin, L., and Hillgartner, F. B. (2001) The homeodomain proteins PBX and MEIS1 are accessory factors that enhance thyroid hormone regulation of the malic enzyme gene in hepatocytes. J. Biol. Chem. 276, 23838-23848.
- 3. Wang, Y., Zhang, Y., and Hillgartner, F. B. (2002) Chicken ovalbumin upstream promoter transcription factor and E-box binding proteins enhance thyroid hormone responsiveness of the malic enzyme gene in avian hepatocytes. Biochem. J. 361, 391-400.
- 4. Yin, L., Zhang, Y., and Hillgartner, F. B. (2002) Sterol regulatory element-binding protein-1 interacts with the nuclear thyroid hormone receptor to enhance acetyl-CoA carboxylase transcription in hepatocytes. J. Biol. Chem. 277,19554-19565.
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