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Znf202 affects high density lipoprotein cholesterol levels and promotes hepatosteatosis in hyperlipidemic mice.

Vrins CL, Out R, van Santbrink P, van der Zee A, Mahmoudi T, Groenendijk M, Havekes LM, van Berkel TJ, Willems van Dijk K, Biessen EA - PLoS ONE (2013)

Bottom Line: The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified.Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated.Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands. c_vrins@hotmail.com

ABSTRACT

Background: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established.

Methodology and principal findings: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression.

Conclusion/significance: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

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Related in: MedlinePlus

The Znf202 specifically binds to the alleged response elements−678 and −564 within the −705/−362 region of the mouse apoE promoter. (A) DNA fragments used in this study containing the putative Znf202 binding sequence. The putative consensus sequences are underlined. (B) EMSA with labeled DNA fragments GnT (lanes 1–10), −678 (lanes 11–20), −564 (lanes 21–30), and PHO (lanes 31–35) described above. Reactions contained the indicated amount (0.1–0.6 µg) of whole cell extract made from either control 911 cells, or from 911 cells overexpressing Znf202. Competition experiments using 50-fold excess of unlabeled GnT oligonucleotide (lanes 4,7 and 10), −678 oligonucleotide (lanes 14,17 and 20), or -564 oligonucleotide (lanes 24,27 and 30) confirmed the specificity of Znf202 binding to both GnT elements. Arrowheads indicate the mobility of unbound DNA and Znf202 protein bound DNA.
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pone-0057492-g002: The Znf202 specifically binds to the alleged response elements−678 and −564 within the −705/−362 region of the mouse apoE promoter. (A) DNA fragments used in this study containing the putative Znf202 binding sequence. The putative consensus sequences are underlined. (B) EMSA with labeled DNA fragments GnT (lanes 1–10), −678 (lanes 11–20), −564 (lanes 21–30), and PHO (lanes 31–35) described above. Reactions contained the indicated amount (0.1–0.6 µg) of whole cell extract made from either control 911 cells, or from 911 cells overexpressing Znf202. Competition experiments using 50-fold excess of unlabeled GnT oligonucleotide (lanes 4,7 and 10), −678 oligonucleotide (lanes 14,17 and 20), or -564 oligonucleotide (lanes 24,27 and 30) confirmed the specificity of Znf202 binding to both GnT elements. Arrowheads indicate the mobility of unbound DNA and Znf202 protein bound DNA.

Mentions: With the Znf202 repressive effect on ApoE promoter activity restricted to the −705/−362 region, we studied the interaction of Znf202 with the two putative GnT sites within this domain at positions −678 and −564 (Fig. 2A) by EMSA. The GnT control probe (Fig. 2B, lanes 1–10) induced a clear mobility shift indicative of the formation of DNA-protein complexes in extracts from Znf202 overexpressing 911 cells (lanes 3, 6, 9). Probe binding was specifically competed by 50-fold excess of unlabeled GnT probe (lane 4, 7, 10). Similar mobility shifts were observed for the [32P]-678 (lanes 11–20) or [32P] −564 probe (lanes 21–30) after Znf202 overexpression. In analogy to the reference probe, the specificity of binding was confirmed by displacement by a 50-fold excess of either unlabeled −678 or −564 probe. Finally, the extracts were unable to form DNA-protein complexes with an irrelevant PHO consensus probe, even at high concentrations (lanes 32 and 33). These in vitro results confirm the repressive role of Znf202 and establish the functionality of our murine Znf202 constructs.


Znf202 affects high density lipoprotein cholesterol levels and promotes hepatosteatosis in hyperlipidemic mice.

Vrins CL, Out R, van Santbrink P, van der Zee A, Mahmoudi T, Groenendijk M, Havekes LM, van Berkel TJ, Willems van Dijk K, Biessen EA - PLoS ONE (2013)

The Znf202 specifically binds to the alleged response elements−678 and −564 within the −705/−362 region of the mouse apoE promoter. (A) DNA fragments used in this study containing the putative Znf202 binding sequence. The putative consensus sequences are underlined. (B) EMSA with labeled DNA fragments GnT (lanes 1–10), −678 (lanes 11–20), −564 (lanes 21–30), and PHO (lanes 31–35) described above. Reactions contained the indicated amount (0.1–0.6 µg) of whole cell extract made from either control 911 cells, or from 911 cells overexpressing Znf202. Competition experiments using 50-fold excess of unlabeled GnT oligonucleotide (lanes 4,7 and 10), −678 oligonucleotide (lanes 14,17 and 20), or -564 oligonucleotide (lanes 24,27 and 30) confirmed the specificity of Znf202 binding to both GnT elements. Arrowheads indicate the mobility of unbound DNA and Znf202 protein bound DNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585336&req=5

pone-0057492-g002: The Znf202 specifically binds to the alleged response elements−678 and −564 within the −705/−362 region of the mouse apoE promoter. (A) DNA fragments used in this study containing the putative Znf202 binding sequence. The putative consensus sequences are underlined. (B) EMSA with labeled DNA fragments GnT (lanes 1–10), −678 (lanes 11–20), −564 (lanes 21–30), and PHO (lanes 31–35) described above. Reactions contained the indicated amount (0.1–0.6 µg) of whole cell extract made from either control 911 cells, or from 911 cells overexpressing Znf202. Competition experiments using 50-fold excess of unlabeled GnT oligonucleotide (lanes 4,7 and 10), −678 oligonucleotide (lanes 14,17 and 20), or -564 oligonucleotide (lanes 24,27 and 30) confirmed the specificity of Znf202 binding to both GnT elements. Arrowheads indicate the mobility of unbound DNA and Znf202 protein bound DNA.
Mentions: With the Znf202 repressive effect on ApoE promoter activity restricted to the −705/−362 region, we studied the interaction of Znf202 with the two putative GnT sites within this domain at positions −678 and −564 (Fig. 2A) by EMSA. The GnT control probe (Fig. 2B, lanes 1–10) induced a clear mobility shift indicative of the formation of DNA-protein complexes in extracts from Znf202 overexpressing 911 cells (lanes 3, 6, 9). Probe binding was specifically competed by 50-fold excess of unlabeled GnT probe (lane 4, 7, 10). Similar mobility shifts were observed for the [32P]-678 (lanes 11–20) or [32P] −564 probe (lanes 21–30) after Znf202 overexpression. In analogy to the reference probe, the specificity of binding was confirmed by displacement by a 50-fold excess of either unlabeled −678 or −564 probe. Finally, the extracts were unable to form DNA-protein complexes with an irrelevant PHO consensus probe, even at high concentrations (lanes 32 and 33). These in vitro results confirm the repressive role of Znf202 and establish the functionality of our murine Znf202 constructs.

Bottom Line: The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified.Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated.Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands. c_vrins@hotmail.com

ABSTRACT

Background: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established.

Methodology and principal findings: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression.

Conclusion/significance: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels.

Show MeSH
Related in: MedlinePlus