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. 2009 May;29(10):2804-15.
doi: 10.1128/MCB.01667-08. Epub 2009 Mar 9.

Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis

Andrew P R Sutherland  1 , Hai Zhang, Ye Zhang, Monia Michaud, Zhifang Xie, Mary-Elizabeth Patti, Michael J Grusby, Weiping J Zhang
Affiliations

Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis

Andrew P R Sutherland et al. Mol Cell Biol. 2009 May.

Abstract

Zbtb20 is a member of the POK family of proteins, which function primarily as transcriptional repressors via interactions mediated by their conserved C(2)H(2) Krüppel type zinc finger and BTB/POZ domains. To define the function of Zbtb20 in vivo, we generated knockout mice by homologous recombination. Zbtb20 null mice display a stark phenotype characterized by postnatal growth retardation, metabolic dysfunction, and lethality. Zbtb20 knockout mice displayed abnormal glucose homeostasis, hormonal responses, and depletion of energy stores, consistent with an energetic deficit. Additionally, increased serum bilirubin and alanine aminotransferase levels were suggestive of liver dysfunction. To identify potential liver-specific Zbtb20 target genes, we performed transcript profiling studies on liver tissue from Zbtb20 knockout mice and wild-type littermate controls. These studies identified sets of genes involved in growth, metabolism, and detoxification that were differentially regulated in Zbtb20 knockout liver. Transgenic mice expressing Zbtb20 in the liver were generated and crossed onto the Zbtb20 knockout background, which resulted in no significant normalization of growth or glucose metabolism but a significant increase in life span compared to controls. These data indicate that the phenotype of Zbtb20 knockout mice results from liver-dependent and -independent defects, suggesting that Zbtb20 plays nonredundant roles in multiple organ systems.

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Figures

FIG. 1.
FIG. 1.
Zbtb20 knockout mouse generation and validation. (A) Schematic demonstration for the gene targeting of the Zbtb20 gene. The probe 5′ is used for Southern blot analysis of EcoRV-digested genomic DNA, which detects a 7.3-kb band for the wild-type (wt) allele, a 5-kb band for the homologously recombined allele with a neomycin resistance cassette (Neo), or a 3.2-kb band for a Neo-deleted allele created by Cre recombinantion. The loxP sites are represented as filled triangles. V, EcoRV; X, XhoI; H, HpaI; E, EcoRI; D, DraIII; C, ClaI; S, SalI; N, NotI; HSV-TK, herpes simplex virus thymidine kinase cassette. (B) Southern blotting identified products specific to the wild-type (WT; 7.3-kb) and knockout (KO; 3.2-kb) alleles in homozygous and heterozygous mice. (C) PCR-amplified products specific to the wild-type (∼100-bp) and knockout (∼300-bp) alleles in homozygous and heterozygous mice. (D) Western blotting was performed on nuclear fractions from the livers of Zbtb20 knockout mice and littermate controls using an anti-Zbtb20 polyclonal antiserum. An antibody against the nuclear protein Sp1 was used as a protein loading control.
FIG. 2.
FIG. 2.
Zbtb20-deficient mice display growth retardation, hypoglycemia, and lethality. (A) Zbtb20 null mice and littermate controls were weighed weekly from 2 to 12 weeks of age. (B) Visual inspection reveals reduced axial growth at 2.5 weeks of age. (C) Blood glucose levels were measured every ∼2 weeks from 2.5 to 12 weeks of age. (D) Survival curves for Zbtb20 null mice and littermate controls (+/+, n = 16; +/−, n = 30; −/−, n = 11).
FIG. 3.
FIG. 3.
Zbtb20 has altered glucose metabolism consistent with nutrient deficiency. (A) Blood glucose levels of Zbtb20 null and littermate controls were measured prior to (fed; P < 0.01) and after (fast; P < 0.005) an overnight fast. (B to D) Glucose tolerance tests (2 mg/kg; +/+, n = 5; +/−, n = 15; −/−, n = 10; P < 0.005) (B), insulin tolerance tests (0.75 U/kg; +/+, n = 5; +/−, n = 5; −/−, n = 5; P < 0.005) (C), and glucagon tolerance tests (30 μg/kg; +/+ n = 4, +/−, n = 24;−/−, n = 5; P < 0.005) (D) were performed on Zbtb20 null and littermate controls. (E) Total glycogen was quantitated in liver from Zbtb20 null mice and littermate controls by a glucose oxidase activity assay (top) (+/+, n = 5; +/−, n = 3; −/−, n = 4, P < 0.05) and periodic acid-Schiff staining of fixed tissue (bottom).
FIG. 4.
FIG. 4.
Transcript profiling of liver tissue from Zbtb20 null mice reveals putative target genes and altered regulation of pathways involved in growth, glucose metabolism, and detoxification. HeatMaps were generated from the transcript profiling data comparing Zbtb20 null and wild-type control liver tissue (+/+, n = 4; −/−, n = 4). The 40 transcripts with the highest upregulation (A) and downregulation (B) in Zbtb20 null liver are shown. Gene ontology analysis revealed transcripts involved in pathways controlling growth (C), glucose metabolism (D), and detoxification (E). WT, wild type; KO, knockout.
FIG. 5.
FIG. 5.
Zbtb20 transgenic mouse generation and validation. (A) Murine Zbtb20 gene cDNA was cloned under the control of regulatory elements from the human ApoE gene. (B) PCR of tail DNA of Zbtb20 transgenic and littermate control animals yields an ∼250-bp band in transgenic animals. (C) Zbtb20 mRNA levels in liver tissue isolated from Zbtb20 transgenic (Tg) and littermate control (WT) mice were measured by quantitative PCR (WT, n = 4; Tg, n = 9). (D) Western blots of total liver lysates from Zbtb20 transgenic and littermate controls. Two distinct bands corresponding to Zbtb20 are detected at ∼90 and ∼100 kDa, respectively, as well as a faster-migrating nonspecific band (NS). (E) Zbtb20 mRNA levels were measured in a panel of tissues isolated from Zbtb20 transgenic and littermate control mice by quantitative PCR (WT, n = 2; Tg, n = 2).
FIG. 6.
FIG. 6.
The Zbtb20 liver transgene partially complements elements of the Zbtb20 null phenotype. (A) Zbtb20 transgenic mice, Zbtb20 null mice, and littermate controls were weighed weekly from 2 to 12 weeks of age. (B) Blood glucose levels were measured every ∼2 weeks from 2.5 to 12 weeks of age for mice fed ad libitum. (C) Survival curves for Zbtb20 null mice and littermate controls (+/+,−, n = 19; +/+,+, n = 12; +/−,−, n = 50; +/−,+, n = 27; −/−,−, n = 16; −/−,+, n = 14; P = 0.017). Total RNA was extracted from liver tissue harvested from a cohort of mice at 4 to 6 weeks of age from the Zbtb20 knockout to Zbtb20 transgenic cross. (D and E) Levels of transcripts for AFP (D) and a number of cytochrome P450 family members (E) were measured by quantitative RT-PCR (+/+,− and +/−,−, n = 10; −/−,−, n = 5; +/+,+ and +/−,+, n = 5; −/−,+, n = 3).
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