ZBED6 counteracts high-fat diet-induced glucose intolerance by maintaining beta cell area and reducing excess mitochondrial activation.

AIMS/HYPOTHESIS: ZBED6 (zinc finger, BED-type containing 6) is known to regulate muscle mass by suppression of Igf2 gene transcription. In insulin-producing cell lines, ZBED6 maintains proliferative capacity at the expense of differentiation and beta cell function. The aim was to study the impact of Zbed6 knockout on beta cell function and glucose tolerance in C57BL/6 mice. METHODS: Beta cell area and proliferation were determined in Zbed6 knockout mice using immunohistochemical analysis. Muscle and fat distribution were assessed using micro-computed tomography. Islet gene expression was assessed by RNA sequencing. Effects of a high-fat diet were analysed by glucose tolerance and insulin tolerance tests. ZBED6 was overexpressed in EndoC-ßH1 cells and human islet cells using an adenoviral vector. Beta cell cell-cycle analysis, insulin release and mitochondrial function were studied in vitro using propidium iodide staining and flow cytometry, ELISA, the Seahorse technique, and the fluorescent probes JC-1 and MitoSox. RESULTS: Islets from Zbed6 knockout mice showed lowered expression of the cell cycle gene Pttg1, decreased beta cell proliferation and decreased beta cell area, which occurred independently from ZBED6 effects on Igf2 gene expression. Zbed6 knockout mice, but not wild-type mice, developed glucose intolerance when given a high-fat diet. The high-fat diet Zbed6 knockout islets displayed upregulated expression of oxidative phosphorylation genes and genes associated with beta cell differentiation. In vitro, ZBED6 overexpression resulted in increased EndoC-ßH1 cell proliferation and a reduced glucose-stimulated insulin release in human islets. ZBED6 also reduced mitochondrial JC-1 J-aggregate formation, mitochondrial oxygen consumption rates (OCR) and mitochondrial reactive oxygen species (ROS) production, both at basal and palmitate + high glucose-stimulated conditions. ZBED6-induced inhibition of OCR was not rescued by IGF2 addition. ZBED6 reduced levels of the mitochondrial regulator PPAR-γ related coactivator 1 protein (PRC) and bound its promoter/enhancer region. Knockdown of PRC resulted in a lowered OCR. CONCLUSIONS/INTERPRETATION: It is concluded that ZBED6 is required for normal beta cell replication and also limits excessive beta cell mitochondrial activation in response to an increased functional demand. ZBED6 may act, at least in part, by restricting PRC-mediated mitochondrial activation/ROS production, which may lead to protection against beta cell dysfunction and glucose intolerance in vivo.

Comprehensive analysis of lncRNAs involved in skeletal muscle development in ZBED6-knockout Bama pigs.

BACKGROUND: The mutation of insulin-like growth factor 2 (IGF2 mutation) that a single-nucleotide substitution (G→A) in the third intron of IGF2 abrogates the interaction with zinc finger BED-type containing 6 (ZBED6) and leads to increased muscle mass in pigs. IGF2 mutation knock-in (IGF2 KI) and ZBED6 knockout (ZBED6 KO) lead to changes in IGF2 expression and increase muscle mass in mice and pigs. Long noncoding RNAs (lncRNAs) may participate in numerous biological processes, including skeletal muscle development. However, the role of the ZBED6-lncRNA axis in skeletal muscle development is poorly characterized. RESULTS: In this study, we assembled transcriptomes using RNA-seq data published in previous studies by our group and identified 11,408 known lncRNAs and 2269 potential lncRNAs in seven tissues, heart, longissimus dorsi, gastrocnemius muscle, liver, spleen, lung and kidney, of ZBED6 KO (lean mass model) and WT Bama pigs. ZBED6 affected the expression of 1570 lncRNAs (differentially expressed lncRNAs [DE-lncRNAs]; log2-fold change ≥ 1, nominal p-value ≤ 0.05) in the seven examined tissues. The expressed lncRNAs (FPKM > 0.1) exhibited tissue-specific patterns in WT pigs. Specifically, 3410 lncRNAs were expressed exclusively in only one tissue. Potential functions of lncRNAs were indirectly predicted by searching their target cis- and trans-regulated protein-coding genes. LncRNAs with tissue-specific expression influence numerous genes related to tissue functions. Weighted gene coexpression network analysis (WGCNA) of 1570 DE-lncRNAs between WT and ZBED6 KO pigs was used to define the following six lncRNA modules specific to different tissues: skeletal muscle, heart, lung, spleen, kidney and liver modules. Furthermore, by conjoint analysis of longissimus dorsi data (tissue-specific expression, muscle module and DE-lncRNAs) and ChIP-PCR revealed NONSUSG002145.1 (adjusted p-values = 0.044), which is coexpressed with the IGF2 gene and binding with ZBED6, may play important roles in ZBED6 KO pig skeletal muscle development. CONCLUSIONS: These findings indicate that the identified lncRNAs may play essential roles in tissue function and regulate the mechanism of ZBED6 action in skeletal muscle development in pigs. To our knowledge, this is the first study describing lncRNAs in ZBED6 KO pigs. These results may open new research directions leading to a better understanding of the global functions of ZBED6 and of lncRNA functions in skeletal muscle development in pigs.

The importance of the ZBED6-IGF2 axis for metabolic regulation in mouse myoblast cells.

The transcription factor ZBED6 acts as a repressor of Igf2 and affects directly or indirectly the transcriptional regulation of thousands of genes. Here, we use gene editing in mouse C2C12 myoblasts and show that ZBED6 regulates Igf2 exclusively through its binding site 5'-GGCTCG-3' in intron 1 of Igf2. Deletion of this motif (Igf2ΔGGCT ) or complete ablation of Zbed6 leads to ~20-fold upregulation of the IGF2 protein. Quantitative proteomics revealed an activation of Ras signaling pathway in both Zbed6-/- and Igf2ΔGGCT myoblasts, and a significant enrichment of mitochondrial membrane proteins among proteins showing altered expression in Zbed6-/- myoblasts. Both Zbed6-/- and Igf2ΔGGCT myoblasts showed a faster growth rate and developed myotube hypertrophy. These cells exhibited an increased O2 consumption rate, due to IGF2 upregulation. Transcriptome analysis revealed ~30% overlap between differentially expressed genes in Zbed6-/- and Igf2ΔGGCT myotubes, with an enrichment of upregulated genes involved in muscle development. In contrast, ZBED6-overexpression in myoblasts led to cell apoptosis, cell cycle arrest, reduced mitochondrial activities, and ceased myoblast differentiation. The similarities in growth and differentiation phenotypes observed in Zbed6-/- and Igf2ΔGGCT myoblasts demonstrates that ZBED6 affects mitochondrial activity and myogenesis largely through its regulation of IGF2 expression. This study adds new insights how the ZBED6-Igf2 axis affects muscle metabolism.

The ZBED6-IGF2 axis has a major effect on growth of skeletal muscle and internal organs in placental mammals.

A single nucleotide substitution in the third intron of insulin-like growth factor 2 (IGF2) is associated with increased muscle mass and reduced subcutaneous fat in domestic pigs. This mutation disrupts the binding of the ZBED6 transcription factor and leads to a threefold up-regulation of IGF2 expression in pig skeletal muscle. Here, we investigated the biological significance of ZBED6-IGF2 interaction in the growth of placental mammals using two mouse models, ZBED6 knock-out (Zbed6-/-) and Igf2 knock-in mice that carry the pig IGF2 mutation. These transgenic mice exhibit markedly higher serum IGF2 concentrations, higher growth rate, increased lean mass, and larger heart, kidney, and liver; no significant changes were observed for white adipose tissues. The changes in body and lean mass were most pronounced in female mice. The phenotypic changes were concomitant with a remarkable up-regulation of Igf2 expression in adult tissues. Transcriptome analysis of skeletal muscle identified differential expression of genes belonging to the extracellular region category. Expression analysis using fetal muscles indicated a minor role of ZBED6 in regulating Igf2 expression prenatally. Furthermore, transcriptome analysis of the adult skeletal muscle revealed that this elevated expression of Igf2 was derived from the P1 and P2 promoters. The results revealed very similar phenotypic effects in the Zbed6 knock-out mouse and in the Igf2 knock-in mouse, showing that the effect of ZBED6 on growth of muscle and internal organs is mediated through the binding site in the Igf2 gene. The results explain why this ZBED6 binding site is extremely well conserved among placental mammals.

Transcriptional modulator ZBED6 affects cell cycle and growth of human colorectal cancer cells.

The transcription factor ZBED6 (zinc finger, BED-type containing 6) is a repressor of IGF2 whose action impacts development, cell proliferation, and growth in placental mammals. In human colorectal cancers, IGF2 overexpression is mutually exclusive with somatic mutations in PI3K signaling components, providing genetic evidence for a role in the PI3K pathway. To understand the role of ZBED6 in tumorigenesis, we engineered and validated somatic cell ZBED6 knock-outs in the human colorectal cancer cell lines RKO and HCT116. Ablation of ZBED6 affected the cell cycle and led to increased growth rate in RKO cells but reduced growth in HCT116 cells. This striking difference was reflected in the transcriptome analyses, which revealed enrichment of cell-cycle-related processes among differentially expressed genes in both cell lines, but the direction of change often differed between the cell lines. ChIP sequencing analyses displayed enrichment of ZBED6 binding at genes up-regulated in ZBED6-knockout clones, consistent with the view that ZBED6 modulates gene expression primarily by repressing transcription. Ten differentially expressed genes were identified as putative direct gene targets, and their down-regulation by ZBED6 was validated experimentally. Eight of these genes were linked to the Wnt, Hippo, TGF-ß, EGF receptor, or PI3K pathways, all involved in colorectal cancer development. The results of this study show that the effect of ZBED6 on tumor development depends on the genetic background and the transcriptional state of its target genes.

Correlation between ZBED6 Gene Upstream CpG Island methylation and mRNA expression in cattle.

DNA methylation is essential for the regulation of gene expression and important roles in muscle development. To assess the extent of epigenetic modifications and gene expression on the differentially methylated region (DMR) in ZBED6, we simultaneously examined DNA methylation and expression in six tissues from two different developmental stages (fetal bovine and adult bovine). The DNA methylation pattern was compared using bisulfite sequencing polymerase chain reaction (BSP) and combined bisulfite restriction analysis (COBRA). The result of quantitative real-time PCR (qPCR) analysis showed that ZBED6 has a broad tissue distribution and is highly expressed in adult bovine (P < 0.05 or P < 0.01). The DNA methylation level was significantly different in liver, lung and spleen between the two cattle groups (P < 0.05 or P < 0.01). The adult bovine group exhibited a significantly higher mRNA level and lower DNA methylation level than the fetal bovine group in liver, lung, and spleen. No significant association was detected between DNA methylation level and muscle, heart, and kidney at two different stages. In this study, the statistical analyses indicated that DNA methylation patterns are associated with mRNA level in some tissues, these results may be a useful parameter to investigate muscle developmental in cattle and as a model for studies in other species, potentially contributing to an improvement of growth performance selection in beef cattle breeding program.

m6A reader YTHDF1 promotes cardiac fibrosis by enhancing AXL translation.

Cardiac fibrosis caused by ventricular remodeling and dysfunction such as post-myocardial infarction (MI) can lead to heart failure. RNA N6-methyladenosine (m6A) methylation has been shown to play a pivotal role in the occurrence and development of many illnesses. In investigating the biological function of the m6A reader YTHDF1 in cardiac fibrosis, adeno-associated virus 9 was used to knock down or overexpress the YTHDF1 gene in mouse hearts, and MI surgery in vivo and transforming growth factor-ß (TGF-ß)-activated cardiac fibroblasts in vitro were performed to establish fibrosis models. Our results demonstrated that silencing YTHDF1 in mouse hearts can significantly restore impaired cardiac function and attenuate myocardial fibrosis, whereas YTHDF1 overexpression could further enhance cardiac dysfunction and aggravate the occurrence of ventricular pathological remodeling and fibrotic development. Mechanistically, zinc finger BED-type containing 6 mediated the transcriptional function of the YTHDF1 gene promoter. YTHDF1 augmented AXL translation and activated the TGF-ß-Smad2/3 signaling pathway, thereby aggravating the occurrence and development of cardiac dysfunction and myocardial fibrosis. Consistently, our data indicated that YTHDF1 was involved in activation, proliferation, and migration to participate in cardiac fibrosis in vitro. Our results revealed that YTHDF1 could serve as a potential therapeutic target for myocardial fibrosis.

Loss of ZBED6 Protects Against Sepsis-Induced Muscle Atrophy by Upregulating DOCK3-Mediated RAC1/PI3K/AKT Signaling Pathway in Pigs.

Sepsis-induced muscle atrophy often increases morbidity and mortality in intensive care unit (ICU) patients, yet neither therapeutic target nor optimal animal model is available for this disease. Here, by modifying the surgical strategy of cecal ligation and puncture (CLP), a novel sepsis pig model is created that for the first time recapitulates the whole course of sepsis in humans. With this model and sepsis patients, increased levels of the transcription factor zinc finger BED-type containing 6 (ZBED6) in skeletal muscle are shown. Protection against sepsis-induced muscle wasting in ZBED6-deficient pigs is further demonstrated. Mechanistically, integrated analysis of RNA-seq and ChIP-seq reveals dedicator of cytokinesis 3 (DOCK3) as the direct target of ZBED6. In septic ZBED6-deficient pigs, DOCK3 expression is increased in skeletal muscle and myocytes, activating the RAC1/PI3K/AKT pathway and protecting against sepsis-induced muscle wasting. Conversely, opposite gene expression patterns and exacerbated muscle wasting are observed in septic ZBED6-overexpressing myotubes. Notably, sepsis patients show increased ZBED6 expression along with reduced DOCK3 and downregulated RAC1/PI3K/AKT pathway. These findings suggest that ZBED6 is a potential therapeutic target for sepsis-induced muscle atrophy, and the established sepsis pig model is a valuable tool for understanding sepsis pathogenesis and developing its therapeutics.

Effect of Zbed6 Single-Allele Knockout on the Growth and Development of Skeletal Muscle in Mice.

ZBED6, a key transcription factor, plays an important role in skeletal muscle and organ growth. ZBED6 knockout (ZBED6-/-) leads to the upregulation of IGF2 in pig and mice muscle, thereby increasing muscle mass. However, the effects and mechanism of Zbed6 single-allele knockout (Zbed6+/-) on mice muscle remain unknown. Here, we reported that Zbed6+/- promotes muscle growth by a new potential target gene rather than Igf2 in mice muscle. Zbed6+/- mice showed markedly higher muscle mass (25%) and a markedly higher muscle weight ratio (18%) than wild-type (WT) mice, coinciding with a larger muscle fiber area (28%). Despite a significant increase in muscle growth, Zbed6+/- mice showed similar Igf2 expression with WT mice, indicating that a ZBED6-Igf2-independent regulatory pathway exists in Zbed6+/- mice muscle. RNA-seq of muscle between the Zbed6+/- and WT mice revealed two terms related to muscle growth. Overlapping the DEGs and C2C12 Chip-seq data of ZBED6 screened out a potential ZBED6 target gene Barx2, which may regulate muscle growth in Zbed6+/- mice. These results may open new research directions leading to a better understanding of the integral functions of ZBED6 and provide evidence of Zbed6+/- promoting muscle growth by regulating Barx2 in mice.

Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs.

BACKGROUND: Chinese indigenous pigs are popular with consumers for their juiciness, flavour and meat quality, but they have lower meat production. Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that promotes skeletal muscle growth by regulating cell proliferation and differentiation. A single nucleotide polymorphism (SNP) within intron 3 of porcine IGF2 disrupts a binding site for the repressor, zinc finger BED-type containing 6 (ZBED6), leading to up-regulation of IGF2 and causing major effects on muscle growth, heart size, and backfat thickness. This favorable mutation is common in Western commercial pig populations, but absent in most Chinese indigenous pig breeds. To improve meat production of Chinese indigenous pigs, we used cytosine base editor 3 (CBE3) to introduce IGF2-intron3-C3071T mutation into porcine embryonic fibroblasts (PEFs) isolated from a male Liang Guang Small Spotted pig (LGSS), and single-cell clones harboring the desired mutation were selected for somatic cell nuclear transfer (SCNT) to generate the founder line of IGF2T/T pigs. RESULTS: We found the heterozygous progeny IGF2C/T pigs exhibited enhanced expression of IGF2, increased lean meat by 18%-36%, enlarged loin muscle area by 3%-17%, improved intramuscular fat (IMF) content by 18%-39%, marbling score by 0.75-1, meat color score by 0.53-1.25, and reduced backfat thickness by 5%-16%. The enhanced accumulation of intramuscular fat in IGF2C/T pigs was identified to be regulated by the PI3K-AKT/AMPK pathway, which activated SREBP1 to promote adipogenesis. CONCLUSIONS: We demonstrated the introduction of IGF2-intron3-C3071T in Chinese LGSS can improve both meat production and quality, and first identified the regulation of IMF deposition by IGF2 through SREBP1 via the PI3K-AKT/AMPK signaling pathways. Our study provides a further understanding of the biological functions of IGF2 and an example for improving porcine economic traits through precise base editing.

Free fatty acid receptor 4 deletion attenuates colitis by modulating Treg Cells via ZBED6-IL33 pathway.

BACKGROUND: Inflammatory bowel disease (IBD) has complex genetic and environmental aspects, and free fatty acid receptors (FFARs) may bridge genetic and dietary aspects. FFAR4 is highly expressed in the intestine and acts primarily as the receptor of long-chain fatty acids, which are major components of the human diet. It is unclear what role, if any, FFAR4 may play in IBD. METHODS: Mouse and human colitis samples, mice with complete FFAR4 knockout, intestine-specific FFAR4 knockout and FFAR4 overexpression and cell culture were used. RNA-sequencing analysis and flow cytometry were performed to examine the mechanisms. FINDINGS: The results showed that FFAR4 expression was upregulated in colitis tissues and that the loss of intestinal FFAR4 ameliorated colitis, whereas intestinal FFAR4 overexpression exacerbated the disease. We identified intestinal epithelial cell deletion of FFAR4 by upregulating ZBED6, which in turn induced L33 transcription, and L33 elevated Treg cell numbers, ameliorating colitis. INTERPRETATION: FFAR4 deletion attenuates colitis by modulating Treg cells via the ZBED6-IL33 pathway. FUNDING: National Natural Science Foundation of China, Innovation and Application Project of Medical and Public Health Technology of Wuxi Science and Technology, Fundamental Research Funds for the Central Universities and the Fund of Wuxi Healthcare Commission.

A Comprehensive Analysis of the Myocardial Transcriptome in ZBED6-Knockout Bama Xiang Pigs.

The ZBED6 gene is a transcription factor that regulates the expression of IGF2 and affects muscle growth and development. However, its effect on the growth and development of the heart is still unknown. Emerging evidence suggests that long noncoding RNAs (lncRNAs) can regulate genes at the epigenetic, transcriptional, and posttranscriptional levels and play an important role in the development of eukaryotes. To investigate the function of ZBED6 in the cardiac development of pigs, we constructed the expression profiles of mRNAs and lncRNAs in myocardial tissue obtained from Bama Xiang pigs in the ZBED6 knockout group (ZBED6-KO) and the wild-type group (ZBED6-WT). A total of 248 differentially expressed genes (DEGs) and 209 differentially expressed lncRNAs (DELs) were detected, and 105 potential cis target genes of DELs were identified. The functional annotation analysis based on the Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases revealed two GO items related to muscle development by the cis target genes of DELs. Moreover, IGF2 was the direct target gene of ZBED6 by ChIP-PCR experiment. Our results explored the mechanism and expression profile of mRNAs and lncRNAs of ZBED6 gene knockout on myocardium tissue development, mining the key candidate genes in that process like IGF2.

Efficient Editing of the ZBED6-Binding Site in Intron 3 of IGF2 in a Bovine Model Using the CRISPR/Cas9 System.

BACKGROUND: Insulin-like growth factor 2 is a growth-promoting factor that plays an important role in the growth and development of mammals. A nucleotide substitution in intron 3 of IGF2-which disrupts the ZBED6-binding site-affects muscle mass, organ size, and fat deposition in pigs. The ZBED6-binding site is also conserved in cattle. METHODS: In the present study, we introduced mutations in the ZBED6-binding site in intron3 of IGF2 in bovine fetal fibroblasts using the CRISPR/Cas9 system, and investigated the effect of disruption of ZBED6 binding on IGF2 expression. RESULTS: Eleven biallelic-mutant single-cell clones were established, three of which contained no foreign DNA residues. Single-cell clones 93 and 135 were used to produce cloned embryos. Dual-luciferase reporter assay in C2C12 cells demonstrated that the mutation in the ZBED6-binding site increases the promoter 3 activity of bovine IGF2. A total of 49 mutant cloned embryos were transplanted into surrogate cows. Unfortunately, all cloned embryos died before birth. IGF2 was found to be hypomethylated in the only fetus born (stillborn), which may have been due to the incomplete reprogramming. CONCLUSIONS: We efficiently constructed IGF2-edited cell lines and cloned embryos, which provided a theoretical basis and experimental materials for beef cattle breeding.

ZBED6 binding motifs correlate with endogenous retroviruses and Syncytin genes.

Retroviruses have infiltrated vertebrate germlines for millions of years as inherited endogenous retroviruses (ERVs). Mammalian genomes host large numbers of ERVs and transposable elements (TEs), including retrotransposons and DNA transposons, that contribute to genomic innovation and evolution as coopted genes and regulators of diverse functions. To explore features distinguishing coopted ERVs and TEs from other integrations, we focus on the potential role of ZBED6 and repeated ERV domestication as repurposed Syncytin genes. The placental mammal-specific ZBED6 is a DNA transposon-derived transcription regulator and we demonstrate that its binding motifs are associated with distinct Syncytins and that ZBED6 binding motifs are 2- to 3-fold more frequent in ERVs than in flanking DNA. Our observations suggest that ZBED6 could contribute an extended regulatory role of genomic expression, utilizing ERVs as platforms for genomic innovation and evolution.

Liver Expression of IGF2 and Related Proteins in ZBED6 Gene-Edited Pig by RNA-Seq.

Zinc finger BED-type containing 6 (ZBED6), a highly conservative transcription factor of placental mammals, has conservative interaction of insulin-like growth factor 2 (IGF2) based on the 16 bp binding sites of ZBED6 on the IGF2 sequence. IGF2 is related to embryo growth and cell proliferation. At the same time, its functions in muscle and adipose in mammals have been widely mentioned in recent studies. To further investigate the mechanism of ZBED6 on IGF2, we detected the expression of IGF2 and related genes in ZBED6 single allele knockout (ZBED6-SKO) pig tissues and analyzed the transcriptome of ZBED6-SKO pig liver. Through RNA-seq, we captured nine up-regulated genes and eight down-regulated genes which related to lipid metabolism. The results showed that the mRNA of IGF2 had an upward trend after the partial knockout of ZBED6 in liver and had no significant difference in protein expression of IGF2. In summary, ZBED6-SKO could affect the secretion of IGF2 in pig liver and its own lipid metabolism. Our research has provided basic information for revealing the regulatory mechanism of the interaction between ZBED6 and IGF2 in mammals.

Knockdown of ZBED6 is not associated with changes in murine preadipocyte proliferation or differentiation.

ZBED6 was identified as a transcription factor that affects muscle mass and fat deposition in pigs. Mechanisms mediating effects on fat mass are unclear. The objective was to determine the effect of ZBED6 mRNA knockdown on 3T3-L1 preadipocyte differentiation and gene expression. Differentiation was associated with increased mRNA abundance of CEBP/α (P < 0.05), CEBP/ß (P < 0.05), CEBP/δ (P < 0.05), FASN (P < 0.05), PPARγ (P < 0.05), and SREBP-1 (P < 0.05), and decreased abundance of PREF-1 (P < 0.05). Knockdown of ZBED6 was not associated with changes in mRNA abundance of selected genes, lipid accumulation, lipid droplet size, or cell number. These results suggest that ZBED6 does not play a major role in preadipocyte differentiation.

Relationship of polymorphisms within ZBED6 gene and growth traits in beef cattle.

ZBED6 is a novel transcription factor that was identified and shown to act as a repressor of IGF2 transcription in skeletal muscle. The aim of this study was to examine the association of the ZBED6 polymorphism with growth traits in beef cattle breed. Three single nucleotide polymorphisms (SNPs) were identified in the bovine ZBED6 by sequencing pooled DNA samples (Pool-Seq) and forced polymerase chain reaction-restriction fragment length polymorphism (Forced PCR-RFLP) methods. Overall, we reported one mutation (SNP1) in the promoter region and two missense mutations (SNP2 and 3) in the coding region (single exon) within the bovine ZBED6 gene, and the haplotype variability and extent of linkage disequilibrium (LD) in 1522 individuals representing four main cattle breeds from China (Nanyang, NY; Qinchuan, QC; Jiaxian, JX; and Chinese Holstein, CH). We also investigated haplotype frequencies and linkage disequilibrium coefficients for three SNPs in all study populations. LD and haplotype structure of ZBED6 were different between breeds. The result of haplotype analysis of three SNPs showed that eight different haplotypes were identified in all breeds. The wild-type haplotype (Hap 1: GCA) and mutant-type haplotype (Hap 8: AGG) shared by all four populations accounted for 15.1, 29.8, 21.7, 57.5% and 9.5, 8.6, 16.7, 0% of all haplotypes were observed in NY, QC, JX and CH, respectively. The statistical analyses indicated that three SNPs were significantly associated with growth traits in NY cattle population (P<0.05 or P<0.01) at five different ages. The results of this study suggest that the ZBED6 gene possibly is a strong candidate gene that affects growth traits in beef cattle breeding program.