Abstract

Simple Summary

   The growth and development of skeletal muscle influences the efficiency
   of animal production. ZBED6 knockout accelerated the growth and
   development of pig and mice by regulating its target gene Igf2.
   However, the effect of, as well as detailed information about, the
   molecular mechanism of Zbed6 single-allele knockout on regulating
   muscle growth and development is still limited. In this study, a model
   of Zbed6 single-allele knockout mice prepared by gene-editing
   technology was used to study the effect and molecular mechanism of
   Zbed6 single-allele knockout on mice. Phenotypes and the RNA-seq
   results of muscle from Zbed6 single-allele knockout and wild-type mice
   showed that Zbed6 single-allele knockout promoted skeletal muscle
   weight and muscle fiber area via another target gene, instead of Igf2.
   This study may help to further explore the function of ZBED6 and its
   other target genes that have not been studied.

Abstract

   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.

   Keywords: ZBED6, single-allele knockout, IGF2, BARX2, mice, skeletal
   muscle

1. Introduction

   Zinc finger BED domain-containing protein 6 (ZBED6), a transcription
   factor, was discovered as a repressor of IGF2 (insulin-like growth
   factor 2) [[38]1]. It is derived and evolved from a domesticated DNA
   transposon that plays an important role distinctively in placental
   mammals [[39]2,[40]3]. This gene is encoded by an intronless gene,
   located in intron 1 of Zc3h11a, and shows a high degree of conservatism
   in 26 kinds of species with an amino acid identity close to 100%, which
   suggests the significance of ZBED6 in animal growth and development
   [[41]1].

   In the earliest time, a quantitative trait locus (QTL) was found to
   significantly affect pig muscle growth, fat deposition, and heart size
   during a conventional breeding process that was located in the IGF2
   gene [[42]4,[43]5]. The mutant of a single base in IGF2 intron 3
   disrupted a binding site of an unknown nucleic factor to IGF2 (G > A),
   resulting in a three-fold upregulation of IGF2 mRNA expression in pig
   muscle, eventually leading to the increase in muscle mass [[44]6]. The
   unknown nucleic factor was named ZBED6 and was identified to be a
   transcription factor that regulates the expression of Igf2 by binding
   with the “GCTCG” motif sequence, thereby regulating the growth and
   development of skeletal muscle [[45]1].

   Since then, multiple studies have extended the approach to several
   mammalians and cell lines. In beef cattle, it was found that the three
   SNPs in the promoter region of the ZBED6 gene were in complete linkage
   disequilibrium (LD) in IGF2, which revealed the significant effect of
   ZBED6 on cattle growth traits [[46]7,[47]8]. A study confirmed that
   Zbed6 knockout mice and Igf2 knockin mice destroyed the binding site of
   ZBED6 and Igf2, which simultaneously resulted a in faster growth speed,
   an increased serum IGF2 level, and increased skeletal muscle mass
   [[48]2]. These results were also proven in either ZBED6 knockout or
   IGF2 intro 3 mutant pigs and revealed the increased effects of ZBED6
   knockout on other internal organs [[49]9,[50]10,[51]11,[52]12]. As for
   specific cells, ZBED6 could not only regulate the proliferation and
   differentiation of mouse C2C12 cells [[53]13,[54]14] but also regulate
   other cell functions, including insulin production of mouse MIN6 cells
   [[55]15], pancreas β Cell proliferation and death [[56]16],
   proliferation and differentiation of mouse fat precursor cells
   [[57]17], and regulation of human colorectal cancer cell cycle and
   growth [[58]18]. In addition, about 2500 binding sites of ZBED6 were
   identified in mice by ChIP sequencing [[59]1], and some genes other
   than IGF2 were proven to be the target genes of ZBED6 that regulate the
   muscle growth of pigs together [[60]10].

   In a previous study by Younis et al. [[61]2], the knockout of Zbed6
   increased the growth and development of skeletal muscle in mice.
   However, the effect exists on Zbed6^+/− mice is unknown not been proved
   in single knockout puberty and adult mice till now. Thus, it is
   important for a single-allele strategy to be performed at both the
   living and transcriptome levels, to explore a more sophisticated
   mechanism and regulation network for ZBED6. The only report related to
   single-allele knockout of ZBED6 reveals that transcriptome differences
   vary from different organs in pigs [[62]19]. There are not any research
   results about whether single-allele knockout causes significant changes
   in skeletal muscle. More phenotypic and transcriptomic information is
   needed to fill in the gap in single knockout genotypes and complete the
   regulation network of ZBED6. In this study, Zbed6^+/− and wild-type
   mice (WT) were characterized in littermates and then slaughtered at the
   age of 8 weeks for muscle dissection to explore the effect of Zbed6^+/−
   in mice skeletal muscle, and the muscle traits of two groups were also
   calculated. RNA-seq analysis of skeletal muscle was performed to
   confirm the transcriptomic differences between Zbed6^+/− and WT mice in
   females to identify some of the ZBED6 target genes functioning in
   Zbed6^+/− skeletal muscle development.

2. Materials and Methods

2.1. Animal Models

   The Zbed6^+/− mouse models in C57BL/6 were provided by Leif Andersson
   (Science for Life Laboratory, Department of Medical Biochemistry and
   Microbiology, Uppsala University). The removal of the whole Zbed6 gene
   was based on DNA homologous recombination aroused by the Cre-lox system
   [[63]2]. The mice and their offspring were kept in the Small Animal
   Experiment Center of the Institute of Animal Science, Chinese Academy
   of Agricultural Sciences (CAAS), Beijing, China. We obtained WT,
   Zbed6^+/−, and Zbed6^−/− mice simultaneously in littermates using
   Zbed6^+/− mice crossing. Different kinds of genotype classes of mice
   were segregated by littermates, and the WT mice were seen as control.
   The body weight of WT and Zbed6^+/− female littermates was recorded at
   the age of 8 weeks. All mice were housed in standard conditions with
   water and food given ad libitum in the Animal Experiment Center of
   CAAS.

2.2. Mice Genotyping

   All mice were labeled with stainless steel ear marks, and 1–3 mm^3
   tails were collected at the age of 4 weeks to extract genomic DNA using
   KAPA Express Extract (KK7102, ROCHE). The tail samples were immersed in
   the mixture of Sup. Protease and Adv. Buffer and then digested for 15
   min at 75 °C. Genotyping was performed by PCR and gel electrophoresis.
   Three oligo-nucleotides were used to detect WT and Zbed6^+/−
   simultaneously. The expected product size is 596 bp for WT. Zbed6^+/−
   has two products with lengths of 596 and 561 bp. The PCR amplification
   protocol included pre-denaturation at 95 °C for 10 min, 35 cycles of
   denaturation at 95 °C for 30 s, and annealing at 63 °C for 30 s
   following an extension stage at 72 °C for 30 s. The amplification was
   ended with end extension stage at 72 °C for 10 min. Primer sequences
   are listed in [64]Supplementary Table S1.

2.3. Animal Slaughter

   Nine female mice of the two groups (WT = 4 and Zbed6^+/− = 5) were fed
   until the age of 8 weeks and euthanized by cervical dislocation. The
   hindlimbs were removed carefully, and the combination of muscle tissue
   of its two hindlimbs was dissected separately and divided to provide
   two specimens. The skeletal muscle of left side was flash-frozen
   immediately and then transferred into −80 °C refrigerator for long-term
   storage for further testing in the future. The skeletal muscle of right
   side was first weighed as muscle mass in this study and then fixed with
   4% paraformaldehyde solution (Solarbio, Beijing, China) for more than
   24 h. Muscle mass and muscle weight ratio (muscle mass/body weight ×
   100%) was compared between Zbed6^+/− and WT groups. All muscle tissues
   with the same purpose were collected from the same place.

2.4. Western Blot Analysis

   Skeletal muscle was taken out from −80 °C refrigerator. Total proteins
   of muscle tissue were extracted using Minute TM Total Protein
   Extraction Kit for Animal Cultured Cells/Tissues (Invent Biotech,
   Plymouth, MN, USA). The proteins were used for concentration analysis
   under BCA Protein Assey Kit (Beyotime, Shanghai, China) and then
   subjected to Western blot analysis with the following antibodies:
   anti-ZBED6 antibody (HPA068807, 1:500, ATLAS, Stockholm, Sweden) and
   anti-Gapdh antibody (HRP-60004, 1:50,000, Proteintech, Chicago, IL,
   USA). The blots were developed using HRP-conjugated secondary
   antibodies. Picture were captured by imaging system (Taon 4600,
   Shanghai, China) and quantified by Image J software (Fiji, National
   Institutes of Health, Bethesda, MD, USA).

2.5. Histochemistry and Analysis of Myofiber

   The chemically fixed muscle was removed from 4% paraformaldehyde
   solution and then put in the dehydration box. The tissue in the
   dehydration box was put in dehydrator (Donatello, DIAPATH) to dehydrate
   with gradient alcohol (Ethanol, 100092683, Sinopharm Group Chemical
   Reagent Co., Ltd., Shanghai, China) and embedded in paraffin to make
   paraffin sections. The slides were created from cross-sections by a
   pathology slicer (RM2016, Shanghai Leica Instrument Co., Ltd.,
   Shanghai, China) and then stained by hematoxylin and eosin (G1003,
   Servicebio, Wuhan, China). The whole slices were scanned and imaged by
   a slice digital scanner (Pannoramic 250FLASH, 3DHISTECH Ltd., Budapest,
   Hungary), the software CaseViewer 2.4 (3DHISTECH Ltd., Budapest,
   Hungary) was used to capture images of HE sections at 10×
   magnification, and Image J was applied for morphological observation.
   At least 20 myofibers were randomly selected from 3 fields per slice to
   detect the average area of the myofiber.

2.6. RNA Isolation and Library Preparation

   The total RNA of all muscle samples from the Zbed6^+/− and WT mice was
   isolated and purified using the RNeasy Mini kit (QIAGEN, Dusseldorf,
   Germany), in accordance with the operation protocol of the kit of the
   manufacturer. The quantity and purity of total RNA were then
   quality-controlled by NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA),
   and the integrity of RNA was checked by Bioanalyzer 2100 (Agilent, CA,
   USA). Only the RNA with concentrations >50 ng/μL, RIN value > 7.0, and
   total RNA > 1μg was submitted to downstream qPCR and RNA-seq analysis.
   cDNA library was then prepared from the qualified RNA using PrimeScript
   RT reagent kit (Takara Biomedical Technology Co., Ltd., Beijing, China)
   for Veriti 96 thermocycler (Applied Biosystems, San Francisco, CA,
   USA).

2.7. Primer Design and Quantitative Real-Time PCR

   Quantitative real-time PCR (qRT-PCR) analysis was performed using ABI
   MicroAmp Optical 384-well reaction plates on an ABI 7900 real-time PCR
   instrument (Applied Biosystems) and Taq Pro Universal SYBR qPCR Master
   Mix (Q712, Vazyme Biotechnology Co., Ltd., Nanjing, China). The qRT-PCR
   protocol included 40 cycles of denaturation at 95 °C for 10 s and
   annealing and extension at 60 °C for 30 s after a pre-denaturation
   stage at 95 °C for 30 s. Gapdh, β-Actin, and Rpl41 were used as the
   housekeeping gene to normalize all the results, which were presented as
   2^−ΔΔCt. The primers used were designed on the online website Primer3
   ([65]https://bioinfo.ut.ee/primer3-0.4.0/, accessed on 15 February
   2023) and are listed in [66]Supplementary Table S1.

2.8. RNA-Seq Analysis

   Transcriptome paired-end sequencing of all RNA samples was conducted at
   LC Sciences (Hangzhou, China) using an Illumina NovaseqTM 6000 (LC Bio
   Technology CO., Ltd. Hangzhou, China) with PE150 sequencing mode
   following standard procedures. To get high-quality clean reads, reads
   were further filtered by Cutadapt
   ([67]https://cutadapt.readthedocs.io/en/stable/, accessed on 15
   February 2023, version: cutadapt-1.9). Then, the sequence quality was
   verified using FastQC
   ([68]http://www.bioinformatics.babraham.ac.uk/projects/fastqc/,
   accessed on 15 February 2023, 0.11.9) including the Q20, Q30, and GC
   content of the clean data. The reference genome assembly of mouse
   (version GRCm39) was downloaded from Ensembl website
   ([69]http://ftp.ensembl.org/pub/release-107/fasta/mus_musculus/dna/Mus_
   musculus.GRCm39.dna.toplevel.fa.gz, accessed on 15 February 2023).
   HISAT2 tools were used for mapping between the acquired clean reads and
   the reference genome. Gene expression of the muscle tissue of all mice
   was calculated by using featureCounts software. Moreover,
   differentially expressed analysis between Zbed6^+/− and WT mice was
   analyzed using the package DESeq2 of R software (version 4.2.0,
   [70]http://cran.r-project.org/, accessed on 15 February 2023). In
   addition, parameter p value < 0.05 and log[2] fold change absolute
   value ≥1 were used as a standard to carry out differential expression
   genes. TPM value, which stands for transcript per million, for all
   annotated transcripts and can be considered as a percentage of
   RPKM/FPKM values, was transferred from read counts in R software. All
   results of differential expression analysis were visualized by R.

2.9. Enrichment Analysis

   Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes
   and Genomes (KEGG) pathway enrichment analysis were performed in the
   DEGs between WT and Zbed6^+/− mice derived from differential expression
   analysis. The enrichment analysis was carried out on the online tool
   KOBAS-intelligence ([71]http://bioinfo.org/kobas, accessed on 15
   February 2023). p value ≤ 0.05 was used as the threshold to identify
   the significant GO terms and KEGG pathway using FDR correction method
   of Benjamini and Hochberg.

2.10. Candidate Gene Selection

   The DEGs of female mice between WT and Zbed6^+/− were overlapped with
   the target genes of ZBED6 acquired using chromatin immunoprecipitation
   (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) in C2C12
   cells that were published to extract for common genes. The candidate
   genes were chosen by querying the function of each common gene on NCBI
   ([72]https://www.ncbi.nlm.nih.gov/, accessed on 15 February 2023). The
   ChIP-seq data of C2C12 cells were also provided by Leif Andersson
   (Science for Life Laboratory, Department of Medical Biochemistry and
   Microbiology, Uppsala University).

2.11. Statistical Analysis

   All the results reported in this study were biologically replicated at
   least three times. The statistical analyses were conducted by GraphPad
   Prism 9.4.1 (GraphPad Software Inc., La Jolla, CA, USA). Data are
   presented as mean ± standard error of the mean (SEM). The p value was
   determined by Student’s t-test and indicated the significant level. p <
   0.05 was considered statistically significant.

3. Results

3.1. Zbed6 Single-Allele Knockout Promotes the Growth of Skeletal Muscle

   The phenotypes of the WT and Zbed6^+/− mice were collected at the age
   of 8 weeks. Nine mice from two groups (n = 4:5) were slaughtered to
   measure their skeletal muscle. The procedure of sample collecting was
   followed as shown in [73]Figure 1A, and the genotyping of mice was
   identified by PCR and subsequent gel electrophoresis. Bands of 596 bp
   represent WT mice, while bands of 561 bp represent Zbed6^+/− mice
   ([74]Figure 1B). The Western blot analysis with an anti-ZBED6 antibody
   showed that a 43.4% decrease in the gray scale value of Zbed6^+/−
   compared to that of WT mice indicates that ZBED6 protein content
   significantly decreased in the skeletal muscle of Zbed6^+/− mice
   ([75]Figure 1C,D).

Figure 1.

   [76]Figure 1
   [77]Open in a new tab

   Schematic overview of the production of mice in this study. (A)
   Skeletal muscle tissues were weighed and collected from WT and
   Zbed6^+/− mice. (B) Genotyping of the different genotype mice was
   identified by polymerase chain reaction (PCR). The 596 bp band
   represents the WT mice, and the 561 bp band represents the Zbed6^+/−.
   (C,D) Western blot analysis of ZBED6 in skeletal muscle from Zbed6^+/−
   mice and WT mice. The results are expressed by the means ± SEMs. WT
   represents wild-type mice, and SKO represents Zbed6^+/− mice. ** p <
   0.01, Student’s t-test. Points in different colors represent actual
   data of mice.

   The measurement of muscle growth showed that the muscle mass of
   Zbed6^+/− increased significantly compared to WT, which means the
   single-allele knockout of ZBED6 also could increase muscle weight in
   mice. In addition, the muscle mass of Zbed6^−/− also increased
   extremely significantly compared to either WT or Zbed6^+/− mice
   ([78]Figure 2A and [79]Supplementary Table S2), consistent with a
   previous study on Zbed6^−/− mice. In addition, Zbed6^+/− mice showed a
   significantly higher muscle weight ratio compared to WT mice
   ([80]Figure 2B).

Figure 2.

   [81]Figure 2
   [82]Open in a new tab

   Phenotypes of slaughtered WT and Zbed6^+/− mice. (A,B) Dissected muscle
   mass and muscle weight ratio of WT and Zbed6^+/− mice. Points in
   different colors represent actual data of muscle traits. (C) H&E
   staining of the skeletal muscle of WT and Zbed6^+/− mice. SKO
   represents Zbed6^+/− mice. Bar, 100 μm. (D) Differences in the average
   area of muscle fiber quantitative analysis in mice. The results were
   expressed by the means ± SEMs. ** p < 0.01, Student’s t-test. Points in
   different colors represent actual data of mice.

   To further investigate the effect of Zbed6 single-allele knockout on
   mice muscle, the muscle tissue was paraffin-fixed for slicing.
   Hematoxylin and eosin (H&E) staining and its quantitative analysis
   clearly revealed that there was a significant increase in the area of
   myofiber in Zbed6^+/− mice compared with the WT group. The image and
   the quantitative analysis of the myofiber showed the same trend as the
   muscle mass, which showed a significant increase between the WT and
   Zbed6^+/− mice ([83]Figure 2C,D and [84]Supplementary Table S3). In
   addition, Zbed6 single-allele knockout in mice can clearly lead to
   significant changes in skeletal muscle.

3.2. Zbed6 Single-Allele Knockout Did Not Increase Igf2 mRNA Expression in
Mice Muscle

   In a previous study, the ZBED6–IGF2 axis had a major effect on the
   muscle growth of placental mammals, such that we first detected the
   change in Igf2 expression to reveal the effect of Zbed6 single-allele
   knockout on Igf2 in mice muscles. According to the results of qRT-PCR,
   with Gapdh, β-Actin, and Rpl41 as internal references, there was no