Bovine myoblast CDKN1C gene super-enhancer and its uses

Abstract

A bovine myoblast CDKN1C gene super-enhancer and uses thereof are provided. The super-enhancer components include Enh22, Enh38, and Enh40, where Enh22 is located at positions 48802495-48803999 on chromosome 29 of the bovine genome, Enh38 is located at positions 48902854-48905629 on chromosome 29 of the bovine genome, and Enh40 is located at positions 48920416-48922207 on chromosome 29 of the bovine genome. The super-enhancer components improve the efficiency of gene editing and transgenic breeding in beef cattle (e.g., without the need for gene sequence cutting and repair) and facilitate the screening of genomic mutations (i.e., molecular genetic markers) associated with dominant traits (e.g., large body size and high meat production) in beef cattle individuals, thereby facilitating the selection and breeding process for superior beef cattle herds.

Description

[Technical Field]

[0001] The present invention relates to a bovine myoblast CDKN1C gene super-enhancer and its uses, which are in the field of molecular breeding of livestock. [Background technology]

[0002] The p57Kip2 protein, encoded by the CDKN1C gene, was identified as the third member of the CIP / Kip family, which also includes p27Kip1 and p21Cip1. Like these proteins, p57Kip2 tightly binds to and inhibits cyclin / cyclin-dependent kinase complexes, thereby regulating cell division cycle progression (Stampone et al., 2018). The CDKN1C gene is known as a cell cycle inhibitor essential for maintaining the balance between cell proliferation and differentiation. As the expression of differentiation marker genes increases, CDKN1C expression also increases, and CDKN1C inhibits cell proliferation for differentiation. In muscle development, CDKN1C acts as a cell cycle inhibitor, involved in inhibiting the G1 / S transition, and plays a role not only in cell cycle regulation but also in muscle cell differentiation and proliferation. Its role in muscle tissue is thought to be more complex and specific. Osborn (2011) demonstrated that CDKN1C promotes muscle progenitor cell differentiation through a positive feedback loop with MyoD (Osborn et al., 2011). In skeletal muscle cells, hypomethylation of long-range regulatory elements within the imprinted control region KvDMR1 allows MyoD to bind, but MyoD cannot bind to the CDKN1C promoter, resulting in the induction of CDKN1C expression (Andresini et al., 2016). Furthermore, Mademtzoglou (2018) found that CDKN1C is not expressed in quiescent muscle stem cells (MuSCs), but is induced in activated and proliferating myoblasts and remains expressed in differentiated myotubes. Primary myoblasts derived from CDKN1C-deficient mice exhibited differentiation defects and accelerated proliferation (Mademtzoglou et al., 2018). This highlights the importance of CDKN1C being regulated by muscle development-related transcription factors during muscle cell differentiation and indicates that CDKN1C activity is essential for regulating muscle stem cell growth. Research on the CDKN1C gene is not limited to model organisms; it has also been studied in cattle. Methylation of KvDMR1 is involved in regulating the imprinting of the bovine CDKN1C gene (Wang et al., 2015).Furthermore, Gao (2021) investigated the effect of SMAD2 / SMAD3 binding to CDKN1C on the proliferation of bovine muscle satellite cells and found that overexpression of SMAD3 significantly increased CDKN1C expression (Gao et al., 2021). These studies reveal the multifaceted functions of CDKN1C in muscle development and highlight its importance in genetic and epigenetic regulation. CDKN1C is negatively correlated with cell proliferation and plays a role in regulating the balance between proliferation and differentiation. These results indicate that the CDKN1C gene plays an important role in the proliferation and differentiation of bovine myoblasts. By inhibiting specific stages of the cell cycle, CDKN1C may promote the transition from myoblasts to mature muscle cells, affecting muscle growth and function. This discovery sheds light on the molecular mechanisms of bovine muscle growth and identifies potential targets for improving cattle breeding and meat quality. Future studies on the role of the CDKN1C gene in bovine myoblast proliferation will help elucidate the regulatory mechanisms of muscle growth and support advances in livestock production and meat quality improvement.

[0003] Super-enhancers are important elements in gene expression regulation, and can increase the frequency of gene transcription by binding to transcription factors and interacting with promoters, thereby promoting the expression of target genes. At present, there are no reports on the transcriptional regulation of the bovine CDKN1C gene, especially on super-enhancers with regulatory effects. Summary of the Invention [Problem to be solved by the invention]

[0004] OBJECT OF THE INVENTION: The object of the present invention is to provide a bovine myoblast CDKN1C gene super-enhancer and uses thereof. [Means for solving the problem]

[0005] Technical Solution: The present invention provides a bovine myoblast CDKN1C gene super enhancer, comprising: The super enhancer components include Enh22, Enh38, and Enh40, where Enh22 is located at positions 48802495-48803999 on chromosome 29 of the bovine genome, Enh38 is located at positions 48902854-48905629 on chromosome 29 of the bovine genome, and Enh40 is located at positions 48920416-48922207 on chromosome 29 of the bovine genome, providing a bovine myoblast CDKN1C gene super enhancer.

[0006] Furthermore, the nucleotide sequence of Enh22 is shown as SEQ ID NO.1, the nucleotide sequence of Enh38 is shown as SEQ ID NO.2, and the nucleotide sequence of Enh40 is shown as SEQ ID NO.3.

[0007] The present invention also provides primer pairs for amplifying the above-mentioned bovine myoblast CDKN1C gene super enhancer, wherein the nucleotide sequences of the primer pair for amplifying Enh22 are represented by SEQ ID NOs. 4 to 5, the nucleotide sequences of the primer pair for amplifying Enh38 are represented by SEQ ID NOs. 6 to 7, and the nucleotide sequences of the primer pair for amplifying Enh40 are represented by SEQ ID NOs. 8 to 9.

[0008] The present invention also provides the use of the bovine myoblast CDKN1C gene super-enhancer described above in regulating muscle growth in cattle.

[0009] The present invention also provides the use of the bovine myoblast CDKN1C gene super enhancer in breeding high-yielding beef cattle.

[0010] Furthermore, a method for improving meat production is to knock down one or more enhancers of Enh22, Enh38, or Enh40 in myoblasts in cattle.

[0011] Furthermore, specific steps of the method include designing sgRNA primers for enhancers Enh22, Enh38, or Enh40, annealing the primers, and ligating them into a CRISPR / Cas9 basic vector to construct a recombinant vector.

[0012] Furthermore, the sgRNA primer nucleotide sequences for enhancer Enh22 are shown in SEQ ID NOs. 10-11.

[0013] Furthermore, the sgRNA primer nucleotide sequences for enhancer Enh38 are shown in SEQ ID NOs. 12-13.

[0014] Furthermore, the sgRNA primer nucleotide sequences for enhancer Enh40 are shown in SEQ ID NOs. 14-15. [Effects of the Invention]

[0015] The beneficial effects are as follows: Compared with the prior art, the present invention has the following distinct and important advantages: In the present invention, we have found that a super-enhancer localized in the bovine CDKN1C gene can improve the efficiency of gene editing and transgenic breeding in beef cattle (e.g., without the need for cutting and repairing gene sequences), and facilitate the screening of genomic mutations (i.e., molecular genetic markers) associated with dominant traits (e.g., large body size and high meat yield) in beef cattle individuals, thereby facilitating the selection and breeding process of superior beef cattle herds. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a micrograph of bovine myoblasts isolated and cultured in an example of the present invention. [Figure 2] FIG. 1 is a schematic diagram of molecular-level screening of super-enhancer candidates in an embodiment of the present invention. [Figure 3] 1 shows the results of identifying the activity of a target super enhancer in bovine myoblasts in an example of the present invention. [Figure 4] 1 shows the effect of the target super enhancer in an embodiment of the present invention on the expression of CDKN1C gene mRNA in bovine myoblasts. [Figure 5] 1 shows the effect of the targeted super enhancer in an embodiment of the present invention on the expression of CDKN1C gene protein in bovine myoblasts. DETAILED DESCRIPTION OF THE INVENTION

[0017] The technical solution of the present invention will be further described below with reference to the drawings.

[0018] Example 1 1. Identification of candidate super-enhancer activity in bovine myoblasts

[0019] 1.1 Super-enhancer candidates First, we used CUT&Tag (H3K27ac and H3K4me1) high-throughput sequencing technology to analyze fetal and adult bovine longissimus dorsi muscles and identify regulatory elements near CDKN1C. Next, we analyzed transposase-accessible chromatin assay by high-throughput sequencing (ATAC-seq) data to identify open region enhancers. Finally, we screened for super-enhancers that interact with the CDKN1C promoter based on Hi-C-loop data. Referring to Figures 2 and 3, the results showed that the 10 enhancer candidates (Enh8, Enh20, Enh22, Enh38, Enh40, Enh59, Enh63, Enh64, Enh71, and Enh72) shown in Table 1 (ARS-UCD1.2) interacted with the CDKN1C gene promoter over a long distance, and among them, Enh8, Enh20, Enh22, Enh38, and Enh40 were components of a super-enhancer.

[0020] [Table 1]

[0021] 1.2 PCR amplification of super-enhancers The longissimus dorsi muscle of a bovine fetus was isolated, and genomic DNA was isolated, extracted, and purified according to the method described by Sambrock et al. (2002). Using the bovine CDKN1C gene sequence (GenBank NC_037356.1) listed in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as a reference sequence, PCR cloning primers were designed using Primer 5.0 to amplify the super-enhancers listed in Table 1. The resulting PCR amplification products of the super-enhancer candidates were used to construct the pGL3-Promoter-Enh recombinant vector.

[0022] [Table 2] TIFF0007752454000004.tif119170

[0023] PCR amplification system: 50 ng / µL of fetal bovine longissimus dorsi genome as template, 4 µL of DNA, 1.5 µL each of 10 pmol / L upstream and downstream primers (primer pairs in Table 2), 2x Max 50 µL, and 43 µL of deionized water, for a total of 100 µL. The PCR amplification reaction program was: (1) 98°C for 2 min, (2) 98°C for 10 s, 55°C for 15 s, 72°C for 20 s, 36 cycles, and (3) 72°C for 5 min. The amplified products were verified by sequencing.

[0024] 1.3 Construction of recombinant vector pGL3-Promoter-Enh

[0025] In October 2022, bovine fetuses around 90 days pregnant were collected from Xiaogan Village, Kunming City, Yunnan Province. Bovine myoblasts were extracted from the collected fetuses using conventional methods and then subcultured for transfection with recombinant vectors.

[0026] The PCR amplification products of the super-enhancer candidates were purified using a SanPrep Column PCR Product Purification Kit (Seiko Bio-Kogyo Co., Ltd., order number B518141) to obtain product S1 for future use. The pGL3-Promoter empty vector (Promega) was linearized by enzymatic digestion with BamH I and Sal I, and the linearized vector was recovered from the digested product using a SanPrep Column DNA Gel Recovery Kit (Seiko Bio-Kogyo Co., Ltd., order number B518131) to obtain product S2. The enzyme digestion system consisted of 50 ng / μL PCR product or 20 μL pGL3-Promoter vector, 10 μL enzyme digestion buffer, 4 μL each of endonucleases (e.g., BamH I and Sal I), and 62 μL deionized water, for a total of 100 μL. The program was 37°C for 20 min. Ligation system: 1 μL of 50 ng / μL Product S2, 1 μL of 50 ng / μL Organism S1, 1 μL of 10× T4 ligase buffer, 0.5 μL of T4 ligase, and 6.5 μL of deionized water, totaling 10 μL. Product S1 and Product S2 were ligated with T4 DNA ligase overnight at 16°C. The ligation products were transformed into competent DH5α cells (Tiangen Biochemical Technology Co., Ltd.). After ampicillin resistance screening, positive single clones were identified by Sanger sequencing. The pGL3-Promoter-Enh recombinant vector was extracted using a SanPrep column plasmid DNA small-scale extraction kit (Seiken Biotechnology Co., Ltd., order number B518191). This vector was then used to transfect bovine myoblasts and identify the activity of the potential super-enhancer.

[0027] 1.4 Transfection of recombinant vectors into cells

[0028] Following the instructions for the transfection reagent TurboFect (Thermo Scientific™), bovine myoblasts were transfected with the pGL3-Promoter-Enh recombinant vector, pRL-TK internal reference vector (Promega), pGL3-Promoter empty vector, and pRL-TK internal reference vector, respectively, to detect the activity of the potential super-enhancers.

[0029] 1.5 Screening for super-enhancers

[0030] The activity of the 10 super-enhancer candidates listed in Table 1 was identified according to the procedure described in the Dual-Luciferase® Reporter Assay System (Promega) manual. Specifically, the activity was determined by the difference in magnitude between the relative fluorescence activity in the presence of the super-enhancer (R1: the ratio of firefly fluorescence to the internal reference pRL-TK Renilla fluorescence after transfection with the pGL3-Promoter-Enh recombinant vector and the pRL-TK internal reference vector) and the relative fluorescence activity in the absence of the super-enhancer (R2: the ratio of firefly fluorescence to the internal reference pRL-TK Renilla fluorescence after transfection with the pGL3-Promoter-NC and the pRL-TK internal reference vector). If the ratio R1 was significantly higher than the ratio R2, the candidate region had super-enhancer activity. In this study, we used CUT&Tag (H3K27ac and H3K4me1) high-throughput sequencing technology to identify regulatory elements near CDKN1C. We analyzed transposase-accessible chromatin assay (ATAC-seq) data to identify super-enhancers in open regions. Based on Hi-C-loop data, we screened for super-enhancers that interact with the CDKN1C promoter. A dual-luciferase vector reporter system was used to identify super-enhancer activity. Because TADs are insulating, based on Hi-C-TAD data, super-enhancers located within TADs were selected for further experimental validation. Finally, we used CRISPRi, real-time fluorescent quantitative PCR, and Western blotting to effectively identify super-enhancer components that can promote CDKN1C gene expression in bovine myoblasts.

[0031] 2. Regulation of CDKN1C gene expression by targeted super-enhancer

[0032] 2.1 Construction of recombinant vector pX330a dCas9-KRAB The double-stranded oligonucleotide primer pair P12–P15 (Table 3) was used as primers and annealed to obtain double-stranded oligonucleotide S3 (NC control group and sgRNA experimental group). The double-stranded oligonucleotide annealing synthesis system consisted of 1 μL each of the upstream and downstream primers corresponding to the 100 μmol / L primer pair P12–P15, and 8 μL of deionized water, for a total of 10 μL. The double-stranded oligonucleotide annealing synthesis program was 95°C for 5 min, followed by slow cooling to room temperature. The pX330a dCas9-KRAB vector (addgene: #92361) was linearized by enzymatic digestion with BbsI. The linearized vector was recovered from the digestion product using a SanPrep column DNA gel recovery kit, yielding product S4 for future use. Enzyme digestion system: 40 µL of 50 ng / µL pX330a dCas9-KRAB vector, 10 µL of enzyme digestion buffer, 1 µL of Bbs I endonuclease, and 49 µL of deionized water, for a total volume of 100 µL. Enzyme digestion program: 95 °C for 3 h.

[0033] The 10 μL ligation system consisted of 1 μL of 50 ng / μL linearized vector, 1 μL of double-stranded oligonucleotide, 1 μL of 10× T4 Ligase buffer, 0.5 μL of T4 ligase, and 6.5 μL of deionized water. The ligation program was performed overnight at 16°C. Products S3 and S4 were ligated with T4 DNA ligase. The ligation products were transformed into DH5α, and after ampicillin resistance screening, positive single clones were confirmed by Sanger sequencing. The plasmid DNA was extracted using a SanPrep column plasmid DNA small-scale extraction kit to obtain the pX330a dCas9-KRAB-NC and pX330a dCas9-KRAB-sgRNA recombinant vectors (collectively referred to as the pX330a dCas9-KRAB recombinant vectors).

[0034] [Table 3]

[0035] 2.2 Transfection of recombinant vectors into cells Using the instructions for the transfection reagent TurboFect (Thermo Scientific™), bovine myoblasts were transfected with pX330a dCas9-KRAB-NC and pX330a dCas9-KRAB-sgRNA, respectively, to inhibit the activity of the target super-enhancer.

[0036] 2.3 RNA isolation, extraction, purification, and reverse transcription Total RNA was extracted from bovine myoblasts transfected with the pX330a dCas9-KRAB recombinant vector using the TRIzol (Takara) method. The extracted RNA was reverse transcribed into cDNA using Takara's PrimeScript™ RT reagent Kit with gDNA Eraser, and used to identify bovine CDKN1C gene expression.

[0037] 2.4 Identification of bovine CDKN1C gene expression Using the bovine CDKN1C gene sequence (GenBank NC_037356.1) from the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as a reference sequence, real-time fluorescent quantitative PCR primers (Table 4) were designed using Primer 5.0 to amplify the CDKN1C gene sequence. The amplification system consisted of 2 μL of 10 ng / μL template cDNA, 1 μL each of upstream and downstream primers corresponding to primer pair P16 or primer pair P17 at 10 pmol / L, 10 μL of 2x SYBR Green qPCR Mix, and 6 μL of deionized water, for a total of 20 μL. The reaction program was: (1) 95°C for 30 s, (2) 95°C for 10 s, 60°C for 30 s, followed by 39 cycles.

[0038] [Table 4]

[0039] The expression level of CDKN1C gene in myoblasts was 2 -ΔΔCt The detection was based on the results. Experimental results showed that the mRNA expression level of the CDKN1C gene in the experimental group transfected with pX330a dCas9-KRAB-sgRNA (experimental interference vector) was significantly lower than that in the negative control group transfected with pX330a dCas9-KRAB-NC (Figure 4). Among these, Enh38 had the highest effect. Western blot analysis showed that the CDKN1C gene protein expression levels in all experimental groups were significantly lower than that in the negative control group transfected with pX330a dCas9-KRAB-NC (Figure 5). The significant decrease in CDKN1C expression indicated the inhibition of targeted super-enhancer activity. These results demonstrate that the components of the targeted super-enhancer can promote CDKN1C gene expression. This super-enhancer was demonstrated to be active in bovine longissimus dorsi muscle tissue. Because the CDKN1C gene is an important gene that affects important economic traits in mammals, the three target super-enhancer components identified in this application can be used as target sites for gene editing and transgenic breeding in beef cattle. Knocking down the above super-enhancers can inhibit the expression of the CDKN1C gene, thereby promoting muscle development and increasing meat production. Alternatively, screening for important mutations that affect the activity of this super-enhancer can also be used in marker-assisted selection of beef cattle.

Claims

1. A bovine myoblast CDKN1C gene super enhancer, the super enhancer is any of Enh22, Enh38, or Enh40, wherein Enh22 is located at positions 48802495-48803999 on chromosome 29 of the bovine genome, Enh38 is located at positions 48902854-48905629 on chromosome 29 of the bovine genome, and Enh40 is located at positions 48920416-48922207 on chromosome 29 of the bovine genome; The nucleotide sequence of Enh22 is represented by SEQ ID NO. 1, the nucleotide sequence of Enh38 is represented by SEQ ID NO. 2, and the nucleotide sequence of Enh40 is represented by SEQ ID NO.

3. A bovine myoblast CDKN1C gene super enhancer.

2. A primer pair for amplifying the bovine myoblast CDKN1C gene super enhancer of claim 1, A primer pair characterized in that the nucleotide sequences of the primer pair for amplifying Enh22 are set forth in SEQ ID NOs. 4-5, the nucleotide sequences of the primer pair for amplifying Enh38 are set forth in SEQ ID NOs. 6-7, and the nucleotide sequences of the primer pair for amplifying Enh40 are set forth in SEQ ID NOs. 8-9.

3. A method for improving meat production, characterized by knocking down one or more enhancers of Enh22, Enh38, or Enh40 in the bovine myoblast CDKN1C gene super enhancer described in claim 1.

4. The method of claim 3, characterized in that the specific steps of the method include designing an sgRNA primer for enhancer Enh22, Enh38, or Enh40, annealing the primer, and ligating it into a CRISPR / Cas9 basic vector to construct a recombinant vector.

5. The method of claim 4, wherein the sgRNA primer nucleotide sequence of enhancer Enh22 is set forth in SEQ ID NO. 10-11.

6. The method of claim 4, wherein the sgRNA primer nucleotide sequence of enhancer Enh38 is set forth in SEQ ID NO. 12-13.

7. The method of claim 4, wherein the sgRNA primer nucleotide sequence of enhancer Enh40 is set forth in SEQ ID NO. 14-15.

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