Bovine myoblast CDKN1C gene super enhancer and its use

Abstract

This invention provides a bovine myoblast CDKN1C gene super-enhancer and its use. [Solution] The components of the super enhancer include Enh22, Enh38, and Enh40, where Enh22 is located at positions 48802495-48803999 of chromosome 29 of the bovine genome, Enh38 is located at positions 48902854-48905629 of chromosome 29 of the bovine genome, and Enh40 is located at positions 48920416-48922207 of chromosome 29 of the bovine genome. The components of the super enhancer described above improve the efficiency of gene editing and genetically modified breeding of beef cattle (e.g., without requiring gene sequence breaks and repairs), facilitate screening of genomic mutations (i.e., molecular genetic markers) associated with dominant traits of individual beef cattle (e.g., large size, high meat yield), thereby promoting the process of selecting and breeding superior beef cattle herds.

Description

Technical Field

[0001] The present invention relates to the bovine myoblast CDKN1C gene super enhancer and its use, and belongs to the field of molecular breeding of livestock.

Background Art

[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. Similar to these proteins, p57Kip2 tightly binds to cyclin / cyclin-dependent kinase complexes, inhibiting them and thereby regulating the progression of the cell division cycle (Stampone et al., 2018). The CDKN1C gene is known as an essential cell cycle inhibitor for maintaining the balance between cell proliferation and differentiation. As the expression of differentiation marker genes increases, the expression of the CDKN1C gene also increases, and CDKN1C inhibits cell proliferation for differentiation. In muscle development, the CDKN1C gene acts as a cell cycle inhibitor, involved in inhibiting G1 / S transition, and plays a role not only in cell cycle regulation but also in muscle cell differentiation and proliferation, suggesting its role in muscle tissue is more complex and specific. Osborn (2011) showed that CDKN1C promotes the differentiation of muscle progenitor cells through a positive feedback loop with MyoD (Osborn et al., 2011). In skeletal muscle cells, hypomethylation of long-range regulatory elements within the imprinting regulatory region KvDMR1 allows MyoD to bind, but MyoD cannot bind to the CDKN1C promoter, thus inducing CDKN1C expression (Andresini et al., 2016). Furthermore, Mademtzoglou (2018) discovered 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 showed 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, indicating that CDKN1C activity is essential for regulating muscle stem cell growth. Studies of the CDKN1C gene are not limited to model organisms; it has also been studied in cattle. KvDMR1 methylation is involved in regulating bovine CDKN1C gene imprinting (Wang et al., 2015).Furthermore, Gao (2021) investigated the effects 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 function 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 a crucial role in the proliferation and differentiation of bovine myoblasts. CDKN1C may influence muscle growth and function by promoting the transition from myoblasts to mature muscle cells by inhibiting specific stages of the cell cycle. This finding elucidates the molecular mechanisms of bovine muscle growth and identifies potential targets for improving cattle breeding and meat quality. Future research into the role of the CDKN1C gene in bovine myoblast proliferation will help elucidate the regulatory mechanisms of muscle growth and support advancements in livestock production and meat quality improvement.

[0003] Super-enhancers are crucial elements in gene expression regulation. They can increase the frequency of gene transcription and promote the expression of target genes by binding to transcription factors and interacting with promoters. Currently, there are no reports on the transcriptional regulation of the bovine CDKN1C gene, particularly regarding super-enhancers with regulatory effects. [Overview of the project] [Problems that the invention aims to solve]

[0004] Objective of the Invention: The objective of the present invention is to provide a bovine myoblast CDKN1C gene super enhancer and its use. [Means for solving the problem]

[0005] Technical solution: The present invention is a bovine myoblast CDKN1C gene super enhancer, 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 bovine myoblast CDKN1C gene super-enhancer described above, wherein the nucleotide sequences of the primer pair for amplifying Enh22 are shown in SEQ ID NO. 4-5, the nucleotide sequences of the primer pair for amplifying Enh38 are shown in SEQ ID NO. 6-7, and the nucleotide sequences of the primer pair for amplifying Enh40 are shown in SEQ ID NO. 8-9.

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

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

[0010] Furthermore, a method to improve meat yield is to knock down one or more enhancers among Enh22, Enh38, or Enh40 in myoblasts.

[0011] Furthermore, the 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 base vector to construct a recombinant vector.

[0012] Furthermore, the sgRNA primer nucleotide sequences of enhancer Enh22 are shown in SEQ ID NO. 10-11.

[0013] Furthermore, the sgRNA primer nucleotide sequences of enhancer Enh38 are shown in SEQ ID NO. 12-13.

[0014] Furthermore, the sgRNA primer nucleotide sequences of enhancer Enh40 are shown in SEQ ID NO. 14-15. [Effects of the Invention]

[0015] The beneficial effects are as follows: Compared to the prior art, the present invention has the following outstanding and important advantages: In this invention, we have found that a super-enhancer localized to the bovine CDKN1C gene improves the efficiency of gene editing and genetically modified breeding of beef cattle (e.g., without requiring gene sequence breaks and repairs), facilitates the screening of genomic mutations (i.e., molecular genetic markers) associated with dominant traits in beef cattle individuals (e.g., large body size and high meat yield), thereby accelerating the process of selecting and breeding superior beef cattle herds. [Brief explanation of the drawing]

[0016] [Figure 1] This is a micrograph of bovine myoblasts isolated and cultured in an embodiment of the present invention. [Figure 2] This is a schematic diagram of molecular-level screening of super-enhancer candidates in the embodiments of the present invention. [Figure 3] This shows the results of identifying the activity of the target super-enhancer in bovine myoblasts in the embodiments of the present invention. [Figure 4] The effect of the target super enhancer in the examples of the present invention on the expression of bovine myoblast CDKN1C gene mRNA. [Figure 5] The effect of the target super enhancer in the examples of the present invention on the expression of bovine myoblast CDKN1C gene protein.

Embodiments for Carrying out the Invention

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

[0018] Example 1 1. Identification of the activity of super enhancer candidates in bovine myoblasts

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

[0020]

Table 1

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

[0022]

Table 2

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

[0024] 1.3 Construction of Recombinant Vector pGL3-Promoter-Enh

[0025] In October 2022, fetal bovine around 90 days of gestation were collected in Xiaogang Village, Kunming City, Yunnan Province. Bovine myoblasts were extracted from the collected fetuses by the conventional method and then subcultured for transfection with the recombinant vector.

[0026] The PCR amplification products of the super-enhancer candidates were purified using the SanPrep Column PCR Product Purification Kit (Biotechnology Co., Ltd., order number B518141) to obtain product S1 for future use. The pGL3-Promoter empty vector (Promega) was enzymatically digested with BamH₂I and Sal₂I to linearize it, and the linearized vector was recovered from the enzymatic digest product using the SanPrep Column DNA Gel Recovery Kit (Biotechnology Co., Ltd., order number B518131) to obtain product S2. Enzymatic digestion system: 50 ng / μL of PCR product or 20 μL of pGL3-Promoter vector, 10 μL of enzymatic digestion buffer, 4 μL each of each endonuclease (e.g., BamH₂I and Sal₂I), and 62 μL of deionized water, totaling 100 μL. Program: 20 min at 37°C. Ligation system: 1 μL of product S2 at 50 ng / μL, 1 μL of biological S1 at 50 ng / μL, 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 DNA ligated overnight at 16°C using T4 DNA ligase. The ligated products were transformed into competent cells DH5α (Tiangen Biochemical Technology Co., Ltd.), and after ampicillin resistance screening, positive single clones were confirmed by Sanger sequencing. The pGL3-Promoter-Enh recombinant vector was obtained by extracting the plasmid DNA using a SanPrep column plasmid DNA small-scale extraction kit (Bioeng Biotechnology Co., Ltd., order number B518191). This vector was used to transfect bovine myoblasts, and the activity of candidate super-enhancers was identified.

[0027] 1.4 Transfection of Recombinant Vectors into Cells

[0028] Following the instructions for the transfection reagent TurboFect (Thermo Scientific®), pGL3-Promoter-Enh recombinant vector, pRL-TK internal reference vector (Promega), pGL3-Promoter empty vector, and pRL-TK internal reference vector were transfected into bovine myoblasts, and the activity of candidate super-enhancers was detected.

[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 operating procedure described in the instructions for the Dual-Luciferase® Reporter Assay System (Promega). Specifically, the activity was determined by the difference in magnitude between the relative fluorescence activity when the super-enhancer was transfected (R1 is the ratio of the fluorescence of fireflies to the fluorescence of the internal reference pRL-TK sea urchin after transfection with the pGL3-Promoter-Enh recombinant vector and the pRL-TK internal reference vector) and the relative fluorescence activity when the super-enhancer was not transfected (R2 is the ratio of the fluorescence of fireflies to the fluorescence of the internal reference pRL-TK sea urchin 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, it indicated that the candidate region had super-enhancer activity. In this invention, we identified regulatory elements near CDKN1C using CUT&Tag (H3K27ac and H3K4me1) high-throughput sequencing technology. We analyzed transposase-accessible chromatin assay (ATAC-seq) data obtained by high-throughput sequencing to identify super-enhancers in the open region. Based on Hi-C-loop data, we screened for super-enhancers that interact with the CDKN1C promoter. We used a dual luciferase vector reporter system to identify the activity of the super-enhancers. Because TADs are insulating, based on Hi-C-TAD data, super-enhancers located within TADs were selected for subsequent experimental validation. Finally, we validated the super-enhancer components capable of promoting CDKN1C gene expression in bovine myoblasts using CRISPRi, real-time fluorescence quantitative PCR, and Western blotting to effectively identify these components.

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

[0032] 2.1 Construction of the recombinant vector pX330a dCas9-KRAB Double-stranded oligonucleotides S3 (NC control group and sgRNA experimental group) were obtained by annealing using double-stranded oligonucleotide primers P12-P15 (Table 3). Double-stranded oligonucleotide annealing synthesis system: 100 μmol / L primers, 1 μL each of upstream and downstream primers corresponding to P12-P15, and 8 μL of deionized water, for a total of 10 μL. Double-stranded oligonucleotide annealing synthesis program: 5 min at 95°C, then slowly cooled to room temperature. The pX330a dCas9-KRAB vector (addgene:#92361) was enzymatically digested with Bbs I to linearize it, and the linearized vector was recovered from the enzymatic digestion product using a SanPrep column DNA gel recovery kit to obtain 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, totaling 100 μL. Enzyme digestion program: 3 hours at 95°C.

[0033] Ligation system 10 μL: 1 μL of 50 ng / μL linearization 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. Ligation program: Ligation was performed overnight at 16°C. Products S3 and S4 were ligated with T4 DNA ligase, respectively. The ligated products were transformed with DH5α, and after ampicillin resistance screening, a positive single clone was confirmed by Sanger sequencing. Extraction was performed using a SanPrep column plasmid DNA small extraction kit to obtain pX330a dCas9-KRAB-NC and pX330a dCas9-KRAB-sgRNA recombinant vectors (collectively referred to as pX330a dCas9-KRAB recombinant vectors).

[0034] [Table 3]

[0035] 2.2 Transfection of Recombinant Vectors into Cells Following the instructions for the transfection reagent TurboFect (Thermo Scientific®), bovine myoblasts were transfected with pX330a dCas9-KRAB-NC and pX330a dCas9-KRAB-sgRNA, respectively, and experiments were conducted to inhibit the activity of the target super-enhancer.

[0036] 2.3 Isolation, extraction, purification, and reverse transcription of RNA 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 degenome reverse transcription kit (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) listed in the NCBI database (http: / / www.ncbi.nlm.nih.gov / ) as a reference sequence, primers for real-time fluorescence quantitative PCR (Table 4) were designed using Primer 5.0, and the CDKN1C gene sequence was amplified. Amplification system: 2 μL of 10 ng / μL template cDNA, 1 μL each of upstream and downstream primers corresponding to 10 pmol / L primer vs. P16 or primer vs. P17, 10 μL of 2×SYBR Green qPCR Mix, and 6 μL of deionized water, total 20 μL. Reaction program: (1) 30 s at 95°C, (2) 10 s at 95°C, 30 s at 60°C, 39 cycles.

[0038] [Table 4]

[0039] The expression level of the CDKN1C gene in myoblasts was increased by 2 -ΔΔCt Detection was performed based on the following. 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 of the negative control group transfected with pX330a dCas9-KRAB-NC (Figure 4). Among these, Enh38 showed the highest effect. Western blotting revealed that the expression levels of the CDKN1C gene protein in all experimental groups were significantly lower than those of the negative control group transfected with pX330a dCas9-KRAB-NC (Figure 5). The significant decrease in CDKN1C expression indicated inhibition of the target super-enhancer activity. These results demonstrate that components of the target super-enhancer can promote CDKN1C gene expression. This super-enhancer was demonstrated to be active in bovine longissimus dorsi muscle tissue. Since the CDKN1C gene is an important gene that affects important economic traits in mammals, the components of the three target super-enhancers identified in this application can be used as target sites for gene editing and genetically modified breeding in beef cattle. By knocking down the above-mentioned super-enhancers, the expression of the CDKN1C gene can be inhibited, thereby promoting muscle development and increasing meat yield. Alternatively, by screening for important mutations that affect the activity of these super-enhancers, they can be used as auxiliary markers for selection in beef cattle.

Claims

1. A bovine myoblast CDKN1C gene super enhancer, The bovine myoblast CDKN1C gene super enhancer is characterized in that the components of the super enhancer include Enh22, Enh38, and Enh40, where Enh22 is located at positions 48802495 to 48803999 of chromosome 29 of the bovine genome, Enh38 is located at positions 48902854 to 48905629 of chromosome 29 of the bovine genome, and Enh40 is located at positions 48920416 to 48922207 of chromosome 29 of the bovine genome.

2. The bovine myoblast CDKN1C gene super-enhancer according to claim 1, characterized in that the nucleotide sequence of Enh22 is shown by SEQ ID NO. 1, the nucleotide sequence of Enh38 is shown by SEQ ID NO. 2, and the nucleotide sequence of Enh40 is shown by SEQ ID NO.

3.

3. A primer pair for amplifying the bovine myoblast CDKN1C gene super-enhancer according to any one of claims 1 to 2, A primer pair characterized in that the nucleotide sequences of the primer pair that amplify Enh22 are shown in SEQ ID NO. 4-5, the nucleotide sequences of the primer pair that amplify Enh38 are shown in SEQ ID NO. 6-7, and the nucleotide sequences of the primer pair that amplify Enh40 are shown in SEQ ID NO. 8-9.

4. Use of the bovine myoblast CDKN1C gene super enhancer according to any one of claims 1 to 2 in regulating the growth of bovine muscle.

5. Use of the bovine myoblast CDKN1C gene super enhancer described in any one of claims 1 to 2 in the breeding of high-yielding beef cattle.

6. The use according to claim 5, characterized in that the method for improving meat yield is to knock down one or more enhancers among Enh22, Enh38, or Enh40 in bovine myoblasts.

7. The use according to claim 6, 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 to a CRISPR / Cas9 basic vector to construct a recombinant vector.

8. The use according to claim 7, characterized in that the sgRNA primer nucleotide sequences of enhancer Enh22 are indicated by SEQ ID NO. 10 to 11.

9. The use according to claim 7, characterized in that the sgRNA primer nucleotide sequences of enhancer Enh38 are indicated by SEQ ID NO. 12-13.

10. The use according to claim 7, characterized in that the sgRNA primer nucleotide sequences of enhancer Enh40 are indicated by SEQ ID NO. 14-15.

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