The role of goat FTO/IGF2BP1-mediated BCL11A m6A modification in intramuscular adipocyte differentiation

Intramuscular fat (IMF) plays a critical role in determining the flavor of goat meat. Studies have shown that N6-methyladenosine (m6A) modification of mRNA exerts a key influence on adipocyte differentiation and lipid metabolism. However, whether m6A−modified mRNA regulates the differentiation of intramuscular adipocytes in goats remains unclear. This study constructed the m6A methylation landscape during the differentiation of goat intramuscular adipocytes using MeRIP-seq and RNA-seq, and identified BCL11A as a key regulator exhibiting synchronized changes in both m6A modification and expression. Functional assays demonstrated that BCL11A promotes lipid accumulation and upregulates adipogenic markers (C/EBPα, PPARγ, SREBP1, FASN). Mechanistically, the demethylase FTO directly targets the m6A site on BCL11A, while the reader protein IGF2BP1 recognizes this modification and reduces BCL11A mRNA stability. FTO promotes adipogenesis, whereas knockdown of IGF2BP1 shortens the half-life of BCL11A and inhibits differentiation. These findings reveal that FTO and IGF2BP1 co-regulate the differentiation of intramuscular adipocytes through post-transcriptional modification of BCL11A, providing novel insights into m6A-mediated lipid deposition in goat muscle.

Goat meat is highly valued for its rich content of essential amino acids, vitamins, and minerals. As a result, while efforts are made to increase mutton yield, there is growing attention on improving its quality. Intramuscular fat (IMF) content is a key indicator for evaluating meat quality (Madruga et al., 2008) and plays a vital role in determining the flavor of goat meat (Thomas et al., 2021; Zhang and Guan, 2019). Studies have shown that adipocyte differentiation is closely associated with intramuscular fat development. IMF deposition is essentially a process in which precursor adipocytes differentiate into mature adipocytes and accumulate lipids (Haczeyni et al., 2018). Therefore, elucidating the molecular mechanisms underlying intramuscular adipocyte differentiation in goats is crucial for enhancing the quality and flavor of goat meat.

N6-methyladenosine (m6A) modification is one of the most prevalent epigenetic modifications in mRNA and plays a crucial regulatory role in various biological processes (An and Duan, 2022; Qiu et al., 2024). The dynamic m6A modification is co-regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) (Liu et al., 2024; Ming et al., 2024; Zhang and Liu, 2022). Beyond reversible methylation and demethylation, m6A-binding proteins recognize m6A-modified bases and activate downstream regulatory pathways, thereby mediating diverse biological functions (Qiao et al., 2022; Shi et al., 2024).

Accumulating evidence indicates that m6A modification significantly influences adipogenesis in animals. For instance, Zhao demonstrated that deficiency of the demethylase FTO impaired the differentiation of 3T3-L1 preadipocytes (Zhao et al., 2014). Similarly, Yao reported that silencing the methyltransferase METTL3 reduced the m6A modification level of JAK1 mRNA in porcine bone marrow mesenchymal stem cells, thereby enhancing its mRNA stability and promoting adipogenic differentiation (Yao et al., 2019). In vivo studies further revealed that knockout of the Bmal1 gene in mouse liver tissue led to increased m6A levels of PPARα mRNA, which was recognized by YTHDF2, resulting in mRNA degradation, reduced stability, and suppressed lipid accumulation (Zhong et al., 2018). These findings collectively highlight the important role of m6A modification in adipocyte differentiation. However, whether m6A methylation is involved in the differentiation of goat intramuscular adipocytes remains unreported.

The B-cell chronic lymphocytic leukemia/lymphoma 11A gene (BCL11A) encodes a zinc-finger transcription factor, also known as the promyelocytic leukemia zinc finger protein (PLZF), which was initially identified in human hematologic malignancies (Hystad et al., 2007). BCL11A functions as either a transcriptional activator or repressor, depending on its binding partners and post-translational modifications such as phosphorylation or sumoylation (Califano et al., 2015; Walker et al., 2015). Studies have shown that BCL11A promotes breast cancer metastasis (Seachrist et al., 2020) and facilitates breast tumorigenesis by activating the Wnt/β-catenin signaling pathway (Zhu et al., 2019). In addition, Li revealed that the transcription factor CTIP1/BCL11A regulates epidermal differentiation and lipid metabolism during skin development (Li et al., 2017). BCL11A is the gene with the most significant difference in the MeRIP-seq integrated analysis. However, after reviewing the literature, it was found that current research on BCL11A mainly focuses on diseases, and there are no reports related to livestock fat. Currently, there are still no studies on BCL11A. Therefore, its selection as a core candidate gene is based on the objective screening results of multi-omics data, and subsequent research on the influence of BCL11A on the differentiation of intramuscular fat cells in goats will be conducted.

To elucidate the role of m6A in goat intramuscular adipocyte differentiation, we integrated transcriptome-wide m6A profiling (MeRIP-seq) and expression data (mRNA-seq). Subsequent functional validation of BCL11A demonstrated that it modulates adipogenic differentiation via a mechanism involving mRNA stability, as supported by luciferase reporter and ACTD assays. This work provides a foundation for understanding the epigenetic regulation of intramuscular fat development.

Cell isolation, culture and induction of differentiation

Goat intramuscular preadipocytes (GIMPA) were isolated from the longissimus dorsi muscle of 7-day old Jianzhou Da-er goats (billy goat, Sichuan jianyang dageda animal husbandry CO., LTD, Sichuan, China). The isolation and culture of cells was consistent with the previous study (Du et al., 2021). DMEM-F12 (Gibco, Carlsbad, CA, USA) containing 10% FBS (fetal bovine serum, Gibco, USA), 1% Penicillin-Streptomycin (Gibco, Carlsbad, CA, USA), and 50 µmol·L−1 oleic acid (Sigma, St Louis, MO, USA) was used to induce adipogenic differentiation of GIMPA to goat intramuscular adipocytes (GIMA).

Total RNA extraction, RNase R treatment and cDNA synthesis

Total RNA was extracted by Trizol reagent (TaKaRa, Tokyo, Japan) according to the instructions. Total RNA (2 µg) was incubated with 3 U/µg of RNase R (Geneseed, Guangzhou, China) for 15 min at 37 °C. After being treated with RNase R, the RNA was equally divided into two parts, one for electrophoresis verification and the other for cDNA synthesis by the Revert Aid First Stand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions; the expression levels of genes were analyzed by qRT-PCR. Then, 1 µg of untreated RNA was taken as a template directly for reverse transcription; RNA and cDNA were stored at −80 °C and −20 °C, respectively.

RNA extraction, library construction, and sequencing

Total RNA was isolated from intramuscular preadipocytes (IMPA1, IMPA2, IMPA3) and intramuscular adipocytes (IMA1, IMA2, IMA3) at 0 and 3 days of induced differentiation. RNA samples were subsequently sent to Shanghai OE Biotech Co., Ltd for sequencing. Library construction was initiated with 100 ng of total RNA. Poly(A)+ mRNA was enriched using Oligo(dT) magnetic beads and then fragmented by ultrasonication. The fragmented RNA was divided into two aliquots: one portion was immunoprecipitated with an m6A-specific antibody for MeRIP-seq library construction, while the other portion was used as input control for standard RNA-seq library preparation. m6A-modified fragments were recovered using antibody-conjugated magnetic beads. Sequencing libraries were constructed following the manufacturer’s instructions. Finally, high-throughput sequencing was performed on an Illumina HiSeq X Ten platform.

Raw data analysis of MeRIP-seq

Paired-end sequencing of the samples was performed on the Illumina platform, generating large volumes of raw data. Clean reads were aligned to the designated reference genome using HISAT2 to obtain unique sequence characteristics of the sequenced samples. Peak detection analysis was conducted using MeTDiff software (screening criteria: P < 0.05, log 2 FC ≥ 1.5) to determine the number, width, and distribution of m6A peaks, and to identify peak-associated genes. Peaks were annotated on a per-sample basis using ChIPseeker, and annotation results were statistically summarized. Motif sequences within the peak regions were analyzed and detected using the MEME-ChIP suite.

Quantitative real-time PCR

The qRT-PCR reaction was carried out in a 20 μL volume containing 1 μL of cDNA, 1 μL each of forward and reverse primers, 7 μL of ddH 2 O, and 10 μL of TB Green™ Premix Ex Taq™ II. mRNA levels were normalized to the endogenous control gene UXT. Relative expression levels were calculated using the 2 ^(−ΔΔCt) method. Primer sequences are listed in Table 1. For each gene in the qRT-PCR experiment, three biological replicates and three technical replicates were conducted.

Vector construction

BCL11A overexpression vector (pcDNA3.1-BCL11A), Methylation site-mutated BCL11A (pcDNA3.1- BCL11A -MT), and control vector (pcDNA3.1) were obtained from Sangon Biotech (Shanghai, China).

The FTO overexpression vector (pcDNA3.1- FTO) and control vector (pcDNA3.1) construct used in this study had been generated by our laboratory prior to this study.

The recombinant dual-luciferase reporter plasmids containing the wild-type (pGLO- BCL11A WT) and methylation site-mutated (pGLO-BCL1 1A MT) sequences of goat BCL11A were constructed by Sangon Biotech (Shanghai, China).

Small RNA synthesis and cell transfection

The interfering RNA of BCL11A, FTO, and IGFBP1 were designed and synthesized by JiMa (Shanghai, China) (Table 2). TurboFect™ Transfection Reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect vector, siRNA, miRNA mimics, and miRNA inhibitor into 80% confluent GIMPAs, according to the manufacturer’s instructions.

Dual-luciferase reporter assay

The luciferase activity was detected after co-transfection of pGLO- BCL11A -WT and pGLO- BCL11A -MT with pcDNA3.1- FTO, respectively. The dual luciferase reporter system (Vazyme, Nanjing, China) was used to detect the binding relation between FTO and pGLO- BCL11A -WT and pGLO- BCL11A -MT.

Bodipy staining and oil red O staining

The cells were washed three times by PBS. After discarding the PBS, the cells were fixed with 4% paraformaldehyde for 30 min, and the formaldehyde was discarded and then washed twice with PBS. The Bodipy stock solution (Thermo fisher Scientific, Waltham, MA, USA) was diluted 1:1000 with PBS under dark conditions to obtain the working solution, and 200 µL of the working solution was added to each well for 30 min at room temperature, and the lipids in the goat intramuscular adipocytes were observed by fluorescence microscope. The Oil red O stock solution (Solarbio, Beijing, China) and ddH 2 O were mixed at a ratio of 3:2 and filtered twice to obtain the Oil red O working solution. Then, 200 µL Oil Red O working solution was added to each well for 30 min at room temperature in the dark for staining, the Oil red O dye was discarded, washed three times with PBS and lipid droplet accumulation was observed and photographed in GIMAs.

Identification and sequencing quality control of intramuscular adipocyte differentiation in goats

Differentiation of goat intramuscular adipocytes was assessed by Oil Red O and BODIPY staining of preadipocytes at day 0 (IMPA) and adipocytes at day 3 (IMA) after oleic acid induction. Observations of cell quantity and lipid droplet morphology revealed that the IMPA group exhibited a limited number of adipocytes with minimal lipid accumulation, indicated by the presence of small lipid droplets, consistent with an undifferentiated state. In contrast, following 3 days of oleic acid induction (IMA group), the number of adipocytes increased significantly, accompanied by the accumulation of larger and more rounded lipid droplets, demonstrating successful differentiation (Figure 1). These differentiated cells were subsequently used for sequencing.

To investigate the biological role of mRNA m6A modification during the differentiation of goat intramuscular preadipocytes, we first performed sequencing and quality control assessments on the obtained data (Table 3). For the MeRIP-seq library, three biological replicates were included per group. Key metrics such as data retention rate and the percentage of bases with Q30 ≥ 30 (Q30%) were calculated (Table 3). The Q30% values for the MeRIP-seq libraries ranged from 93.82% to 94.08%, while those for the RNA-seq libraries ranged from 95.87% to 96.1%. These results confirm that the sequencing data were of high quality and suitable for subsequent analysis.

Analysis of mRNA m6A modification characteristics and alignment of sequencing reads with the reference genome

Clean reads were aligned to the designated reference genome using HISAT2 to obtain their genomic positions. The alignment statistics are summarized in Supplementary Table S1. For the MeRIP-seq libraries, the overall mapping rates ranged from 96.05% to 96.26%, and the uniquely mapped rates were between 90.82% and 91.49%. For the RNA-seq libraries, the overall mapping rates were 95.30% to 95.78%, with uniquely mapped rates of 83.78% to 84.62%. These mapping rates, all exceeding 95% overall, are considered high quality for downstream analysis.

Analysis of the sequencing data revealed 9042 methylated genes in the IMPA group and 9106 methylated genes in the IMA group, with 8369 genes being common to both groups (Figure 2A). These methylated genes contained 17,566 and 17,490 distinct m6A peaks in the IMPA and IMA groups, respectively, of which 2638 peaks were shared (Figure 2B). Compared with other chromosomes, more m6A peaks were observed on chromosomes 3, 5, 7, 11, 18, 19, and 25 (Figure 2C). Further analysis indicated that m6A peaks were predominantly enriched in the 3′UTR and coding sequence (CDS) regions (Figures 2D, E). In addition, motif analysis identified the GGAAG/GGAAA consensus sequence in both the IMPA and IMA groups (Figure 2F).

Differential enrichment of mRNA m6A modification during goat intramuscular adipocyte differentiation

A total of 638 differentially methylated mRNAs (P < 0.05, |log2FC| ≥ 1.5), corresponding to 705 differential peaks (304 hyper-methylated and 401 hypo-methylated), were identified (Figure 3A). The top 10 differentially methylated mRNAs with either significant hyper-methylation or hypo-methylation are listed in Supplementary Table S2. Compared to the IMPA group, the IMA group exhibited more hypo-methylated peaks than hyper-methylated peaks, with the highest number of differential peaks observed on chromosome 18, suggesting that chromosome 18 is the most active site for m6A methylation during goat intramuscular adipocyte differentiation (Figure 3B). Analysis of peak distribution per gene revealed that the majority of genes (84.9%) contained only one m6A peak (Figure 3C). Furthermore, the density of m6A modification along mRNA molecules was calculated, showing that longer gene transcripts tended to contain more m6A peaks (Figure 3D). To investigate the biological processes associated with m6A modification during goat intramuscular adipocyte differentiation, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses (Figure 3E). KEGG pathway analysis revealed that these differentially methylated genes were primarily enriched in pathways related to cancer pathogenesis, Herpes simplex virus 1 infection, and Rap1 signaling. Additionally, several lipid metabolism-related pathways were identified, including the TGF-β signaling pathway, Rap1 signaling pathway, calcium signaling pathway, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and Ras signaling pathway (Figure 3F).

Analysis of differentially expressed mRNAs in goat IMPA and IMA by RNA-seq

A total of 3,037 differentially expressed mRNAs were identified between the IMPA and IMA groups (P < 0.05 and |Log2(fold change)|> 1.5). Among these, the expression levels of 1,706 genes were significantly upregulated, while 1,331 genes were downregulated (Figure 4A). Hierarchical clustering analysis established distinct expression profiles, confirming significant differences in mRNA expression between the IMPA and IMA groups (Figure 4B). Gene Ontology (GO) analysis indicated that these genes were primarily associated with cell division, actin filament binding, and nucleosomes (Figure 4C). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these genes were predominantly enriched in pathways including systemic lupus erythematosus, pathways in cancer, the PI3K-Akt signaling pathway, the Wnt signaling pathway, the Rap1 signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, and the Apelin signaling pathway (Figure 4D).

Comprehensive analysis of RNA-Seq and MeRIP-Seq data and verification of sequencing results

To further investigate the potential functions of mRNA m6A modification during the differentiation of goat intramuscular adipocytes, we performed an integrated analysis of RNA-seq and MeRIP-seq data. A total of 635 mRNAs (P < 0.05 and |Log 2 (fold change)| > 1.5) exhibited concurrent differential methylation and expression (Figure 5A). Among these, 224 genes showed concurrent upregulation in both methylation and expression levels, 129 genes exhibited upregulated methylation but downregulated expression, 225 genes demonstrated concurrent downregulation in both methylation and expression, and 57 genes displayed downregulated methylation but upregulated expression (Figure 5A). Gene Ontology (GO) analysis suggested that these genes may be primarily associated with voltage-gated potassium channel activity, the voltage-gated potassium channel complex, calmodulin binding, calmodulin-dependent protein kinase activity, the Tie signaling pathway, and ATP binding (Figure 5B). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that these genes were predominantly enriched in pathways including the Ras signaling pathway, calcium signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway, and the Apelin signaling pathway (Figure 5C). To validate the reliability of the RNA-seq results, four differentially expressed mRNAs (BCL11A, FBXO40, ELAVL2, C1QL2) were selected for verification by RT-qPCR using UXT as the reference gene. The results showed that BCL11A and FBXO40 were significantly upregulated during intramuscular adipocyte differentiation (Figure 5D), while ELAVL2 and C1QL2 were markedly downregulated. These findings are consistent with the RNA-seq data, confirming the credibility of the RNA-seq analysis (Figure 5D). Furthermore, to verify the reliability of the MeRIP-seq results, two differentially expressed mRNAs modified by m6A methylation were selected for MeRIP-qPCR analysis. The results indicated elevated methylation levels of BCL11A and SLC22A31, which aligns with the trends observed in the sequencing data (Figure 5E).

BCL11A is a zinc-finger transcription factor that encodes a Krüppel-like zinc finger protein. However, there have been relatively few studies investigating its role in lipid metabolism. Recent research has revealed that the transcription factor CTIP1/BCL11A regulates epidermal differentiation and lipid metabolism during skin development. Combined with our sequencing data, this finding suggests that BCL11A may be involved in the regulation of adipogenesis. Therefore, we plan to further explore the gene function and mechanistic roles of BCL11A.

BCL11A relies on m6A modification to promote the differentiation of intramuscular adipocytes in goats

The sequencing results indicated that both the m6A methylation and mRNA expression levels of BCL11A were upregulated following the differentiation of goat intramuscular adipocytes. We performed MeRIP-qPCR and RT-qRT-PCR on the same set of sequencing samples for validation, and the results were consistent with the sequencing data (Supplementary Figures 1A, B).

Goat intramuscular adipocytes were transfected with a BCL11A overexpression vector, followed by induction of differentiation using oleic acid for 3 days. The overexpression efficiency was assessed by qRT-PCR, which revealed that the expression level of BCL11A in the OE- BCL11A group was approximately 2500-fold higher than that in the control group, indicating successful establishment of BCL11A overexpression in goat intramuscular adipocytes (Figure 6A). Bodipy and Oil Red O staining results demonstrated a significant increase in lipid droplet content in adipocytes following BCL11A overexpression (Figure 6B). Consistent with this, quantitative analysis of the Oil Red O staining (by measuring OD values) showed a highly significant increase in the OD value in the OE- BCL11A group (P < 0.01; Figure 6C). Furthermore, overexpression of the goat BCL11A gene led to a highly significant upregulation in the expression of adipogenic marker genes C/EBPα, PPARγ, SREBP1 and FASN (P < 0.05; Figure 6D).

Goat intramuscular adipocytes were transfected with BCL11A siRNA and subsequently induced to differentiate with oleic acid for 3 days. Transfection efficiency was evaluated by qRT-PCR, which showed that the expression level of BCL11A in the si- BCL11A group was downregulated by approximately 65% compared to the control group (Figure 6E). Bodipy and Oil Red O staining results revealed a significant reduction in lipid droplet content in the adipocytes following BCL11A knockdown (Figure 6F). Quantitative analysis of the Oil Red O staining (by measuring OD values) indicated a highly significant decrease in the OD value in the si- BCL11A group (P < 0.01; Figure 6G). Moreover, knockdown of the goat BCL11A gene resulted in a highly significant downregulation in the expression of adipogenic marker genes C/EBPα, C/EBPβ, PPARγ, SREBP1, and FASN (P < 0.05; Figure 6H).

Using the goat BCL11A sequence (XM_018055277.1) obtained from the NCBI database, the complete coding sequence (CDS) was submitted to SRAMP for prediction. Our analysis identified the adenine (A) base at position 161 within the CDS of the BCL11A gene as the site with the highest prediction confidence for methylation (Supplementary Figure 2).

In the subsequent experiments, compared with the control group, the number and size of lipid droplets did not show significant changes after the m6A site mutation of BCL11A (Figure 6I). The qRT-PCR analysis revealed that there was no significant difference in the expression levels of fat formation marker genes (C/EBPα, C/EBPβ, PPARγ, SREBP1, and FASN) compared with the control group (Figure 6J).

FTO-mediated modification of BCL11A m6A can promote the differentiation of intramural adipocytes in goats

We used the RBPsuit software to predict the proteins that potentially interact with BCL11A. We found that FTO might be an interacting protein of BCL11A. Therefore, we focused on this part to investigate the regulatory effect of FTO on BCL11A. To investigate whether FTO regulates the expression of the BCL11A gene, we first overexpressed FTO in goat intramuscular adipocytes. The results showed a significant downregulation of BCL11A expression (Figure 7A). Conversely, knockdown of FTO led to a highly significant upregulation of BCL11A expression levels (Figure 7B). Subsequently, a 121 bp nucleotide sequence flanking and including the methylation site was cloned into the pmirGLO vector, designated as the wild-type (pmirGLO-BCL11A WT). The adenine (A) at the predicted position 161 was mutated to thymine (T), and the mutated sequence was also cloned into the pmirGLO vector, designated as the mutant type (pmirGLO-BCL11A mut) (Figure 7C). Dual-luciferase reporter assays revealed that the luciferase activity in the pmirGLO-BCL11A WT + OE-FTO group was significantly lower than that in the pmirGLO-BCL11A WT + NC group (P < 0.01). However, after the mutation of the methylation site from A to T, the luciferase activity in the pmirGLO-BCL11A mut + OE-FTO group was restored compared to the pmirGLO-BCL11A mut + NC group (Figure 7D). Collectively, these findings demonstrate that the demethylase FTO inhibits the expression of the goat BCL11A gene by targeting the adenine base at the methylation site (position 161) within its CDS region.

Goat intramuscular adipocytes were transfected with an FTO overexpression vector and subsequently induced to differentiate with oleic acid for 3 days. qRT-PCR analysis confirmed the overexpression efficiency, revealing an approximately 58-fold increase in FTO expression in the OE- FTO group compared to the control group (Figure 7E). Bodipy and Oil Red O staining results demonstrated a significant increase in lipid droplet content in adipocytes following FTO overexpression (Figure 7F). Quantitative measurement of the OD values indicated a significant upregulation in the OE- FTO group (P < 0.05; Figure 7G). Furthermore, overexpression of the goat FTO gene resulted in a highly significant upregulation in the expression of adipogenic marker genes C/EBPα, C/EBPβ, PPARγ, SREBP1, and FASN (P < 0.05; Figure 7H). These results collectively indicate that FTO overexpression promotes the differentiation of goat intramuscular adipocytes.

Goat intramuscular adipocytes were transfected with FTO siRNA and subsequently induced to differentiate with oleic acid for 3 days. Transfection efficiency was determined by qRT-PCR, which showed that the expression level of FTO in the si- FTO group was downregulated by approximately 60% compared to the control group (Figure 7I). Staining results demonstrated that the lipid droplet content in adipocytes was reduced following FTO knockdown (Figure 7J). Quantitative analysis of OD values indicated a highly significant decrease in the si- FTO group (P < 0.01; Figure 7K). Furthermore, knockdown of the goat FTO gene resulted in a highly significant downregulation in the expression of adipogenic marker genes C/EBPα, C/EBPβ, PPARγ, SREBP1, and FASN (P < 0.05; Figure 7L). Collectively, these results demonstrate that FTO knockdown inhibits the differentiation of goat intramuscular adipocytes.

The m6A binding protein IGF2BP1 affects the differentiation of intramuscular adipocytes in goats by reducing the mRNA stability of BCL11A

To identify the m6A-binding proteins responsible for the biological functions of BCL11A following m6A methylation, this study utilized the RBPsuite software to predict m6A-binding proteins of the goat BCL11A gene. The prediction results indicated that the binding protein (reader) IGF2BP1 interacts with BCL11A, with 23 potential binding sequences identified (Figure 8A). Among these, the motif AAGCACCCGUU exhibited the highest confidence level and represented the strongest binding site to BCL11A (Figure 8B). Studies have shown that IGF2BP1, as an m6A-binding protein of the IGF2BPs family, primarily influences the stability and biological functions of methylated mRNA. To determine whether IGF2BP1 affects the stability of BCL11A mRNA, four siRNAs targeting goat IGF2BP1 were synthesized, of which siIGF2BP1–3 exhibited the highest interference efficiency and was therefore selected for subsequent experiments (Figure 8C). Subsequently, an mRNA stability assay was conducted using actinomycin D. The results demonstrated that knockdown of IGF2BP1 significantly reduced BCL11A mRNA stability (P < 0.05) and shortened its half-life (Figure 8D).

In this study, MeRIP−seq demonstrated that m 6 A peaks from both IMPA and IMA samples were preferentially enriched within 3′UTR regions, a distribution pattern differing from that reported in mice. In human cells, however, canonical m 6 A modifications are predominantly enriched around stop codons and 3′UTRs (Dominissini et al., 2012), implying that the divergent genomic distribution of m 6 A peaks arises from inherent species−specific epigenetic features. Functionally, the 3′UTR governs mRNA subcellular localization, transcript stability and translational efficiency; it also participates in protein–protein interactions (PPIs) and facilitates the functional transformation of genetic information from mRNA to protein. Accordingly, m 6 A deposition within 3′UTRs is capable of altering the translational output of target transcripts (Mayr, 2019). Motif analysis of m 6 A peaks using MEME−ChIP further identified the GGAAG motif in the IMPA group and the GGAAA motif in the IMA group. Such divergent conserved motifs imply distinct binding preferences for m 6 A reader proteins, which potentially mediate the epigenetic regulation of goat intramuscular preadipocyte differentiation. Compared with the IMPA group, differentially methylated peaks in IMA were widely scattered across multiple chromosomes, with the highest enrichment detected on chromosome 18, indicative of preferential m 6 A modification events occurring on this chromosome. Quantitative statistics showed that approximately 84.9% of modified genes carried a single m 6 A peak, whereas the remaining genes possessed two or more peaks. In addition, genes with longer transcript fragments tended to harbor more m 6 A peaks, consistent with a previous report in livestock species (Wu et al., 2018). To decipher the biological functions of m 6 A modification during intramuscular adipogenesis, GO and KEGG enrichment analyses were conducted on m 6 A−modified genes. KEGG results revealed prominent enrichment in the TGF−β signaling pathway, Rap1 signaling pathway, and glycosphingolipid biosynthesis (Ganglio series). The TGF−β superfamily, including TGF−β1–3, activin/inhibin, GDFs, myostatin and BMP members, has been well documented to modulate food intake, lipid turnover and glucose homeostasis (Wen et al., 2022). Rap1 signaling serves as a core intracellular transduction cascade participating in cell proliferation, differentiation, apoptosis and metabolic homeostasis; notably, its extensive crosstalk with the MAPK pathway—a master regulator of adipogenic programming—supports its potential role in controlling intramuscular preadipocyte maturation (Li et al., 2023). Consistently, pharmacological suppression of glycosphingolipid synthesis was previously proven to repress hepatic fatty acid and cholesterol metabolic genes and alter systemic body weight in mice (Chatterjee et al., 2020), which echoes our enrichment results.Collectively, our genome−wide profiling confirms unique m 6 A topological features in goat intramuscular preadipocytes: differential m 6 A modifications preferentially accumulate on chromosome 18, and most target transcripts bear only one methylation peak. Functional enrichment further links these m 6 A−modified transcripts to multiple adipogenesis−related signaling cascades. These lines of evidence strongly support the essential involvement of m 6 A RNA methylation in governing goat intramuscular preadipocyte differentiation.

Integrated analysis combining MeRIP-seq and RNA-seq showed that m 6 A methylation reshapes the biological functions of target genes. Such epigenetic modification is accompanied by significant upregulation or downregulation of corresponding gene expression. KEGG enrichment screened three core pathways for genes with both altered m 6 A level and transcriptional abundance: PI3K-Akt, Rap1 and Apelin signaling pathways. Apelin signaling is a well-established regulator of lipid metabolism (Liu et al., 2022). Hormones and cytokines bind to specific receptors to trigger downstream signaling networks. This process activates AMPK and PI3K-AKT-mTOR cascades and suppresses JNK and NF-κB pathways simultaneously. Crosstalk among these downstream pathways jointly governs energy homeostasis, glucose turnover and lipid metabolism (Ji et al., 2019). The PI3K-Akt pathway bidirectionally modulates lipid-metabolizing genes across diverse tissues in vivo. It controls multiple steps of lipid metabolism, including lipid synthesis, uptake, transportation and decomposition. Abnormal activation of PI3K-Akt leads to disturbed lipid homeostasis. This dysfunction is closely related to obesity, type 2 diabetes, cardiovascular diseases and malignant tumors (Cui et al., 2018). In summary, transcripts with divergent methylation and expression are tightly correlated with cell differentiation and lipid metabolism. Most of these functional transcripts exert biological effects relying on adipocytokine and PI3K-Akt signaling. These findings lay a solid foundation for further mechanistic exploration in goat intramuscular adipogenesis.

To elucidate the regulatory role of the BCL11A gene in goat intramuscular adipocyte differentiation through m6A methylation site-dependent modification, we transfected the goat BCL11A overexpression vector into preadipocytes. Oil Red O and Bodipy staining demonstrated that BCL11A overexpression promoted lipid droplet accumulation. Detection of adipogenic marker genes revealed that the expression levels of C/EBPα, FASN, PPARγ, and SREBP1 were extremely significantly upregulated. Following interference with the goat BCL11A gene, morphological staining showed a reduction in cellular lipid droplet accumulation. Additionally, the expression levels of adipocyte differentiation marker genes C/EBPα, PPARγ, SREBP1, and FASN were extremely significantly downregulated, while C/EBPβ was significantly downregulated. However, when we mutated the m6A methylation modification sites of the BCL11A gene, there were no significant alterations in the expression of adipogenic marker genes and lipid synthesis marker genes (C/EBPα, C/EBPβ, PPARγ, SREBP1, and FASN). This observation differed substantially from the previous results of BCL11A overexpression. Thus, it is hypothesized that BCL11A may regulate goat intramuscular adipocyte differentiation through m6A methylation site-dependent modification and through the coordinated action of C/EBPα, C/EBPβ, PPARγ, SREBP1 and FASN.

Research has established the demethylase FTO as the first candidate gene confirmed to be associated with obesity. Claussnitzer further showed that FTO can suppress thermogenesis in preadipocytes (Claussnitzer et al., 2015). Notably, additional studies have indicated that increasing FTO activity enhances adipogenic capacity, while decreasing FTO activity diminishes adipogenic capacity (Merkestein et al., 2015). The Wnt/β-catenin signaling pathway is modulated by FTO to facilitate adipogenesis (Chen et al., 2017). Kajimura reported that FTO inhibits the expression of uncoupling protein 1 in brown adipocytes via its demethylase activity, thereby suppressing the conversion of adipose tissue to brown adipose tissue (Kajimura et al., 2009). In another study, FTO was also shown to upregulate C/EBPβ levels and inhibit the differentiation of adipose tissue into brown adipose tissue, thereby suppressing the browning of white adipose tissue (Tews et al., 2011).

To verify whether the BCL11A methylation sites are targeted by FTO, we constructed a dual-luciferase reporter vector containing the BCL11A methylation mutation sites. This experiment confirmed that FTO has a targeted binding relationship with the BCL11A methylation sites. Subsequently, the FTO overexpression vector was transfected into goat intramuscular preadipocytes. Oil Red O and Bodipy staining showed that OE- FTO promoted lipid droplet accumulation in goat intramuscular adipocytes. Meanwhile, detection of adipocyte differentiation marker genes revealed that the expression levels of adipogenic marker genes C/EBPα, PPARγ, SREBP1, and FASN were extremely significantly upregulated, and C/EBPβ was significantly upregulated. These results suggest that the goat FTO gene positively regulates the differentiation of intramuscular preadipocytes.

During m6A modification, the effects of m6A methylation on mRNA are ultimately mediated through recognition and binding to specific m6A-binding proteins (Cai et al., 2019). IGF2BP1 belongs to the IGF2BPs family, and studies have reported that IGF2BP1 modulates the stability of methylated gene mRNAs (Shi et al., 2023). IGF2BP2 enhances GLUT1 expression via direct binding and stabilization of GLUT1 mRNA, thus promoting glycolysis and proliferation of PDAC cells (Huang et al., 2019). Additionally, studies have demonstrated that the m6A-binding protein IGF2BP1 regulates MGAT5 mRNA stability via m6A methylation modification, thereby sustaining the stemness of liver cancer stem cells (Yang et al., 2021).

Actinomycin D is a transcription inhibitor that intercalates into DNA. It forms a highly stable complex with DNA, preventing the unwinding of the DNA double helix and thereby inhibiting the activity of DNA-dependent RNA polymerase. Actinomycin D can suppress the synthesis of new mRNA, and the stability of mRNA is evaluated by measuring the abundance of mRNA after transcriptional inhibition (Koba and Konopa, 2005). At low concentrations, actinomycin D inhibits transcription without significantly affecting DNA replication or protein synthesis (Zucenka and Griskevicius, 2024). Other transcription inhibitors, such as 5,6-dichloro-1β-D-ribofuranosylbenzimidazole (DRB), which directly interacts with RNA polymerase II, have also been successfully used in similar assays (Kuo et al., 2023).

This study analyzed the role of the FTO-BCL11A-IGF2BP1 pathway in regulating the differentiation of goat intramuscular preadipocytes through sequencing and in vitro cell function tests. However, all the experimental conclusions were only verified in the in vitro cell system. Due to the complex microenvironment of live muscle tissues, the true physiological role of this regulatory axis in the process of intramuscular fat deposition in animals still requires further verification through live animal models (genetically edited individuals).

In this experiment, we treated cells with actinomycin D and calculated the half-life of BCL11A. The results showed that interfering with IGF2BP1 could inhibit the stability of BCL11A mRNA, a gene with differential m6A modification. The demethylase FTO targets the differentially methylated gene BCL11A in goats, and by influencing the stability of BCL11A mRNA, the m6A reader protein IGF2BP1 ultimately affects intramuscular adipocyte differentiation (Figure 9).