Characterization of the 5′ regulatory region of the porcine CEBPB gene

An in silico analysis of the 5′ regulatory sequence of the porcine CEBPB gene was conducted to identify putative promoter elements and transcription factor binding sites associated with adipogenesis. The analysis was performed using the DNASTAR Lasergene software suite (https://www.dnastar.com/software/). Multiple regulatory motifs were identified, including a TATA box, CRE-like elements, glucocorticoid response elements (GRE), a coding sequence start (CDS), and the transcription start site (TSS) region (Fig. 1a). To excise this regulatory element-rich region, two out of four designed single guide RNAs (gRNAs) were selected for CRISPR/Cas9 targeting, resulting in the generation of two cleavage sites flanking the region of interest (Fig. 1b; Table S1).

Fig. 1

Schematic representation of the 5′ regulatory region of the CEBPB gene showing predicted transcription factor binding sites relevant to adipogenesis, including TATA box, CRE-like elements, GRE, CDS, and TSS. The positions of gRNA5 and gRNA7 used to delete the promoter region are indicated (a). Genomic structure of the porcine CEBPB gene with the locations of the designed gRNAs used for CRISPR/Cas9-mediated editing (b)

Introduction of a deletion in the CEBPB locus

CRISPR/Cas9-mediated genome editing was employed to introduce a targeted deletion within the CEBPB locus. Guide RNAs were cloned into the pX330 plasmid vector, and MSCs were transfected via nucleofection. Transfection efficiency was determined using the pmaxGFP control plasmid (Lonza, Basel, Switzerland) by counting GFP-positive MSCs in randomly selected microscopic fields. The mean efficiency was estimated at approximately 60% (Fig. S1). Following transfection, single MSC colonies were genotyped by PCR. Primers were designed to amplify genomic DNA regions flanking the gRNA target sites. In wild-type cells, the expected PCR product was 818 base pairs (bp), whereas cells carrying the intended deletion yielded a 243-bp fragment, indicating a 575-bp deletion in the CEBPB promoter region (Fig. 2a). Based on this genotyping approach, a total of eleven clones with targeted deletions were identified—one homozygous and ten heterozygous. The editing efficiency at the CEBPB locus reached 22.9%, as determined by the proportion of edited clones (n = 11) among all screened samples (n = 48). Sanger sequencing of PCR amplicons confirmed the presence of a 575-bp deletion within the 5′ regulatory region of the CEBPB gene (Fig. 2b). No additional mutations, insertions or deletions (indels), or partial deletions were detected apart from the intended 575-bp deletion in the target region.

Fig. 2

Representative electrophoresis image of PCR genotyping from single-cell-derived colonies, showing detection of the CEBPB promoter deletion. L – 100 bp DNA ladder (Thermo Fisher Scientific); Lanes 2, 4–10, 12, 14 – wild-type colony (818 bp); lane 1 – colony with a homozygous deletion (243 bp); lanes 3,11,13 – colonies with a heterozygous deletion; lane 15 - negative control (non-transfected cells); lane 16 - negative control (no DNA template) (a). Sanger sequencing chromatogram confirming a 575 bp deletion in the 5′ regulatory region of the CEBPB gene. The sequence shows the junction between the excised DNA ends, with partial gRNA5 and gRNA7 recognition sequences indicated (b)

Expression profile of the CEBPB gene in wide-type MSC (MSCWT) and in MSC with deletion in promoter region (MSCDEL)

To assess whether the deletion introduced in the promoter region of the CEBPB gene affected its transcriptional activity, relative mRNA expression levels were compared between wild-type cells (MSCWT) and genetically modified cells (MSCDEL) carrying either homozygous or heterozygous deletions. A significant overall difference in CEBPB transcript levels was observed among cell types (Wilcoxon signed-rank test, p = 0.004). Post hoc analysis revealed that MSCWT exhibited significantly higher CEBPB expression than MSCDEL with heterozygous deletions (p = 0.025) and homozygous deletions (p = 0.025) (Fig. 3a). CEBPB expression in wild-type cells was approximately 11-fold higher than in heterozygous cells. Due to the markedly reduced proliferation of MSCs carrying a homozygous deletion, these cells could not be used for adipogenic differentiation analysis. Therefore, further differentiation studies were conducted only on MSCDEL cells with heterozygous deletions, in comparison to MSCWT. The expression profile of CEBPB was evaluated over the course of adipogenic differentiation, from day 0 (undifferentiated MSCs) to day 10. In MSCWT, CEBPB exhibited a characteristic peak in transcript levels on day 4 of differentiation. In contrast, MSCDEL cells showed significantly reduced CEBPB expression across all examined time points (MANOVA, p < 0.001) (Fig. 3b). These results demonstrate that the targeted promoter deletion effectively disrupted CEBPB expression, confirming the functional importance of this regulatory region during adipogenic differentiation.

Fig. 3

Relative transcript levels of CEBPB gene in wide-type MSC (MSCWT) and in MSC with deletion in promoter region (MSCDEL) in homozygous and heterozygous cells, Wilcoxon test, p < 0.004 (a) and during subsequent days of adipogenesis for heterozygous cells (b). The error bars represent 95% confidence intervals. **: significantly higher in MSCWT than in MSCDEL, MANOVA test, p < 0.001

Expression patterns of adipogenesis-related genes in MSCWT and MSCDEL

To assess the functional role of CEBPB in porcine adipogenesis, the expression profiles of key adipogenic marker genes were analyzed over the course of differentiation in MSCWT and MSCDEL (heterozygous) cells. In MSCWT, the highest expression level of GATA2 was observed at day 0, followed by a gradual decrease throughout the differentiation process. In contrast, MSCDELexhibited significantly lower GATA2 expression compared to MSCWT at all time points except day 10 (MANOVA, p < 0.001) (Fig. 4a). CEBPA expression in MSCWT progressively increased during adipogenesis, reaching its peak at day 10. In  MSCDEL, CEBPA transcript levels were significantly lower than those in MSCWT across all days of differentiation (MANOVA, p < 0.001) (Fig. 4b). A significant induction of PPARG expression was detected in MSCWT starting from day 6. In MSCDEL, PPARG expression was significantly reduced relative to MSCWT at all time points, except on day 6 (MANOVA, p < 0.001) (Fig. 4c). The expression of FABP4, a marker of mature adipocytes, was also altered. MSCDEL exhibited significantly lower FABP4 transcript levels than MSCWT at days 0, 6, 8, and 10, while significantly higher levels were observed at days 2 and 4 (MANOVA, p < 0.001) (Fig. 4d).

Fig. 4

Relative transcript levels of GATA2 (a), CEBPA (b), PPARG (c) and FABP4 (d) during subsequent days of adipogenesis in MSCWT and MSCDEL. Error bars represent 95% confidence intervals. **: significantly higher in MSCWT than in MSCDEL, p < 0.001; ^^: significantly lower in MSCWT than in MSCDEL, MANOVA test, p < 0.001

These results demonstrate that disruption of CEBPB expression interferes with the normal transcriptional cascade of adipogenesis, suppressing the expression of upstream transcription factors (GATA2, CEBPA, PPARG) as well as FABP4, a gene characteristic of terminal adipocyte differentiation. The downregulation of these markers corresponded with the observed phenotypic effect—cells with the CEBPB promoter deletion failed to accumulate lipid droplets, indicating an absence of adipogenic differentiation at the cellular level (Fig. S2).

Assessment of the proliferative status of MSCWT and MSCDEL

Due to the observed reduction in proliferation in CEBPB-modified cells, additional analyses were conducted to assess the expression of three proliferation-associated marker genes: CCND1, MCM2, and PCNA. A significant decrease in transcript levels of all three genes was detected in MSCDEL cells —both heterozygous and homozygous—compared to unmodified MSCWT cells (Wilcoxon signed-rank test, p = 0.004) (Fig. 5). These results indicate that CEBPB plays a dual role in porcine mesenchymal stem cells, being essential not only for adipogenic differentiation but also for the regulation of cell proliferation.

Fig. 5

Relative transcript levels of CCDN1 (a), MCM2 (b) and PCNA (c) genes in wide-type MSC (MSCWT) and in MSC with deletion in promoter region of CEBPB gene (MSCDEL) in homozygous and heterozygous status, Wilcoxon test, p < 0.004