A Primate-Specific Endogenous Retroviral Envelope Protein Regulates Human Cardiomyocyte Development by Inhibiting SFRP2

Research Background and Significance

Endogenous Retroviruses (ERVs) are remnants of ancient viral infections that integrated into the host genome and were inherited through the germline to modern human genomes during evolution. These ERVs account for 5%-8% of the human genome. Although most ERV genes have lost the ability to encode complete proteins due to mutations, some non-coding ERVs function in early embryonic development. Additionally, ERVs can act as enhancers or promoters to regulate the expression of nearby genes, participating in various physiological functions of organisms. Studies have shown that proteins derived from ERVs play important roles in early extraembryonic development, but it is unclear whether ERVs also participate in somatic cell development. This study explored the function of the primate-specific ERVH48-1 (Suppressyn) in somatic cell development, investigating its regulatory mechanism on mesoderm and cardiomyocyte differentiation during early embryonic development, thus providing a new perspective on the role of ERVs in human development.

Research Origin

This study was jointly conducted by research teams from institutions including Guangzhou Medical University, Hong Kong Academy of Sciences, and the University of Macau. The article was published in the journal “Cell Stem Cell” on September 5, 2024, with Ran Zhang et al. as the first authors and Xiaofei Zhang, Dajiang Qin, and others as the corresponding authors.

Research Process

Overview of Experimental Design and Procedures

Using human pluripotent stem cells (hPSCs) as a model, this study investigated the role of the ERVH48-1 gene in embryonic development by knocking it out. The research process included the following steps:

1. Expression of ERVH48-1 at Different Embryonic Stages: Using single-cell RNA sequencing and immunofluorescence, the research team found that ERVH48-1 is highly expressed during the development of mesoderm and endoderm in embryos, particularly in the early stages of cardiomyocyte differentiation.

2. Effects of Knocking Out ERVH48-1: By using CRISPR-Cas9 technology to knock out ERVH48-1, it was found that the absence of this gene inhibits hPSC differentiation into mesoderm and cardiomyocytes, diverting cell fate towards ectoderm-like cells.

3. Study of the Signal Peptide Function of ERVH48-1: By constructing signal peptide-deficient mutants of ERVH48-1, the research team discovered that the N-terminal signal peptide of ERVH48-1 is crucial for its localization to the cell membrane and function.

4. Interaction Between ERVH48-1 and SFRP2: ERVH48-1 interacts with SFRP2 (Secreted Frizzled-Related Protein 2), promoting its polyubiquitination and accelerating its degradation, thereby blocking SFRP2’s inhibition of the Wnt/β-catenin signaling pathway, regulating cardiomyocyte differentiation.

5. Signal Peptide Substitution Experiment: To further validate the role of SFRP2, the research team constructed an SFRP2 chimera with the ERVH48-1 signal peptide, finding that this chimera could restore Wnt/β-catenin signaling and cardiomyocyte differentiation in ERVH48-1 deficient cells.

Data Analysis and Methods

The study used extensive data analysis methods, including single-cell RNA sequencing, fluorescence-activated cell sorting (FACS), immunofluorescence, co-immunoprecipitation (Co-IP), and proteomics analysis. These data supported the role of ERVH48-1 in regulating the Wnt signaling pathway through SFRP2 degradation. For statistical analysis, the research team used one-way analysis of variance (ANOVA) and Sidak correction to ensure the significance of the results.

Research Results

1. Impact of ERVH48-1 on Cardiomyocyte Fate: Knocking out ERVH48-1 significantly inhibited the differentiation of hPSCs into cardiomyocytes, whereas normal cardiomyocyte development was observed in wild-type cells. Immunofluorescence and gene expression analysis showed that loss of ERVH48-1 led to decreased expression of cardiomyocyte-specific marker genes (such as TNNT2), and cardiomyocyte beating was also inhibited.

2. Regulation of Wnt Signaling by ERVH48-1 via SFRP2: ERVH48-1 interacts with SFRP2 and promotes its degradation, reducing its secretion and thus relieving its inhibition on the Wnt/β-catenin signaling pathway. Experimental results showed that loss of ERVH48-1 led to decreased β-catenin levels inside cells, ultimately affecting cardiomyocyte differentiation.

3. Role of the Signal Peptide: The signal peptide of ERVH48-1 is vital for its membrane localization and function. Deleting the signal peptide of ERVH48-1 resulted in the loss of its ability to promote SFRP2 degradation, leading to ineffective activation of the Wnt signaling pathway and obstructed cardiomyocyte differentiation.

4. Success of Chimera Experiment: The fusion of the ERVH48-1 signal peptide with SFRP2 created a chimera that partially restored the cardiomyocyte differentiation function of ERVH48-1 knockout cells, further proving that the function of ERVH48-1 is achieved by regulating SFRP2.

Research Conclusion and Significance

This study revealed the important role of ERVH48-1 in primate-specific embryonic somatic cell development, particularly in mesoderm and cardiomyocyte differentiation processes. ERVH48-1 promotes the differentiation of hPSCs into cardiomyocytes by inhibiting the activity of SFRP2 and activating the Wnt/β-catenin signaling pathway. This research not only revealed the potential role of ERVs in somatic cell development but also expanded our understanding of the functional diversity of ERVs.

Research Highlights

1. ERV Function in Somatic Cell Development: Previous studies mainly focused on the impact of ERVs on extraembryonic development and the immune system, while this study is the first to reveal the critical regulatory role of ERVH48-1 in somatic cell development.

2. Interaction Between ERVH48-1 and SFRP2: The study found that ERVH48-1 regulates SFRP2 degradation, influencing the Wnt signaling pathway, providing new insights into how ERVs regulate cell fate through protein interactions.

3. Potential Application Prospects: The regulatory role of ERVH48-1 in cardiomyocyte differentiation offers potential targets for future regenerative medicine, potentially playing a role in stem cell therapy for heart diseases.

Summary

This study not only revealed the novel function of ERVH48-1 in cardiomyocyte development but also emphasized the potential role of ERVs in somatic cell development. ERVH48-1 regulates the cardiomyocyte differentiation process by controlling the degradation of SFRP2 and activating the Wnt/β-catenin signaling pathway. This finding provides new directions for the future application of ERVs in developmental biology, genomics, and regenerative medicine.