Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular...

Area of Science:

• Molecular Biology
• Developmental Biology
• Cell Biology

Background:

• Post-translational modifications, such as phosphorylation, rapidly diversify protein function.
• OCT4 (Octamer-binding transcription factor 4) is crucial for embryonic stem cell (ESC) pluripotency and preimplantation development.
• Its activity is regulated by post-transcriptional modifications, with several phosphorylation sites previously identified.

Purpose of the Study:

• To precisely determine the function of serine 12 phosphorylation on the OCT4 transcription factor in ESCs.
• To identify the specific kinase responsible for OCT4 phosphorylation at serine 12.
• To investigate the role of this modification in maintaining pluripotency and self-renewal.

Main Methods:

• Utilized CRISPR/Cas9 genome engineering to create isogenic mouse ESCs (mESCs) with serine 12 ablated.
• Employed chemical inhibitors to probe kinase activity.
• Developed and used a specific antibody for OCT4 phosphorylated at serine 12.
• Combined molecular and cellular biology techniques.

Main Results:

• Identified cyclin-dependent kinase (CDK) 7 as the upstream kinase phosphorylating OCT4 at serine 12.
• Generation of mESCs lacking endogenous serine 12 phosphorylation on OCT4 showed no discernible effect on pluripotency or self-renewal.
• These findings suggest potential functional compensation by other phosphorylation events.

Conclusions:

• While CDK7 phosphorylates OCT4 at serine 12, this specific modification is not essential for maintaining pluripotency and self-renewal in ESCs.
• Precise genome engineering of specific amino acid modifications, like OCT4 serine 12, is a valuable approach to elucidate the endogenous function of post-translational modifications.
• The study highlights the complex regulatory network governing OCT4 function in stem cells.