Elucidation of epigenetic action mechanisms of Strip2 involved in differentiation and tumorigenic potential of pluripotent stem cells

• Applicant: Professor Dr. Agapios Sachinidis, Ph.D.
• Subject Area: Anatomy and Physiology
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 450480255

Recently, we demonstrated that Strip2 (Fam40b) plays an important role in the differentiation of murine embryonic stem cells (ESCs). Constitutive knockdown (KD) of Strip2 in ESCs (Strip2 KD ESCs) led to an inhibition of differentiation process in particular towards mesodermal cells and their derivatives. We could show that Strip2 is located in the cytosol but also in the nucleus and nuclear bodies of the ESCs. Based on transcriptome and proteome findings of undifferentiated and differentiated Strip2 KD ESCs versus Strip2 ESCs (control ESCs) we proposed that Strip2 is a key regulator for the initiation of the differentiation of ESCs. Our findings showed protein-protein interactions of Strip2 with the co-repressor proteins of the NuRD/Trim28/HDACs/SETDB1 histone methyltransferase complex. The complex is activated by binding of Krueppel-associated box (KRAB) zinc finger proteins (KRAB Zfps) to specific DNA motifs thereby causing methylation of H3 to Lysin-9 residues (H3K9). Notably, our Chip-seq data showed that Strip2 binds to a similar sequence motif (20 base pairs) like the KRAB Zfps). Finally, we demonstrated that differentiated Strip2 KD ESCs (4-day and 16-day KD embryoid bodies, EBs) share tumorigenic gene/protein and histone methylation (mainly H3K9) and acetylation signatures. Histone methylation and acetylation is mediated by the DNA methyltransferases (DNMTs) and by histone acetyl transferases (HATs). We found several DNMTs and HATs to be upregulated in differentiated Strip2 KD ESCs (4-day and16-day KD EBs) and in addition HAT and Dnmt activity was significantly higher than in control 4-day and 16-day Strip2 EBs. The tumorigenic potential of the cells from the 16-day Strip2 KD EBs was very high in contrast to 16-day control EBs which were non-tumorigenic. We propose that Strip2 is an epigenetic modifier of the NuRD/Trim28/HDACs/SETDB1 histone methyltransferase complex which specifically tri-methylates Lys-9 in H3 histones thereby generating H3K9me3. We propose that Strip2 prevents methylation of H3 thereby promoting differentiation and developmental processes. We will use small molecule inhibitors of the DNMT and HAT to test whether inhibition will improve the differentiation potential of the Strip2 deficient ESCs and 16-day EBs and will diminish/attenuate their tumorigenicity. Applying “gain” and “loss” of function methodologies as well as CRISPR technologies we will prove the role of Strip2 for the differentiation processes also for human induced pluripotent stem cells (hiPSCs). The project will contribute to the elucidation of novel fundamental epigenetic mechanisms which are involved in regulation of differentiation processes and the tumorigenicity of pluripotent stem cells (ESCs and hiPSCs). The project will also contribute to the development of novel therapeutic concepts that target to increase the degree of differentiation of tumor cells through epigenetically active drugs and thereby to reduce the malignancy of tumor cells.

• DFG Programme: Research Grants