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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Published on: October 28, 2011
PRC1 catalytic unit RING1B regulates early neural differentiation of human pluripotent stem cells
Divya Desai1, Aparna Khanna2, Prasad Pethe3
1Department of Biological Sciences, NMIMS Sunandan Divatia School of Science, SVKM's NMIMS (deemed to-be University), Mumbai, 56, India.
Insights
Polycomb Repressive Complex 1 (PRC1) component RING1B regulates gene expression during human neural differentiation. It prevents premature neuronal gene activation, being removed to allow differentiation.
Area of Science:
- Developmental Biology
- Epigenetics
- Stem Cell Biology
Background:
- Polycomb group (PcG) proteins, including Polycomb repressive complex (PRC) complexes, are key histone modifiers controlling gene expression.
- RING1B, a core catalytic subunit of PRC1, mediates H2AK119 monoubiquitination, a mark associated with gene repression.
- The precise role of PRC1 in early human neural specification remains largely uncharacterized.
Purpose of the Study:
- To investigate the function of PRC1 in neuronal differentiation using human pluripotent stem cells.
- To elucidate the dynamic role of RING1B and H2AK119ub1 during the transition from pluripotent stem cells to neural progenitors and mature neurons.
Main Methods:
- Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) were differentiated into neural progenitor cells.
- Expression levels of key neural markers (NESTIN, TUJ1, NCAD, PAX6) and PRC1 components (RING1B, BMI1) were analyzed.
- Chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) was employed to assess the genomic localization of RING1B and H2AK119ub1 at specific gene promoters.
Main Results:
- RING1B and BMI1 expression significantly increased during neural progenitor differentiation.
- RING1B and H2AK119ub1 were detected at the promoters of early neuronal genes (TUJ1, NCAM, NESTIN) during initial differentiation stages.
- RING1B occupancy decreased at these early neuronal gene promoters as differentiation progressed, coinciding with their activation.
- RING1B remained significantly associated with promoters of mature neuronal genes (IRX3, GSX2, SOX1, NEUROD1, FOXG1) in neural progenitors.
Conclusions:
- PRC1 component RING1B occupies neuronal gene promoters in human pluripotent stem cells, potentially inhibiting precocious gene expression.
- Upon receiving neuronal inductive signals, RING1B is removed from neuronal gene promoters, accompanied by the loss of the repressive H2AK119ub1 modification.
- These findings highlight a crucial regulatory role for PRC1 in controlling the timing of gene activation during human neurodevelopment.
Background:
Polycomb group (PcG) proteins are histone modifiers which control gene expression by assembling into large repressive complexes termed - Polycomb repressive complex (PRC); RING1B, core catalytic subunit of PRC1 that performs H2AK119 monoubiquitination leading to gene repression. The role of PRC1 complex during early neural specification in humans is unclear; we have tried to uncover the role of PRC1 in neuronal differentiation using human pluripotent stem cells as an in vitro model.
Results:
We differentiated both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) towards neural progenitor stage evident from the expression of NESTIN, TUJ1, NCAD, and PAX6. When we checked the total expression of RING1B and BMI1, we saw that they were significantly upregulated in differentiated neural progenitors compared to undifferentiated cells. Further, we used Chromatin Immunoprecipitation coupled with qPCR to determine the localization of RING1B, and the repressive histone modification H2AK119ub1 at the promoters of neuronal specific genes. We observed that RING1B localized to and catalyzed H2AK119ub1 modification at promoters of TUJ1, NCAM, and NESTIN during early differentiation and later RING1B was lost from its promoter leading their expression; while functional RING1B persisted significantly on mature neuronal genes such as IRX3, GSX2, SOX1, NEUROD1 and FOXG1 in neural progenitors.
Conclusion:
The results of our study show that PRC1 catalytic component RING1B occupies neuronal gene promoters in human pluripotent stem cells and may prevent their precocious expression. However, when neuronal inductive signals are given, RING1B is not only removed from neuronal gene promoters, but the inhibitory H2AK119ub1 modification is also lost.
