Abstract
Neuronal differentiation is an intricate and a complex process which involves crosstalk among various signaling pathways, growth factors, transcription factors, and epigenetic modifiers. During different stages of neuronal development, there are various histone modifiers which drive the expression of lineage-specific genes. Polycomb group proteins are one of the histone modifiers that control transcriptional repression of specific genes in development, differentiation, and functionality of various tissues. Chromatin immunoprecipitation (ChIP) is a widely used technique to investigate the interaction of proteins and DNA; ChIP combined with quantitative real-time PCR (qPCR) gives a quantitative data about the occupancy of specific protein on a particular stretch of DNA, and this can help us investigate how a protein regulates expression of a specific gene. In this chapter, we describe a protocol for ChIP coupled to qPCR during early neuronal differentiation to identify the specific genomic targets regulated by components of Polycomb repressive complex 1.
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Abbreviations
- BMI1/PCGF4:
-
B Lymphoma Mo-MLV insertion region 1 homolog, Polycomb ring finger protein 4
- ChIP:
-
Chromatin immunoprecipitation
- H2AK119ub1:
-
Monoubiquitination of lysine 119 on histone H2A
- hESC:
-
Human embryonic stem cells
- hiPSC:
-
Human-induced pluripotent stem cells
- NPC:
-
Neuronal progenitor cells
- PcG:
-
Polycomb group protein(s)
- PRC1:
-
Polycomb repressive complex 1
- RING1B:
-
Really interesting new gene 1B/RING-type E3 ubiquitin transferase RING1
References
Dang L, Tropepe V (2006) Neural induction and neural stem cell development. Regen Med 1:635–652. https://doi.org/10.2217/17460751.1.5.635
Kriegstein A, Alvarez-Buylla A (2009) The glial nature of embryonic and adult neural stem cells. Annu Rev Neurosci 32:149–184. https://doi.org/10.1146/annurev.neuro.051508.135600
Kondo T, Isono K, Kondo K et al (2014) Polycomb potentiates Meis2 activation in midbrain by mediating interaction of the promoter with a tissue-specific enhancer. Dev Cell 28:94–101. https://doi.org/10.1016/j.devcel.2013.11.021
Luis NM, Morey L, Di Croce L, Benitah SA (2012) Polycomb in stem cells: PRC1 branches out. Cell Stem Cell 11:16–21. https://doi.org/10.1016/j.stem.2012.06.005
Yan Y, Chen H, Costa M (2009) Chromatin immunoprecipitation assays. Methods Mol Biol:567. https://doi.org/10.1007/978-1-60327-414-2
Haring M, Offermann S, Danker T et al (2007) Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods 3:1–16. https://doi.org/10.1186/1746-4811-3-11
Bertani S, Kan A, Sauer F (2008) Chromatin immunoprecipitation from human embryonic stem cells. J Vis Exp:1–6. https://doi.org/10.3791/780
Acknowledgments
This work is supported by DST-SERB, India, and NMIMS (deemed-to-be) University.
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Desai, D., Pethe, P. (2021). ChIP-qPCR for Polycomb Group Proteins During Neuronal Differentiation of Human Pluripotent Stem Cells. In: Turksen, K. (eds) Embryonic Stem Cell Protocols . Methods in Molecular Biology, vol 2520. Humana, New York, NY. https://doi.org/10.1007/7651_2021_400
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DOI: https://doi.org/10.1007/7651_2021_400
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2436-4
Online ISBN: 978-1-0716-2437-1
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