Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance

Objective The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. Materials and Methods We used bioinform...

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Bibliographic Details
Published in:Experimental hematology 2011-08, Vol.39 (8), p.866-879.e1
Main Authors: O’Connor, Michael D, Wederell, Elizabeth, Robertson, Gordon, Delaney, Allen, Morozova, Olena, Poon, Steven S.S, Yap, Damian, Fee, John, Zhao, Yongjun, McDonald, Helen, Zeng, Thomas, Hirst, Martin, Marra, Marco A, Aparicio, Samuel A.J.R, Eaves, Connie J
Format: Article
Language:English
Subjects:
Advanced Basic Science
Blotting, Western
Cell Cycle Proteins - genetics
Cell Cycle Proteins - metabolism
Cell Differentiation - genetics
Cell Line, Tumor
Cells, Cultured
Gene Expression Profiling
Gene Library
Hematology, Oncology and Palliative Medicine
Humans
Immunoprecipitation
Intracellular Signaling Peptides and Proteins - genetics
Intracellular Signaling Peptides and Proteins - metabolism
Neoplasm Proteins - genetics
Neoplasm Proteins - metabolism
Oligonucleotide Array Sequence Analysis
Pluripotent Stem Cells - metabolism
Protein Binding
Retinoblastoma Protein - genetics
Retinoblastoma Protein - metabolism
Retinoblastoma-Binding Protein 4 - genetics
Retinoblastoma-Binding Protein 4 - metabolism
Reverse Transcriptase Polymerase Chain Reaction
RNA Interference
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Summary:Objective The molecular mechanisms that maintain human pluripotent stem (PS) cells are not completely understood. Here we sought to identify new candidate PS cell regulators to facilitate future improvements in their generation, expansion, and differentiation. Materials and Methods We used bioinformatic analyses of multiple serial-analysis-of-gene-expression libraries (generated from human PS cells and their differentiated derivatives), together with small interfering RNA (siRNA) screening to identify candidate pluripotency regulators. Validation of candidate regulators involved promoter analyses, Affymetrix profiling, real-time PCR, and immunoprecipitation. Results Promoter analysis of genes differentially expressed across multiple serial-analysis-of-gene-expression libraries identified E2F motifs in the promoters of many PS cell-specific genes (e.g., POU5F1 , NANOG , SOX2 , FOXD3 ). siRNA analyses identified two retinoblastoma binding proteins (RBBP4, RBBP9) as required for maintenance of multiple human PS cell types. Both RBBPs were bound to RB in human PS cells, and E2F motifs were present in the promoters of genes whose expression was altered by decreasing RBBP4 and RBBP9 expression. Affymetrix and real-time PCR studies of siRNA-treated human PS cells showed that reduced RBBP4 or RBBP9 expression concomitantly decreased expression of POU5F1 , NANOG , SOX2 , and/or FOXD3 plus certain cell cycle genes (e.g., CCNA2 , CCNB1 ), while increasing expression of genes involved in organogenesis (particularly neurogenesis). Conclusions These results reveal new candidate positive regulators of human PS cells, providing evidence of their ability to regulate expression of pluripotency, cell cycle, and differentiation genes in human PS cells. These data provide valuable new leads for further elucidating mechanisms of human pluripotency.
ISSN:0301-472X
1873-2399
DOI:10.1016/j.exphem.2011.05.008