title: Retinoblastoma-binding proteins 4 and 9 are important for human pluripotent stem cell maintenance.
abstract: 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.
  
  
  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.
authors.affiliation: Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, Canada. m.oconnor@uws.edu.au
authors.forename: Michael D
authors.initials: MD
authors.lastname: O'Connor
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Wederell
authors.forename: Gordon
authors.initials: G
authors.lastname: Robertson
authors.forename: Allen
authors.initials: A
authors.lastname: Delaney
authors.forename: Olena
authors.initials: O
authors.lastname: Morozova
authors.forename: Steven S S
authors.initials: SS
authors.lastname: Poon
authors.forename: Damian
authors.initials: D
authors.lastname: Yap
authors.forename: John
authors.initials: J
authors.lastname: Fee
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Zhao
authors.forename: Helen
authors.initials: H
authors.lastname: McDonald
authors.forename: Thomas
authors.initials: T
authors.lastname: Zeng
authors.forename: Martin
authors.initials: M
authors.lastname: Hirst
authors.forename: Marco A
authors.initials: MA
authors.lastname: Marra
authors.forename: Samuel A J R
authors.initials: SA
authors.lastname: Aparicio
authors.forename: Connie J
authors.initials: CJ
authors.lastname: Eaves
mesh_terms: D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011485:Protein Binding; D034622:RNA Interference; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D056505:Retinoblastoma-Binding Protein 4 / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; C082637:RBBP4 protein, human; C485185:RBBP9 protein, human; D016160:Retinoblastoma Protein; D056505:Retinoblastoma-Binding Protein 4