Abstract
Retinoblastoma binding protein 9 (RBBP9) is required for maintaining the expression of both pluripotency and cell cycle genes in human pluripotent stem cells (hPSCs). An siRNA-based study from our group showed it does so by influencing cell cycle progression through the RB/E2F pathway. In non-pluripotent cells, RBBP9 is also known to have serine hydrolase (SH) activity, acting on currently undefined target proteins. The role of RBBP9 SH activity in hPSCs, and during normal development, is currently unknown. To begin assessing whether RBBP9 SH activity might contribute to hPSC maintenance, hPSCs were treated with ML114—a selective chemical inhibitor of RBBP9 SH activity. Stem cells treated with ML114 showed significantly reduced population growth rate, colony size and progression through the cell cycle, with no observable change in cell morphology or decrease in pluripotency antigen expression—suggesting no initiation of hPSC differentiation. Consistent with this, hPSCs treated with ML114 retained the capacity for tri-lineage differentiation, as seen through teratoma formation. Subsequent microarray and Western blot analyses of ML114-treated hPSCs suggest the nuclear transcription factor Y subunit A (NFYA) may be a candidate effector of RBBP9 SH activity in hPSCs. These data support a role for RBBP9 in regulating hPSC proliferation independent of differentiation, whereby inhibition of RBBP9 SH activity de-couples decreased hPSC proliferation from initiation of differentiation.
Highlights
The retinoblastoma (RB) binding protein 9 (RBBP9—previously termed Bog [1,2] and RBBP10 [3]) is expressed in human pluripotent stem cells and in a range of human cancer cells [1,4,5,6]
The data presented here show ML114 treatment decreases human pluripotent stem cells (hPSCs) proliferation. These findings are consistent with published work that shows inactivation of Retinoblastoma binding protein 9 (RBBP9) serine hydrolase (SH) activity via mutation of the active site serine decreases proliferation
As modulating proliferation is an important step in generating induced pluripotent stem cells [47], modulating RBBP9 SH activity during reprogramming strategies might improve the efficiency of induced pluripotent stem cell production
Summary
The retinoblastoma (RB) binding protein 9 (RBBP9—previously termed Bog [1,2] and RBBP10 [3]) is expressed in human pluripotent stem cells (hPSCs) and in a range of human cancer cells [1,4,5,6]. Comparison of protein sequences identified a conserved serine residue hypothesized to be the putative nucleophilic serine (Ser75) within a GXSXG motif common to other SHs [4,7,8,9]. This finding is supported by X-ray crystallography that suggests RBBP9 is a member of the ‘domain of unknown function’ superfamily (DUF1234) of serine proteases [7]. While a number of these studies made use of commercially available SH probes, the lack of specificity of these probes makes them poorly suited to assessing RBBP9 SH activity in a cellular context where other SH enzymes are expressed (such as hPSCs)
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