Ramming deterministic and highly efficient. Defining the dynamics of these mutually exclusive MBD family-NuRD complexes along with their regulatory target genes in hPSC should shed further light on the mechanisms of somatic cell reprogramming. Recent evidence suggests that the repressive activity of NuRD is opposed by signaling pathways that support expression of pluripotent factors, hence maintaining a stochastic ground state in which ESC self-renew but are transcriptionally poised for lineage-specific differentiation. We found that several serine residues on SFRS2 were preferentially phosphorylated in pluripotent cells. The question of which signaling axis ) mediates phosphorylation on SFRS2, and whether this activity represents a general mechanism to reinforce expression of pluripotent-specific gene isoforms in hPSC, is worthy of future study. Non-coding RNA has emerged as an important post-transcriptional regulatory pathway in pluripotent cells, with functional links established between specific micro- or long noncoding – RNAs and master regulatory transcription factors. The alternatively spliced MBD2 
isoforms harbor differences in both their 3UTR and protein-coding sequences. As a result, the somatic cell-specific MBD2a isoform is targeted by MLN1117 cost miR-302 family members. To our knowledge the 3-UTR of MBD2c is not subject to miR-mediated suppression, although we did observe modest regulation of SFRS2 by miR-301 family members in vitro. These results are consistent with the notion that the microRNA machinery may act synergistically with SB-366791 splicing factors and gene isoforms to either enforce a self-renewing ground state or rapidly translate lineage commitment signals into appropriate transcriptional programs. Further analysis will be required to determine the full extent of microRNA-mediated regulation of proteome diversity and whether lncRNA or other non-coding sequences are also involved. Collectively these data may allow a quantitative assessment of the network topology including the relative contribution of each node comprising a putative feedback loop linking the core pluripotent genes with the alternative splicing apparatus and specific gene isoforms. In summary, we delineate genetic, biochemical, and functional links consistent with a general model, in which the master regulators of pluripotency act in concert with splicing factors and the microRNA machinery to mediate protein diversity via alternative splicing, ultimately enforcing a pluripotent ground state. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Stem Cell. Author manuscript; available 
in PMC 2015 July 03. Lu et al. Page 8 Experimental Procedures Cell culture hPSCs for proteomics and phosphoproteomics were maintained in mTeSR media on 6-well plates pre-coated with matrigel as previously described. Chromatin Immunoprecipitation ChIP was performed in H1 ESC using anti-OCT4. Coimmunoprecipitation Protein CoIP was performed in 293T cells using anti-FLAG gel. Genome-wide expression and alternative splicing data Gene expression and alternative splicing profiling was performed using Affymetrix arrays. All array data have been deposited in the GEO database under the accession# GSE55673. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19846406 Manuscript Proteomic data Samples were processed for protein expression and phosphorylation analyses by 3D RP-SAX-RP LC-MS/MS. Native mass spectrometry data are available for download at: http://blaispath.Ramming deterministic and highly efficient. Defining the dynamics of these mutually exclusive MBD family-NuRD complexes along with their regulatory target genes in hPSC should shed further light on the mechanisms of somatic cell reprogramming. Recent evidence suggests that the repressive activity of NuRD is opposed by signaling pathways that support expression of pluripotent factors, hence maintaining a stochastic ground state in which ESC self-renew but are transcriptionally poised for lineage-specific differentiation. We found that several serine residues on SFRS2 were preferentially phosphorylated in pluripotent cells. The question of which signaling axis ) mediates phosphorylation on SFRS2, and whether this activity represents a general mechanism to reinforce expression of pluripotent-specific gene isoforms in hPSC, is worthy of future study. Non-coding RNA has emerged as an important post-transcriptional regulatory pathway in pluripotent cells, with functional links established between specific micro- or long noncoding – RNAs and master regulatory transcription factors. The alternatively spliced MBD2 isoforms harbor differences in both their 3UTR and protein-coding sequences. As a result, the somatic cell-specific MBD2a isoform is targeted by miR-302 family members. To our knowledge the 3-UTR of MBD2c is not subject to miR-mediated suppression, although we did observe modest regulation of SFRS2 by miR-301 family members in vitro. These results are consistent with the notion that the microRNA machinery may act synergistically with splicing factors and gene isoforms to either enforce a self-renewing ground state or rapidly translate lineage commitment signals into appropriate transcriptional programs. Further analysis will be required to determine the full extent of microRNA-mediated regulation of proteome diversity and whether lncRNA or other non-coding sequences are also involved. Collectively these data may allow a quantitative assessment of the network topology including the relative contribution of each node comprising a putative feedback loop linking the core pluripotent genes with the alternative splicing apparatus and specific gene isoforms. In summary, we delineate genetic, biochemical, and functional links consistent with a general model, in which the master regulators of pluripotency act in concert with splicing factors and the microRNA machinery to mediate protein diversity via alternative splicing, ultimately enforcing a pluripotent ground state. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Cell Stem Cell. Author manuscript; available in PMC 2015 July 03. Lu et al. Page 8 Experimental Procedures Cell culture hPSCs for proteomics and phosphoproteomics were maintained in mTeSR media on 6-well plates pre-coated with matrigel as previously described. Chromatin Immunoprecipitation ChIP was performed in H1 ESC using anti-OCT4. Coimmunoprecipitation Protein CoIP was performed in 293T cells using anti-FLAG gel. Genome-wide expression and alternative splicing data Gene expression and alternative splicing profiling was performed using Affymetrix arrays. All array data have been deposited in the GEO database under the accession# GSE55673. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19846406 Manuscript Proteomic data Samples were processed for protein expression and phosphorylation analyses by 3D RP-SAX-RP LC-MS/MS. Native mass spectrometry data are available for download at: http://blaispath.