Sridhar Rao

A protein interaction network for pluripotency of embryonic stem cells

Embryonic stem (ES) cells are pluripotent and of therapeutic potential in regenerative medicine. Understanding pluripotency at the molecular level should illuminate fundamental properties of stem cells and the process of cellular reprogramming. Through cell fusion the embryonic cell phenotype can be imposed on somatic cells, a process promoted by the homeodomain protein Nanog, which is central to the maintenance of ES cell pluripotency. Nanog is thought to function in concert with other factors such as Oct4 (ref. 8) and Sox2 (ref. 9) to establish ES cell identity. Here we explore the protein network in which Nanog operates in mouse ES cells. Using affinity purification of Nanog under native conditions followed by mass spectrometry, we have identified physically associated proteins. In an iterative fashion we also identified partners of several Nanog-associated proteins (including Oct4), validated the functional relevance of selected newly identified components and constructed a protein interaction network. The network is highly enriched for nuclear factors that are individually critical for maintenance of the ES cell state and co-regulated on differentiation. The network is linked to multiple co-repressor pathways and is composed of numerous proteins whose encoding genes are putative direct transcriptional targets of its members. This tight protein network seems to function as a cellular module dedicated to pluripotency.

Differential Roles of Sall4 Isoforms in Embryonic Stem Cell Pluripotency

ABSTRACT Murine embryonic stem (ES) cells are defined by continuous self-renewal and pluripotency. A diverse repertoire of protein isoforms arising from alternative splicing is expressed in ES cells without defined biological roles. Sall4, a transcription factor essential for pluripotency, exists as two isoforms (Sall4a and Sall4b). Both isoforms can form homodimers and a heterodimer with each other, and each can interact with Nanog. By genomewide location analysis, we determined that Sall4a and Sall4b have overlapping, but not identical binding sites within the ES cell genome. In addition, Sall4b, but not Sall4a, binds preferentially to highly expressed loci in ES cells. Sall4a and Sall4b binding sites are distinguished by both epigenetic marks at target loci and their clustering with binding sites of other pluripotency factors. When ES cells expressing a single isoform of Sall4 are generated, Sall4b alone could maintain the pluripotent state, although it could not completely suppress all differentiation markers. Sall4a and Sall4b collaborate in maintenance of the pluripotent state but play distinct roles. Our work is novel in establishing such isoform-specific differences in ES cells.

PU.1 and Spi-B Are Required for Normal B Cell Receptor–Mediated Signal Transduction

PU.1 and Spi-B have previously been implicated in the regulation of genes encoding B cell receptor (BCR) signaling components. Spi-B-/- B lymphocytes respond poorly to BCR stimulation; PU.1-/- mice, however, lack B cells, precluding an analysis of BCR responses. We now show that PU.1+/- Spi-B-/- B cells exhibit more extensive defects than Spi-B-/- B cells, indicating that both PU.1 and Spi-B are required for normal BCR signaling. Strikingly, BCR cross-linking results in substantially reduced protein tyrosine phosphorylation in mutant B cells. Further analysis shows that Igalpha is phosphorylated and syk is recruited and becomes phosphorylated but that BLNK and PLCgamma phosphorylation are defective in mutant cells. Our data support the existence of a novel component coupling syk to downstream targets.

A technique for porcine hepatocyte harvest and description of differentiated metabolic functions in static culture.

Current bioartificial liver devices are based on the use of a large mass of hepatocytes exhibiting differentiated metabolic function. The pig has become a source of interest for the acquisition of such cells-however, harvesting a large mass of highly viable cells has met with difficulty. This study describes a technique for harvesting large quantities of hepatocytes at viabilities greater than 90% and also describes several features documenting differentiated function. Pigs, 6 to 10 kg body weight, underwent in situ two-step whole liver perfusion (ethylene glycol tetraacetic acid and collagenase) and ex vivo cell harvest. Harvests yielded an average of 19.5 billion cells with an average viability of 94.6%. Hepatocytes were then entrapped in type I collagen (3 x 10(5) cells/well) and cultured in serum-free media for 5 days. Pig hepatocytes produced stable amounts of albumin and maintained cytochrome P-450 and glucuronidation activity over 5 days, as shown by the metabolism of lidocaine and 4-methylumbelliferone. These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiated function over at least 5 days of culture, and therefore should prove to be an excellent source of hepatocytes for bioartificial liver devices.

PU.1/Spi-B Regulation of c-rel Is Essential for Mature B Cell Survival

PU.1(+/-)Spi-B(-/-) mice exhibit reduced numbers of immature and mature B lymphocytes, which exhibit severe defects in response to BCR-mediated stimulation and poor survival. We found that expression of c-rel, a member of the Rel/NF-kappa B family, is dramatically reduced in PU.1(+/-)Spi-B(-/-) splenic B cells. Analysis of the murine c-rel promoter identified three PU.1/Spi-B binding sites critical for c-rel promoter activity. Furthermore, reintroduction of Rel protein restored wild-type B cell numbers to mice reconstituted with PU.1(+/-)Spi-B(-/-) bone marrow. These findings are the first to demonstrate that a member of the Rel/NF-kappa B family is directly regulated by Ets proteins and dissect the molecular basis for the function of two Ets factors, PU.1 and Spi-B, in promoting B lymphocyte survival.

A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets

Summary Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

SPI-B Activates Transcription via a Unique Proline, Serine, and Threonine Domain and Exhibits DNA Binding Affinity Differences from PU.1

SPI-B is a B lymphocyte-specific Ets transcription factor that shares a high degree of similarity with PU.1/SPI-1. In direct contrast to PU.1 −/−mice that die in utero and lack monocytes, neutrophils, B cells, and T cells, Spi-B −/− mice are viable and exhibit a severe B cell proliferation defect. Since PU.1 is expressed at wild type levels in Spi-B −/− B cells, the mutant mice provide genetic evidence that SPI-B and PU.1 have at least some non-redundant roles in B lymphocytes. To begin to understand the molecular basis for these defects, we delineated functional domains of SPI-B for comparison to those of PU.1. By using a heterologous co-transfection system, we identified two independent transactivation domains in the N terminus of SPI-B. Interestingly, only one of these domains (amino acids 31–61), a proline/serine/threonine-rich region, unique among Ets proteins, is necessary for transactivation of the immunoglobulin λ light chain enhancer. This transactivation motif is in marked contrast to PU.1, which contains acidic and glutamine-rich domains. In addition, we describe a functional PU.1 site within the c-FES promoter which SPI-B fails to bind efficiently and transactivate. Finally, we show that SPI-B interacts with the PU.1 cofactors Pip, TBP, c-Jun and with lower affinity to nuclear factor interleukin-6β and retinoblastoma. Taken together, these data suggest that SPI-B binds DNA with a different affinity for certain sites than PU.1 and harbors different transactivation domains. We conclude that SPI-B may activate unique target genes in B lymphocytes and interact with unique, although currently unidentified, cofactors.

Spi-B can functionally replace PU.1 in myeloid but not lymphoid development

Mature macrophages, neutrophils and lymphoid cells do not develop in PU.1−/− mice. In contrast, mice lacking the highly related protein Spi‐B generate all hematopoietic lineages but display a B‐cell receptor signaling defect. These distinct phenotypes could result from functional differences between PU.1 and Spi‐B or their unique temporal and tissue‐specific expression (PU.1: myeloid and B cells; Spi‐B: B cells only). To address this question, we introduced the Spi‐B cDNA into the murine PU.1 locus by homologous recombination. In the absence of PU.1, Spi‐B rescued macrophage and granulocyte development when assayed by in vitro differentiation of embryonic stem cells. Adherent, CD11b+/F4/80+ cells capable of phagocytosis were detected in PU.1Spi‐B/Spi‐B embryoid bodies, and myeloid colonies were present in hematopoietic progenitor assays. Despite its ability to rescue myeloid differentiation, Spi‐B did not rescue lymphoid development in a RAG‐2−/− complementation assay. These results demonstrate an important difference between PU.1 and Spi‐B. Careful comparison of these Ets factors will delineate important functional domains of PU.1 involved in lymphocyte lineage commitment and/or maturation.

Unraveling the transcriptional network controlling ES cell pluripotency

Embryonic stem cells (ES cells) are powerful tools for genetic engineering and hold significant potential for regenerative medicine. Recent work provides new insights into ES cell pluripotency and delineates separate transcriptional pathways in ES cells for maintenance of the undifferentiated state and for self-renewal.

THE ETS FACTORS PU.1 AND SPI-B REGULATE THE TRANSCRIPTION IN VIVO OF P2Y10, A LYMPHOID RESTRICTED HEPTAHELICAL RECEPTOR

To investigate the in vivo functions of PU.1 and Spi-B, two highly related Ets transcription factors, we previously generatedPU.1 +/+ Spi-B −/− andPU.1 +/− Spi-B −/− mice and demonstrated a significant decrease in B-cell receptor (BCR) signaling in mutants. Major components of BCR signaling appear to be expressed at normal levels in these mice, implying that PU.1 and Spi-B cooperate in the transcription of additional target genes important for antigen receptor signaling. We used subtractive hybridization to identify novel in vivo PU.1/Spi-B target genes and determined that the expression of a heptahelical receptor, P2Y10, is dramatically reduced inPU.1 +/− Spi-B −/−B-cells. Further analysis shows that P2Y10 expression is restricted to lymphoid cells and parallels that of Spi-B in B-lymphocytes. Lastly, the P2Y10 promoter contains a PU.1/Spi-B binding site functionally required for efficient transcription in B-cells. Thus,P2Y10 is likely to be a direct in vivotranscriptional target for PU.1 and Spi-B and provides a unique model to explore transcriptional regulation by this Ets factor subfamily. Furthermore, P2Y10 suggests an intriguing connection between heterotrimeric G-proteins and BCR signaling.