Nicholas P. Mullin

An Oct4-Centered Protein Interaction Network in Embryonic Stem Cells

Summary Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-β, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.

Crystal structure of a complement control protein that regulates both pathways of complement activation and binds heparan sulfate proteoglycans

Vaccinia virus complement control protein (VCP) inhibits both pathways of complement activation through binding the third and fourth components. A homolog of mammalian regulators of complement activation, its ability to bind heparin endows VCP with additional activities of significance to viral infectivity. The structure of VCP reveals a highly extended molecule with a putative heparin recognition site at its C-terminal end. A second cluster of positive charges provides a possibly overlapping binding site for both heparin and complement components. Experiments suggested by the structure indicate that VCP can bind heparin and control complement simultaneously. This, the structure of any intact regulator of complement activation, along with attendant functional insights, will stimulate the design of new therapeutic inhibitors of complement.

Reduced Oct4 Expression Directs a Robust Pluripotent State with Distinct Signaling Activity and Increased Enhancer Occupancy by Oct4 and Nanog

Summary Embryonic stem cell (ESC) pluripotency is governed by a gene regulatory network centered on the transcription factors Oct4 and Nanog. To date, robust self-renewing ESC states have only been obtained through the chemical inhibition of signaling pathways or enforced transgene expression. Here, we show that ESCs with reduced Oct4 expression resulting from heterozygosity also exhibit a stabilized pluripotent state. Despite having reduced Oct4 expression, Oct4+/− ESCs show increased genome-wide binding of Oct4, particularly at pluripotency-associated enhancers, homogeneous expression of pluripotency transcription factors, enhanced self-renewal efficiency, and delayed differentiation kinetics. Cells also exhibit increased Wnt expression, enhanced leukemia inhibitory factor (LIF) sensitivity, and reduced responsiveness to fibroblast growth factor. Although they are able to maintain pluripotency in the absence of bone morphogenetic protein, removal of LIF destabilizes pluripotency. Our findings suggest that cells with a reduced Oct4 concentration range are maintained in a robust pluripotent state and that the wild-type Oct4 concentration range enables effective differentiation.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

ABSTRACT Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

A New C-Type Lectin Similar to the Human Immunoreceptor DC-SIGN Mediates Symbiont Acquisition by a Marine Nematode

ABSTRACT Although thiotrophic symbioses have been intensively studied for the last three decades, nothing is known about the molecular mechanisms of symbiont acquisition. We used the symbiosis between the marine nematode Laxus oneistus and sulfur-oxidizing bacteria to study this process. In this association a monolayer of symbionts covers the whole cuticle of the nematode, except its anterior-most region. Here, we identify a novel Ca2+-dependent mannose-specific lectin that was exclusively secreted onto the posterior, bacterium-associated region of L. oneistus cuticle. A recombinant form of this lectin induced symbiont aggregation in seawater and was able to compete with the native lectin for symbiont binding in vivo. Surprisingly, the carbohydrate recognition domain of this mannose-binding protein was similar both structurally and functionally to a human dendritic cell-specific immunoreceptor. Our results provide a molecular link between bacterial symbionts and host-secreted mucus in a marine symbiosis and suggest conservation in the mechanisms of host-microbe interactions throughout the animal kingdom.

A novel C-type lectin secreted by a tissue-dwelling parasitic nematode

Many parasitic nematodes live for surprisingly long periods in the tissues of their hosts, implying sophisticated mechanisms for evading the host immune system. The nematode Toxocara canis survives for years in mammalian tissues, and when cultivated in vitro, secretes antigens such as TES-32. From the peptide sequence, we cloned TES-32 cDNA, which encodes a 219 amino-acid protein that has a domain characteristic of host calcium-dependent (C-type) lectins, a family of proteins associated with immune defence. Homology modelling predicted that TES-32 bears remarkable structural similarity to mammalian immune-system lectins. Native TES-32 acted as a functional lectin in affinity chromatography. Unusually, it bound both mannose- and galactose-type monosaccharides, a pattern precluded in mammalian lectins by a constraining loop adjacent to the carbohydrate-binding site. In TES-32, this loop appeared to be less obtrusive, permitting a broader range of ligand binding. The similarity of TES-32 to host immune cell receptors suggests a hitherto unsuspected strategy for parasite immune evasion.

OCT4/SOX2-independent Nanog autorepression modulates heterogeneous Nanog gene expression in mouse ES cells.

NANOG, OCT4 and SOX2 form the core network of transcription factors supporting embryonic stem (ES) cell self‐renewal. While OCT4 and SOX2 expression is relatively uniform, ES cells fluctuate between states of high NANOG expression possessing high self‐renewal efficiency, and low NANOG expression exhibiting increased differentiation propensity. NANOG, OCT4 and SOX2 are currently considered to activate transcription of each of the three genes, an architecture that cannot readily account for NANOG heterogeneity. Here, we examine the architecture of the Nanog‐centred network using inducible NANOG gain‐ and loss‐of‐function approaches. Rather than activating itself, Nanog activity is autorepressive and OCT4/SOX2‐independent. Moreover, the influence of Nanog on Oct4 and Sox2 expression is minimal. Using Nanog:GFP reporters, we show that Nanog autorepression is a major regulator of Nanog transcription switching. We conclude that the architecture of the pluripotency gene regulatory network encodes the capacity to generate reversible states of Nanog transcription via a Nanog‐centred autorepressive loop. Therefore, cellular variability in self‐renewal efficiency is an emergent property of the pluripotency gene regulatory network.

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self‐renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self‐renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP‐Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple‐repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog‐Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self‐renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self‐renewal.

The pluripotency rheostat Nanog functions as a dimer

The defining activity of the homeodomain protein Nanog is the ability to confer cytokine-independent self-renewal upon ES (embryonic stem) cells in which it is overexpressed. However, the biochemical basis by which Nanog achieves this function remains unknown. In the present study, we show that Nanog dimerizes through a functionally critical domain. Co-immunoprecipitation of Nanog molecules tagged with distinct epitopes demonstrates that Nanog self-associates through a region in which every fifth residue is tryptophan. In vitro binding experiments establish that this region participates directly in self-association. Moreover, analytical ultracentrifugation indicates that, in solution, Nanog is in equilibrium between monomeric and dimeric forms with a K(d) of 3 muM. The functional importance of Nanog dimerization is established by ES cell colony-forming assays in which deletion of the tryptophan-repeat region eliminates the capacity of Nanog to direct LIF (leukaemia inhibitory factor)-independent self-renewal.

The X-inactivation trans-activator Rnf12 is negatively regulated by pluripotency factors in embryonic stem cells

X-inactivation, the molecular mechanism enabling dosage compensation in mammals, is tightly controlled during mouse early embryogenesis. In the morula, X-inactivation is imprinted with exclusive silencing of the paternally inherited X-chromosome. In contrast, in the post-implantation epiblast, X-inactivation affects randomly either the paternal or the maternal X-chromosome. The transition from imprinted to random X-inactivation takes place in the inner cell mass (ICM) of the blastocyst from which embryonic stem (ES) cells are derived. The trigger of X-inactivation, Xist, is specifically downregulated in the pluripotent cells of the ICM, thereby ensuring the reactivation of the inactive paternal X-chromosome and the transient presence of two active X-chromosomes. Moreover, Tsix, a critical cis-repressor of Xist, is upregulated in the ICM and in ES cells where it imposes a particular chromatin state at the Xist promoter that ensures the establishment of random X-inactivation upon differentiation. Recently, we have shown that key transcription factors supporting pluripotency directly repress Xist and activate Tsix and thus couple Xist/Tsix control to pluripotency. In this manuscript, we report that Rnf12, a third X-linked gene critical for the regulation of X-inactivation, is under the control of Nanog, Oct4 and Sox2, the three factors lying at the heart of the pluripotency network. We conclude that in mouse ES cells the pluripotency-associated machinery exerts an exhaustive control of X-inactivation by taking over the regulation of all three major regulators of X-inactivation: Xist, Tsix, and Rnf12.