A transit-amplifying progenitor with biphasic behavior contributes to epidermal renewal.

Transit-amplifying (TA) cells are progenitors that undergo an amplification phase followed by transition into an extinction phase. A long postulated epidermal TA progenitor with biphasic behavior has not yet been experimentally observed in vivo. Here, we identify such a TA population using clonal analysis of Aspm-CreER genetic cell-marking in mice, which uncovers contribution to both homeostasis and injury repair of adult skin. This TA population is more frequently dividing than a Dlx1-CreER-marked long-term self-renewing (e.g. stem cell) population. Newly developed generalized birth-death modeling of long-term lineage tracing data shows that both TA progenitors and stem cells display neutral competition, but only the stem cells display neutral drift. The quantitative evolution of a nascent TA cell and its direct descendants shows that TA progenitors indeed amplify the basal layer before transition and that the homeostatic TA population is mostly in extinction phase. This model will be broadly useful for analyzing progenitors whose behavior changes with their clone age. This work identifies a long-missing class of non-self-renewing biphasic epidermal TA progenitors and has broad implications for understanding tissue renewal mechanisms.

Binary organization of epidermal basal domains highlights robustness to environmental exposure.

Adulte interfollicular epidermis (IFE) renewal is likely orchestrated by physiological demands of its complex tissue architecture comprising spatial and cellular heterogeneity. Mouse tail and back skin display two kinds of basal IFE spatial domains that regenerate at different rates. Here, we elucidate the molecular and cellular states of basal IFE domains by marker expression and single-cell transcriptomics in mouse and human skin. We uncover two paths of basal cell differentiation that in part reflect the IFE spatial domain organization. We unravel previously unrecognized similarities between mouse tail IFE basal domains defined as scales and interscales versus human rete ridges and inter-ridges, respectively. Furthermore, our basal IFE transcriptomics and gene targeting in mice provide evidence supporting a physiological role of IFE domains in adaptation to differential UV exposure. We identify Sox6 as a novel UV-induced and interscale/inter-ridge preferred basal IFE-domain transcription factor, important for IFE proliferation and survival. The spatial, cellular, and molecular organization of IFE basal domains underscores skin adaptation to environmental exposure and its unusual robustness in adult homeostasis.

Polymorphism and electronic structure of polyimine and its potential significance for prebiotic chemistry on Titan.

The chemistry of hydrogen cyanide (HCN) is believed to be central to the origin of life question. Contradictions between Cassini-Huygens mission measurements of the atmosphere and the surface of Saturn's moon Titan suggest that HCN-based polymers may have formed on the surface from products of atmospheric chemistry. This makes Titan a valuable "natural laboratory" for exploring potential nonterrestrial forms of prebiotic chemistry. We have used theoretical calculations to investigate the chain conformations of polyimine (pI), a polymer identified as one major component of polymerized HCN in laboratory experiments. Thanks to its flexible backbone, the polymer can exist in several different polymorphs, which are relatively close in energy. The electronic and structural variability among them is extraordinary. The band gap changes over a 3-eV range when moving from a planar sheet-like structure to increasingly coiled conformations. The primary photon absorption is predicted to occur in a window of relative transparency in Titan's atmosphere, indicating that pI could be photochemically active and drive chemistry on the surface. The thermodynamics for adding and removing HCN from pI under Titan conditions suggests that such dynamics is plausible, provided that catalysis or photochemistry is available to sufficiently lower reaction barriers. We speculate that the directionality of pI's intermolecular and intramolecular =N-H(…)N hydrogen bonds may drive the formation of partially ordered structures, some of which may synergize with photon absorption and act catalytically. Future detailed studies on proposed mechanisms and the solubility and density of the polymers will aid in the design of future missions to Titan.

Acetonitrile cluster solvation in a cryogenic ethane-methane-propane liquid: Implications for Titan lake chemistry.

The atmosphere of Titan, Saturn's largest moon, exhibits interesting UV- and radiation-driven chemistry between nitrogen and methane, resulting in dipolar, nitrile-containing molecules. The assembly and subsequent solvation of such molecules in the alkane lakes and seas found on the moon's surface are of particular interest for investigating the possibility of prebiotic chemistry in Titan's hydrophobic seas. Here we characterize the solvation of acetonitrile, a product of Titan's atmospheric radiation chemistry tentatively detected on Titan's surface [H. B. Niemann et al., Nature 438, 779-784 (2005)], in an alkane mixture estimated to match a postulated composition of the smaller lakes during cycles of active drying and rewetting. Molecular dynamics simulations are employed to determine the potential of mean force of acetonitrile (CH3CN) clusters moving from the alkane vapor into the bulk liquid. We find that the clusters prefer the alkane liquid to the vapor and do not dissociate in the bulk liquid. This opens up the possibility that acetonitrile-based microscopic polar chemistry may be possible in the otherwise nonpolar Titan lakes.

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Runx1 and p21 synergistically limit the extent of hair follicle stem cell quiescence in vivo.

Mechanisms of tissue stem cell (SC) quiescence control are important for normal homeostasis and for preventing cancer. Cyclin-dependent kinase inhibitors (CDKis) are known inhibitors of cell cycle progression. We document CDKis expression in vivo during hair follicle stem cell (HFSC) homeostasis and find p21 (cyclin-dependent kinase inhibitor 1a, Cdkn1a), p57, and p15 up-regulated at quiescence onset. p21 appears important for HFSC timely onset of quiescence. Conversely, we find that Runx1 (runt related transcription factor 1), which is known for promoting HFSC proliferation, represses p21, p27, p57, and p15 transcription in HFSC in vivo. Intriguingly, in cell culture, tumors, and normal homeostasis, Runx1 and p21 interplay modulates proliferation in opposing directions under the different conditions. Unexpectedly, Runx1 and p21 synergistically limit the extent of HFSC quiescence in vivo, which antagonizes the role of p21 as a cell cycle inhibitor. Importantly, we find in cultured keratinocytes that Runx1 and p21 bind to the p15 promoter and synergistically repress p15 mRNA transcription, thereby restraining cell cycle arrest. This documents a surprising ability of a CDKi (p21) to act as a direct transcriptional repressor of another CDKi (p15). We unveil a robust in vivo mechanism that enforces quiescence of HFSCs, and a context-dependent role of a CDKi (p21) to limit quiescence of SCs, potentially by directly down-regulating mRNA levels of (an)other CDKi(s).

Activation of Src and transformation by an RPTPα splice mutant found in human tumours.

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection.

The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.

Multiplexed microcolumn-based process for efficient selection of RNA aptamers.

We describe a reusable microcolumn and process for the efficient discovery of nucleic acid aptamers for multiple target molecules. The design of our device requires only microliter volumes of affinity chromatography resin-a condition that maximizes the enrichment of target-binding sequences over non-target-binding (i.e., background) sequences. Furthermore, the modular design of the device accommodates a multiplex aptamer selection protocol. We optimized the selection process performance using microcolumns filled with green fluorescent protein (GFP)-immobilized resin and monitoring, over a wide range of experimental conditions, the enrichment of a known GFP-binding RNA aptamer (GFPapt) against a random RNA aptamer library. We validated the multiplex approach by monitoring the enrichment of GFPapt in de novo selection experiments with GFP and other protein preparations. After only three rounds of selection, the cumulative GFPapt enrichment on the GFP-loaded resin was greater than 10(8) with no enrichment for the other nonspecific targets. We used this optimized protocol to perform a multiplex selection to two human heat shock factor (hHSF) proteins, hHSF1 and hHSF2. High-throughput sequencing was used to identify aptamers for each protein that were preferentially enriched in just three selection rounds, which were confirmed and isolated after five rounds. Gel-shift and fluorescence polarization assays showed that each aptamer binds with high-affinity (KD < 20 nM) to the respective targets. The combination of our microcolumns with a multiplex approach and high-throughput sequencing enables the selection of aptamers to multiple targets in a high-throughput and efficient manner.

Extracellular domain dependence of PTPalpha transforming activity.

Two isoforms of the transmembrane protein tyrosine phosphatase PTPalpha, which differ by nine amino acids in their extracellular regions, are expressed in a tissue-specific manner. Over-expression of the shorter isoform transforms rodent cells, and it has previously been reasonable to assume that this was a direct consequence of its dephosphorylation and activation of Src. Transformation by the longer wild-type isoform has not previously been studied. We tested the activities of both isoforms in NIH3T3 cells and found that, while both dephosphorylated and activated Src similarly, only the shorter isoform induced focus formation or anchorage-independent growth. Differences in phosphorylation of PTPalpha at its known regulatory sites, Grb2 binding to PTPalpha, phosphorylation level of focal adhesion kinase by PTPalpha, or overall localization were excluded as possible explanations for the differences in transforming activities. The results suggest that transformation by PTPalpha involves at least one function other than, or in addition to, its activation of Src and that this depends on PTPalpha's extracellular domain. Previous studies have suggested that PTPalpha might be a useful target in breast and colon cancer therapy, and the results presented here suggest that it may be advantageous to develop isoform-specific therapeutic reagents.

Chromatin Modifiers Alter Recombination Between Divergent DNA Sequences.

Recombination between divergent DNA sequences is actively prevented by heteroduplex rejection mechanisms. In baker's yeast, such antirecombination mechanisms can be initiated by the recognition of DNA mismatches in heteroduplex DNA by MSH proteins, followed by recruitment of the Sgs1-Top3-Rmi1 helicase-topoisomerase complex to unwind the recombination intermediate. We previously showed that the repair/rejection decision during single-strand annealing recombination is temporally regulated by MSH (MutShomolog) protein levels and by factors that excise nonhomologous single-stranded tails. These observations, coupled with recent studies indicating that mismatch repair (MMR) factors interact with components of the histone chaperone machinery, encouraged us to explore roles for epigenetic factors and chromatin conformation in regulating the decision to reject vs. repair recombination between divergent DNA substrates. This work involved the use of an inverted repeat recombination assay thought to measure sister chromatid repair during DNA replication. Our observations are consistent with the histone chaperones CAF-1 and Rtt106, and the histone deacetylase Sir2, acting to suppress heteroduplex rejection and the Rpd3, Hst3, and Hst4 deacetylases acting to promote heteroduplex rejection. These observations, and double-mutant analysis, have led to a model in which nucleosomes located at DNA lesions stabilize recombination intermediates and compete with MMR factors that mediate heteroduplex rejection.

Apoptosis of estrogen-receptor negative breast cancer and colon cancer cell lines by PTP alpha and src RNAi.

We show that siRNA-mediated suppression of protein tyrosine phosphatase alpha (PTP alpha) reduces Src activity 2 to 4-fold in breast, colon and other human cancer cell lines. Src and PTP alpha RNAi induced apoptosis in estrogen receptor (ER)-negative breast cancer and colon cancer cells, but not in immortalized noncancerous breast cells, ER-positive breast cancer cells or other cancer cell types tested. RNAi of other Src family members (Fyn and Yes) or of PTP1B, a phosphatase previously suggested to be an activator of Src in breast cancer, had no effect. Although further tests with primary tumor tissues are required, the unexpected correlation between ER status and Src/PTP alpha dependence in breast cancer cell lines may be important for planning therapeutic strategies, and the insensitivity of normal breast cells to the RNAi highlights the potential of PTP alpha, which may be easier to target than Src, as a therapeutic target in ER-negative breast cancer.

Unfair competition governs the interaction of pCPI-17 with myosin phosphatase (PP1-MYPT1).

The small phosphoprotein pCPI-17 inhibits myosin light-chain phosphatase (MLCP). Current models postulate that during muscle relaxation, phosphatases other than MLCP dephosphorylate and inactivate pCPI-17 to restore MLCP activity. We show here that such hypotheses are insufficient to account for the observed rapidity of pCPI-17 inactivation in mammalian smooth muscles. Instead, MLCP itself is the critical enzyme for pCPI-17 dephosphorylation. We call the mutual sequestration mechanism through which pCPI-17 and MLCP interact inhibition by unfair competition: MLCP protects pCPI-17 from other phosphatases, while pCPI-17 blocks other substrates from MLCP's active site. MLCP dephosphorylates pCPI-17 at a slow rate that is, nonetheless, both sufficient and necessary to explain the speed of pCPI-17 dephosphorylation and the consequent MLCP activation during muscle relaxation.

Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.

The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1(CreER) and Slc1a3(CreER) genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other's territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

Physical and functional interaction between the transcriptional cofactor CBP and the KH domain protein Sam68.

CBP is a multifunctional transcriptional cofactor with tumor suppressor activity. The CH3 domain of CBP binds numerous transcription factors and several viral oncoproteins. We identified the Src substrate and RNA-binding protein Sam68 as novel CH3-binding protein. Sam68 binds the CH3 domain in part through a conserved FXD/EXXXL motif that is shared among several CH3-binding proteins, including the adenoviral oncoprotein E1A and the tumor suppressor p53. Sam68 and CBP interact in vivo and colocalize in nuclear sub-domains. Sam68 has potent transcriptional repression activity that is independent of its RNA binding activity, which suggests that RNA processing and regulation of gene expression by Sam68 are separable functions. Consistent with this, CBP did not stimulate the ability of Sam68 to promote Rev response element-containing mRNA export. Interestingly, Sam68 can regulate RNA processing in the absence of a Rev response element, suggesting that Sam68 functions through a novel RNA element. Together, these findings reveal a previously unidentified function for Sam68 as a transcriptional repressor and suggest that Sam68 might link cellular signaling pathways with components of the transcriptional machinery.

Sam68 exerts separable effects on cell cycle progression and apoptosis.

BACKGROUND: The RNA-binding protein Sam68 has been implicated in a number of cellular processes, including transcription, RNA splicing and export, translation, signal transduction, cell cycle progression and replication of the human immunodeficiency virus and poliovirus. However, the precise impact it has on essential cellular functions remains largely obscure. RESULTS: In this report we show that conditional overexpression of Sam68 in fibroblasts results in both cell cycle arrest and apoptosis. Arrest in G1 phase of the cell cycle is associated with decreased levels of cyclins D1 and E RNA and protein, resulting in dramatically reduced Rb phosphorylation. Interestingly, cell cycle arrest does not require the specific RNA binding ability of Sam68. In marked contrast, induction of apoptosis by Sam68 absolutely requires a fully-functional RNA binding domain. Moreover, the anti-cancer agent trichostatin A potentiates Sam68-driven apoptosis. CONCLUSIONS: For the first time we have shown that Sam68, an RNA binding protein with multiple apparent functions, exerts functionally separable effects on cell proliferation and survival, dependent on its ability to bind specifically to RNA. These findings shed new light on the ability of signal transducing RNA binding proteins to influence essential cell function. Moreover, the ability of a class of anti-cancer therapeutics to modulate its ability to promote apoptosis suggests that Sam68 status may impact some cancer treatments.

Macrostate data clustering.

We develop an effective nonhierarchical data clustering method using an analogy to the dynamic coarse graining of a stochastic system. Analyzing the eigensystem of an interitem transition matrix identifies fuzzy clusters corresponding to the metastable macroscopic states (macrostates) of a diffusive system. A "minimum uncertainty criterion" determines the linear transformation from eigenvectors to cluster-defining window functions. Eigenspectrum gap and cluster certainty conditions identify the proper number of clusters. The physically motivated fuzzy representation and associated uncertainty analysis distinguishes macrostate clustering from spectral partitioning methods. Macrostate data clustering solves a variety of test cases that challenge other methods.

Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers.

During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55. DOI: http://dx.doi.org/10.7554/eLife.01695.001.

Discovering aptamers by cell-SELEX against human soluble growth factors ectopically expressed on yeast cell surface.

SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (K(D)  = ∼ 1 nM) and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens.

RAPID-SELEX for RNA aptamers.

Aptamers are high-affinity ligands selected from DNA or RNA libraries via SELEX, a repetitive in vitro process of sequential selection and amplification steps. RNA SELEX is more complicated than DNA SELEX because of the additional transcription and reverse transcription steps. Here, we report a new selection scheme, RAPID-SELEX (RNA Aptamer Isolation via Dual-cycles SELEX), that simplifies this process by systematically skipping unnecessary amplification steps. Using affinity microcolumns, we were able to complete a multiplex selection for protein targets, CHK2 and UBLCP1, in a third of the time required for analogous selections using a conventional SELEX approach. High-throughput sequencing of the enriched pools from both RAPID and SELEX revealed many identical candidate aptamers from the starting pool of 5 × 10(15) sequences. For CHK2, the same sequence was preferentially enriched in both selections as the top candidate and was found to bind to its respective target. These results demonstrate the efficiency and, most importantly, the robustness of our selection scheme. RAPID provides a generalized approach that can be used with any selection technology to accelerate the rate of aptamer discovery, without compromising selection performance.