Gene Resistance to Transcriptional Reprogramming following Nuclear Transfer Is Directly Mediated by Multiple Chromatin-Repressive Pathways.

Understanding the mechanism of resistance of genes to reactivation will help improve the success of nuclear reprogramming. Using mouse embryonic fibroblast nuclei with normal or reduced DNA methylation in combination with chromatin modifiers able to erase H3K9me3, H3K27me3, and H2AK119ub1 from transplanted nuclei, we reveal the basis for resistance of genes to transcriptional reprogramming by oocyte factors. A majority of genes is affected by more than one type of treatment, suggesting that resistance can require repression through multiple epigenetic mechanisms. We classify resistant genes according to their sensitivity to 11 chromatin modifier combinations, revealing the existence of synergistic as well as adverse effects of chromatin modifiers on removal of resistance. We further demonstrate that the chromatin modifier USP21 reduces resistance through its H2AK119 deubiquitylation activity. Finally, we provide evidence that H2A ubiquitylation also contributes to resistance to transcriptional reprogramming in mouse nuclear transfer embryos.

Transcription factor SOX15 regulates stem cell pluripotency and promotes neural fate during differentiation by activating the neurogenic gene Hes5.

SOX2 and SOX15 are Sox family transcription factors enriched in embryonic stem cells (ESCs). The role of SOX2 in activating gene expression programs essential for stem cell self-renewal and acquisition of pluripotency during somatic cell reprogramming is well-documented. However, the contribution of SOX15 to these processes is unclear and often presumed redundant with SOX2 largely because overexpression of SOX15 can partially restore self-renewal in SOX2-deficient ESCs. Here, we show that SOX15 contributes to stem cell maintenance by cooperating with ESC-enriched transcriptional coactivators to ensure optimal expression of pluripotency-associated genes. We demonstrate that SOX15 depletion compromises reprogramming of fibroblasts to pluripotency which cannot be compensated by SOX2. Ectopic expression of SOX15 promotes the reversion of a postimplantation, epiblast stem cell state back to a preimplantation, ESC-like identity even though SOX2 is expressed in both cell states. We also uncover a role of SOX15 in lineage specification, by showing that loss of SOX15 leads to defects in commitment of ESCs to neural fates. SOX15 promotes neural differentiation by binding to and activating a previously uncharacterized distal enhancer of a key neurogenic regulator, Hes5. Together, these findings identify a multifaceted role of SOX15 in induction and maintenance of pluripotency and neural differentiation.

Long-term association of a transcription factor with its chromatin binding site can stabilize gene expression and cell fate commitment.

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.

Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine: IMPLICATIONS FOR THE PHARMACOLOGY OF ADENOSINE ANTIMETABOLITES.

Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mm deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (kcat) and Km values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors.

9-(2'-Deoxy-2'-Fluoro-ß-d-Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance.

Current chemotherapy against African sleeping sickness, a disease caused by the protozoan parasite Trypanosoma brucei, is limited by toxicity, inefficacy, and drug resistance. Nucleoside analogues have been successfully used to cure T. brucei-infected mice, but they have the limitation of mainly being taken up by the P2 nucleoside transporter, which, when mutated, is a common cause of multidrug resistance in T. brucei We report here that adenine arabinoside (Ara-A) and the newly tested drug 9-(2'-deoxy-2'-fluoro-ß-d-arabinofuranosyl) adenine (FANA-A) are instead taken up by the P1 nucleoside transporter, which is not associated with drug resistance. Like Ara-A, FANA-A was found to be resistant to cleavage by methylthioadenosine phosphorylase, an enzyme that protects T. brucei against the antitrypanosomal effects of deoxyadenosine. Another important factor behind the selectivity of nucleoside analogues is how well they are phosphorylated within the cell. We found that the T. brucei adenosine kinase had a higher catalytic efficiency with FANA-A than the mammalian enzyme, and T. brucei cells treated with FANA-A accumulated high levels of FANA-A triphosphate, which even surpassed the level of ATP and led to cell cycle arrest, inhibition of DNA synthesis, and the accumulation of DNA breaks. FANA-A inhibited nucleic acid biosynthesis and parasite proliferation with 50% effective concentrations (EC50s) in the low nanomolar range, whereas mammalian cell proliferation was inhibited in the micromolar range. Both Ara-A and FANA-A, in combination with deoxycoformycin, cured T. brucei-infected mice, but FANA-A did so at a dose 100 times lower than that of Ara-A.

From the covalent linkage of drugs to novel inhibitors of ribonucleotide reductase: synthesis and biological evaluation of valproic esters of 3'-C-methyladenosine.

We synthesized a series of serum-stable covalently linked drugs derived from 3'-C-methyladenosine (3'-Me-Ado) and valproic acid (VPA), which are ribonucleotide reductase (RR) and histone deacetylase (HDAC) inhibitors, respectively. While the combination of free VPA and 3'-Me-Ado resulted in a clear synergistic apoptotic effect, the conjugates had lost their HDAC inhibitory effect as well as the corresponding apoptotic activity. Two of the analogs, 2',5'-bis-O-valproyl-3'-C-methyladenosine (A160) and 5'-O-valproyl-3'-C-methyladenosine (A167), showed promising cytotoxic activities against human hematological and solid cancer cell lines. A167 was less potent than A160 but had interesting features as an RR inhibitor. It inhibited RR activity by competing with ATP as an allosteric effector and concomitantly reduced the intracellular deoxyribonucleoside triphosphate (dNTP) pools. A167 represents a novel lead compound, which in contrast to previously used RR nucleoside analogs does not require intracellular kinases for its activity and therefore holds promise against drug resistant tumors with downregulated nucleoside kinases.

Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding.

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k(cat)) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.

Low complexity domains, condensates, and stem cell pluripotency.

Biological reactions require self-assembly of factors in the complex cellular milieu. Recent evidence indicates that intrinsically disordered, low-complexity sequence domains (LCDs) found in regulatory factors mediate diverse cellular processes from gene expression to DNA repair to signal transduction, by enriching specific biomolecules in membraneless compartments or hubs that may undergo liquid-liquid phase separation (LLPS). In this review, we discuss how embryonic stem cells take advantage of LCD-driven interactions to promote cell-specific transcription, DNA damage response, and DNA repair. We propose that LCD-mediated interactions play key roles in stem cell maintenance and safeguarding genome integrity.

ATP-binding cassette protein ABCF1 couples transcription and genome surveillance in embryonic stem cells through low-complexity domain.

OCT4 and SOX2 confer pluripotency by recruiting coactivators to activate stem cell­specific transcription. However, the composition of coactivator complexes and their roles in maintaining stem cell fidelity remain unclear. Here, we report the ATP-binding cassette subfamily F member 1 (ABCF1) as a coactivator for OCT4/SOX2 critical for stem cell self-renewal. The intrinsically disordered low-complexity domain (LCD) of ABCF1 contributes to phase separation in vitro and transcriptional activation of pluripotency genes by mediating multivalent interactions with SOX2 and co-dependent coactivators XPC and DKC1. These LCD-driven transcription factor­coactivator interactions critical for pluripotency gene expression are disrupted by DNA damage, likely due to LCD-dependent binding of ABCF1 to damage-generated intracellular DNA fragments instead of SOX2. This study identifies a transcriptional coactivator that uses its LCD to form selective multivalent interactions to regulate stem cell self-renewal and exit from pluripotency when genome integrity is compromised.

Multipotency of mouse trophoblast stem cells.

BACKGROUND: In a number of disease processes, the body is unable to repair injured tissue, promoting the need to develop strategies for tissue repair and regeneration, including the use of cellular therapeutics. Trophoblast stem cells (TSCs) are considered putative stem cells as they differentiate into other subtypes of trophoblast cells. To identify cells for future therapeutic strategies, we investigated whether TSCs have properties of stem/progenitor cells including self-renewal and the capacity to differentiate into parenchymal cells of fetal organs, in vitro and in vivo. METHODS: TSCs were isolated using anti-CD117 micro-beads, from embryonic day 18.5 placentas. In vitro, CD117+ TSCs were cultured, at a limiting dilution in growth medium for the development of multicellular clones and in specialized medium for differentiation into lung epithelial cells, cardiomyocytes, and retinal photoreceptor cells. CD117+ TSCs were also injected in utero into lung, heart, and the sub-retinal space of embryonic day 13.5 fetuses, and the organs were harvested for histological assessment after a natural delivery. RESULTS: We first identified CD117+ cells within the labyrinth zone and chorionic basal plate of murine placentas in late pregnancy, embryonic day 18.5. CD117+ TSCs formed multicellular clones that remained positive for CD117 in vitro, consistent with self-renewal properties. The clonal cells demonstrated multipotency, capable of differentiating into lung epithelial cells (endoderm), cardiomyocytes (mesoderm), and retinal photoreceptor cells (ectoderm). Finally, injection of CD117+ TSCs in utero into lungs, hearts, and the sub-retinal spaces of fetuses resulted in their engraftment on day 1 after birth, and the CD117+ TSCs differentiated into lung alveolar epithelial cells, heart cardiomyocytes, and retina photoreceptor cells, corresponding with the organs in which they were injected. CONCLUSIONS: Our findings demonstrate that CD117+ TSCs have the properties of stem cells including clonogenicity, self-renewal, and multipotency. In utero administration of CD117+ TSCs engraft and differentiate into resident cells of the lung, heart, and retina during mouse development.

Oligomerization status directs overall activity regulation of the Escherichia coli class Ia ribonucleotide reductase.

Ribonucleotide reductase (RNR) is a key enzyme for the synthesis of the four DNA building blocks. Class Ia RNRs contain two subunits, denoted R1 (alpha) and R2 (beta). These enzymes are regulated via two nucleotide-binding allosteric sites on the R1 subunit, termed the specificity and overall activity sites. The specificity site binds ATP, dATP, dTTP, or dGTP and determines the substrate to be reduced, whereas the overall activity site binds dATP (inhibitor) or ATP. By using gas-phase electrophoretic mobility macromolecule analysis and enzyme assays, we found that the Escherichia coli class Ia RNR formed an inhibited alpha(4)beta(4) complex in the presence of dATP and an active alpha(2)beta(2) complex in the presence of ATP (main substrate: CDP), dTTP (substrate: GDP) or dGTP (substrate: ADP). The R1-R2 interaction was 30-50 times stronger in the alpha(4)beta(4) complex than in the alpha(2)beta(2) complex, which was in equilibrium with free alpha(2) and beta(2) subunits. Studies of a known E. coli R1 mutant (H59A) showed that deficient dATP inhibition correlated with reduced ability to form alpha(4)beta(4) complexes. ATP could also induce the formation of a generally inhibited alpha(4)beta(4) complex in the E. coli RNR but only when used in combination with high concentrations of the specificity site effectors, dTTP/dGTP. Both allosteric sites are therefore important for alpha(4)beta(4) formation and overall activity regulation. The E. coli RNR differs from the mammalian enzyme, which is stimulated by ATP also in combination with dGTP/dTTP and forms active and inactive alpha(6)beta(2) complexes.

Adenosine kinase mediates high affinity adenosine salvage in Trypanosoma brucei.

African sleeping sickness is caused by Trypanosoma brucei. This extracellular parasite lacks de novo purine biosynthesis, and it is therefore dependent on exogenous purines such as adenosine that is taken up from the blood and other body fluids by high affinity transporters. The general belief is that adenosine needs to be cleaved to adenine inside the parasites in order to be used for purine nucleotide synthesis. We have found that T. brucei also can salvage this nucleoside by adenosine kinase (AK), which has a higher affinity to adenosine than the cleavage-dependent pathway. The recombinant T. brucei AK (TbAK) preferably used ATP or GTP to phosphorylate both natural and synthetic nucleosides in the following order of catalytic efficiencies: adenosine > cordycepin > deoxyadenosine > adenine arabinoside (Ara-A) > inosine > fludarabine (F-Ara-A). TbAK differed from the AK of the related intracellular parasite Leishmania donovani by having a high affinity to adenosine (K m = 0.04-0.08 microm depending on [phosphate]) and by being negatively regulated by adenosine (K i = 8-14 microm). These properties make the enzyme functionally related to the mammalian AKs, although a phylogenetic analysis grouped it together with the L. donovani enzyme. The combination of a high affinity AK and efficient adenosine transporters yields a strong salvage system in T. brucei, a potential Achilles' heel making the parasites more sensitive than mammalian cells to adenosine analogs such as Ara-A. Studies of wild-type and AK knockdown trypanosomes showed that Ara-A inhibited parasite proliferation and survival in an AK-dependent manner by affecting nucleotide levels and by inhibiting nucleic acid biosynthesis.

Enzymatically active mammalian ribonucleotide reductase exists primarily as an alpha6beta2 octamer.

Ribonucleotide reductase synthesizes deoxyribonucleotides, which are essential building blocks for DNA synthesis. The mammalian ribonucleotide reductase is described as an alpha(2)beta(2) complex consisting of R1 (alpha) and R2 (beta) proteins. ATP stimulates and dATP inhibits enzyme activity by binding to an allosteric site called the activity site on the R1 protein. Despite the opposite effects by ATP and dATP on enzyme activity, both nucleotides induce formation of R1 oligomers. By using a new technique termed Gas-phase Electrophoretic-Mobility Macromolecule Analysis (GEMMA), we have found that the ATP/dATP-induced R1 oligomers have a defined size (hexamers) and can interact with the R2 dimer to form an enzymatically active protein complex (alpha(6)beta(2)). The newly discovered alpha(6)beta(2) complex can either be in an active or an inhibited state depending on whether ATP or dATP is bound. Our results suggest that this protein complex is the major form of ribonucleotide reductase at physiological levels of R1-R2 protein and nucleotides.