Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations.

The intestinal microbiota undergoes diurnal compositional and functional oscillations that affect metabolic homeostasis, but the mechanisms by which the rhythmic microbiota influences host circadian activity remain elusive. Using integrated multi-omics and imaging approaches, we demonstrate that the gut microbiota features oscillating biogeographical localization and metabolome patterns that determine the rhythmic exposure of the intestinal epithelium to different bacterial species and their metabolites over the course of a day. This diurnal microbial behavior drives, in turn, the global programming of the host circadian transcriptional, epigenetic, and metabolite oscillations. Surprisingly, disruption of homeostatic microbiome rhythmicity not only abrogates normal chromatin and transcriptional oscillations of the host, but also incites genome-wide de novo oscillations in both intestine and liver, thereby impacting diurnal fluctuations of host physiology and disease susceptibility. As such, the rhythmic biogeography and metabolome of the intestinal microbiota regulates the temporal organization and functional outcome of host transcriptional and epigenetic programs.

Deterministic direct reprogramming of somatic cells to pluripotency.

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Inquiring into the differential action of interferons (IFNs): an IFN-alpha2 mutant with enhanced affinity to IFNAR1 is functionally similar to IFN-beta.

Alpha and beta interferons (IFN-alpha and IFN-beta) are multifunctional cytokines that exhibit differential activities through a common receptor composed of the subunits IFNAR1 and IFNAR2. Here we combined biophysical and functional studies to explore the mechanism that allows the alpha and beta IFNs to act differentially. For this purpose, we have engineered an IFN-alpha2 triple mutant termed the HEQ mutant that mimics the biological properties of IFN-beta. Compared to wild-type (wt) IFN-alpha2, the HEQ mutant confers a 30-fold higher binding affinity towards IFNAR1, comparable to that measured for IFN-beta, resulting in a much higher stability of the ternary complex as measured on model membranes. The HEQ mutant, like IFN-beta, promotes a differentially higher antiproliferative effect than antiviral activity. Both bring on a down-regulation of the IFNAR2 receptor upon induction, confirming an increased ternary complex stability of the plasma membrane. Oligonucleotide microarray experiments showed similar gene transcription profiles induced by the HEQ mutant and IFN-beta and higher levels of gene induction or repression than those for wt IFN-alpha2. Thus, we show that the differential activities of IFN-beta are directly related to the binding affinity for IFNAR1. Conservation of the residues mutated in the HEQ mutant within IFN-alpha subtypes suggests that IFN-alpha has evolved to bind IFNAR1 weakly, apparently to sustain differential levels of biological activities compared to those induced by IFN-beta.

Upregulation of a small subset of genes drives type I interferon-induced antiviral memory.

Interferons (IFNs) stand in the frontline of defense against viral infections. In this study, we aimed at characterizing the gene expression profile specific to the antiviral effect out of the hundreds of genes involved also in other IFN activities. We found that the IFN-induced antiviral state is maintained for a prolonged time even after IFN occlusion. This was achieved through the active expression of a small set of <40 genes long after IFN was occluded, from which two groups are distinguished: one includes genes participating in direct inhibition of viral replication, such as Mx and OAS; the second group is related to antigen presentation, including all genes involved in the proteasome-to-immunoproteasome switch and class I MHC genes. Transcription of these genes continued after IFN removal and was Stat1 independent, suggesting the involvement of other signaling elements in addition to the canonical signal transduction pathway. Not less important were genes whose upregulation, in cases by many fold, is terminated once IFN is removed. Among these are viral sensing genes, such as retinoic acid-inducible gene-I protein (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and toll-like receptor (TLR), cytokines, and apoptotic-related genes. Our findings provide a systemwide depiction of prolonged intracellular antiviral protection without the need for ongoing IFN stimulation.

Variations in the unstructured C-terminal tail of interferons contribute to differential receptor binding and biological activity.

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.

Single-cell transcriptome conservation in cryopreserved cells and tissues.

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.

Mutational analysis of the IFNAR1 binding site on IFNalpha2 reveals the architecture of a weak ligand-receptor binding-site.

Type I interferons activate cellular responses by forming a ternary complex with two receptor components, IFNAR1 and IFNAR2. While the binding of the IFNAR2 receptor to interferon is of high affinity and well characterized, the binding to IFNAR1 is weak, transient, and poorly understood. Here, we mapped the complete binding region of IFNAR1 on IFNalpha2 by creating a panel of 21 single alanine mutant proteins, and determined their binding affinities. The IFNAR1 binding site on IFNalpha2 maps to the center of the B and C helices, opposite to the binding site for IFNAR2. No hot spots for binding were found in the interface, with individual mutations having an up to fivefold effect on binding. Of the nine residues that affected binding, three adjacent conserved residues, located on the B helix, conferred an increase in the binding affinity to IFNAR1, as well as an increase in the biological activity of the interferon mutant. This suggests that binding of alpha interferons to the IFNAR1 receptor is sub-optimal. A correlation between binding affinity and biological activity was found, albeit not across the whole range of affinities. In WISH cells, but not DAUDI cells, the anti-proliferative activity was markedly affected by fluctuations in the IFNalpha2 affinity towards the IFNAR1 receptor. On the other hand, the antiviral activity of interferons on WISH cells seems to change in accordance to the binding affinity towards IFNAR1 only as long as the binding affinity is not beyond twofold of the wild-type. In accordance, the biological roles of the two interferon-receptor subunits are discussed.

The stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities.

Type I interferons (IFNs) signal for their diverse biological effects by binding a common receptor on target cells, composed of the two transmembrane IFNAR1 and IFNAR2 proteins. We have previously differentially enhanced the antiproliferative activity of IFN by increasing the weak binding affinity of IFN to IFNAR1. In this study, we further explored the affinity interdependencies between the two receptor subunits and the role of IFNAR1 in differential IFN activity. For this purpose, we generated a panel of mutations targeting the IFNAR2 binding site on the background of the IFNalpha2 YNS mutant, which increases the affinity to IFNAR1 by 60-fold, resulting in IFNAR2-to-IFNAR1 binding affinity ratios ranging from 1000:1 to 1:1000. Both the antiproliferative and antiviral potencies of the interferon mutants clearly correlated to the in situ binding IC(50) values, independently of the relative contributions of the individual receptors, thus relating to the integral lifetime of the complex. However, the antiproliferative potency correlated throughout the entire range of affinities, as well as with prolonged IFNAR1 receptor down-regulation, whereas the antiviral potency reached a maximum at binding affinities equivalent to that of wild-type IFNalpha2. Our data suggest that (i) the specific activity of interferon is related to the ternary complex binding affinity and not to affinity toward individual receptor components and (ii) although the antiviral pathway is strongly dependent on pSTAT1 activity, the cytostatic effect requires additional mechanisms that may involve IFNAR1 down-regulation. This differential interferon response is ultimately mediated through distinct gene expression profiling.

An interferon alpha2 mutant optimized by phage display for IFNAR1 binding confers specifically enhanced antitumor activities.

All alpha-interferons (IFNalpha) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFNalpha2. Here, we constructed a phage display library by randomizing three positions on IFNalpha2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFNalpha2, and 3-fold tighter compared with IFNbeta. Binding of YNS to IFNAR2 was comparable with wild-type IFNalpha2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFNalpha2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFNalpha2 showed visible tumors. Histological analysis of these tumors showed a significant anti-angiogenic effect of YNS, compared with wild-type IFNalpha2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency.