Autoreactivity to self-antigens LL37 and ADAMTSL5 influences the clinical response to risankizumab in psoriatic patients.

The autoantigens LL37 and ADAMTSL5 contribute to induce pathogenetic T-cells responses in a subset of psoriatic patients. Whether the presence of LL37-and/or ADAMTS5-reactive T-cells influences the clinical response to treatment is still unknown. The aim of the study is to evaluate the clinical responses to the anti-IL-23 risankizumab in LL37 and/or ADAMTSL5-reactive patients in comparison with non-reactive ones and to assess whether genetics (HLA-Cw06.02) or BMI influences the response to treatment. Patients were screened at baseline for the presence of circulating LL37 or/and ADAMTSL5-reactive T-cells and were treated as per protocol with risankizumab. Effectiveness data (PASI scores) were collected at weeks 4, 16, 28, 40 and 52. Data were also analyzed based on HLA-Cw06.02 status and BMI. The overall response to treatment of patients with autoreactivity to LL37 or ADAMTSL5 did not differ compared to the non-reactive cohort as measured as PASI75/90/100 at different time points; however, subjects that had autoreactive T-cells to both LL37 and ADAMTS5 demonstrated suboptimal response to treatment starting at week16. HLA-Cw06:02+ patients demonstrated faster response to risankizumab at week 4 compared to HLA-Cw06:02-. Additionally, the response to treatment was influenced by the BMI with slower responses seen in overweight and obese patients at week 4 and week16. In conclusion, while the presence of either LL37-and ADAMTS5-reactive circulating T-cells do not influence the clinical response to risankizumab, the presence of the double reactivity to both LL37 and ADAMTS5 decreases the clinical responses. Moreover, we evidenced that HLA-Cw06+ respond faster to IL-23 inhibition and that BMI, associated to autoreactivity, can influence the speed in response.

Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.

Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations.

In multicellular organisms, transcription regulation is one of the central mechanisms modelling lineage differentiation and cell-fate determination. Transcription requires dynamic chromatin configurations between promoters and their corresponding distal regulatory elements. It is believed that their communication occurs within large discrete foci of aggregated RNA polymerases termed transcription factories in three-dimensional nuclear space. However, the dynamic nature of chromatin connectivity has not been characterized at the genome-wide level. Here, through a chromatin interaction analysis with paired-end tagging approach using an antibody that primarily recognizes the pre-initiation complexes of RNA polymerase II, we explore the transcriptional interactomes of three mouse cells of progressive lineage commitment, including pluripotent embryonic stem cells, neural stem cells and neurosphere stem/progenitor cells. Our global chromatin connectivity maps reveal approximately 40,000 long-range interactions, suggest precise enhancer-promoter associations and delineate cell-type-specific chromatin structures. Analysis of the complex regulatory repertoire shows that there are extensive colocalizations among promoters and distal-acting enhancers. Most of the enhancers associate with promoters located beyond their nearest active genes, indicating that the linear juxtaposition is not the only guiding principle driving enhancer target selection. Although promoter-enhancer interactions exhibit high cell-type specificity, promoters involved in interactions are found to be generally common and mostly active among different cells. Chromatin connectivity networks reveal that the pivotal genes of reprogramming functions are transcribed within physical proximity to each other in embryonic stem cells, linking chromatin architecture to coordinated gene expression. Our study sets the stage for the full-scale dissection of spatial and temporal genome structures and their roles in orchestrating development.

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.

Sox2 is required for embryonic development of the ventral telencephalon through the activation of the ventral determinants Nkx2.1 and Shh.

The Sox2 transcription factor is active in stem/progenitor cells throughout the developing vertebrate central nervous system. However, its conditional deletion at E12.5 in mouse causes few brain developmental problems, with the exception of the postnatal loss of the hippocampal radial glia stem cells and the dentate gyrus. We deleted Sox2 at E9.5 in the telencephalon, using a Bf1-Cre transgene. We observed embryonic brain defects that were particularly severe in the ventral, as opposed to the dorsal, telencephalon. Important tissue loss, including the medial ganglionic eminence (MGE), was detected at E12.5, causing the subsequent impairment of MGE-derived neurons. The defect was preceded by loss of expression of the essential ventral determinants Nkx2.1 and Shh, and accompanied by ventral spread of dorsal markers. This phenotype is reminiscent of that of mice mutant for the transcription factor Nkx2.1 or for the Shh receptor Smo. Nkx2.1 is known to mediate the initial activation of ventral telencephalic Shh expression. A partial rescue of the normal phenotype at E14.5 was obtained by administration of a Shh agonist. Experiments in Medaka fish indicate that expression of Nkx2.1 is regulated by Sox2 in this species also. We propose that Sox2 contributes to Nkx2.1 expression in early mouse development, thus participating in the region-specific activation of Shh, thereby mediating ventral telencephalic patterning induction.

Sox2 and Mitf cross-regulatory interactions consolidate progenitor and melanocyte lineages in the cranial neural crest.

The cellular origin and molecular mechanisms regulating pigmentation of head and neck are largely unknown. Melanocyte specification is controlled by the transcriptional activity of Mitf, but no general logic has emerged to explain how Mitf and progenitor transcriptional activities consolidate melanocyte and progenitor cell fates. We show that cranial melanocytes arise from at least two different cellular sources: initially from nerve-associated Schwann cell precursors (SCPs) and later from a cellular source that is independent of nerves. Unlike the midbrain-hindbrain cluster from which melanoblasts arise independently of nerves, a large center of melanocytes in and around cranial nerves IX-X is derived from SCPs, as shown by genetic cell-lineage tracing and analysis of ErbB3-null mutant mice. Conditional gain- and loss-of-function experiments show genetically that cell fates in the neural crest involve both the SRY transcription factor Sox2 and Mitf, which consolidate an SCP progenitor or melanocyte fate by cross-regulatory interactions. A gradual downregulation of Sox2 in progenitors during development permits the differentiation of both neural crest- and SCP-derived progenitors into melanocytes, and an initial small pool of nerve-associated melanoblasts expands in number and disperses under the control of endothelin receptor B (Ednrb) and Wnt5a signaling.

Essential role of Sox2 for the establishment and maintenance of the germ cell line.

Sox2 is a pluripotency-conferring gene expressed in primordial germ cells (PGCs) and postnatal oocytes, but the role it plays during germ cell development and early embryogenesis is unknown. Since Sox2 ablation causes early embryonic lethality shortly after blastocyst implantation, we generated mice bearing Sox2-conditional deletion in germ cells at different stages of their development through the Cre/loxP recombination system. Embryos lacking Sox2 in PGCs show a dramatic decrease of germ cell numbers at the time of their specification. At later stages, we found that Sox2 is strictly required for PGC proliferation. On the contrary, Sox2 deletion in meiotic oocytes does not impair postnatal oogenesis and early embryogenesis, indicating that it is not essential for oocyte maturation or for zygotic development. We also show that Sox2 regulates Kit expression in PGCs and binds to discrete transcriptional regulatory sequences of this gene, which is known to be important for PGCs survival and proliferation. Sox2 also stimulates the expression of Zfp148, which is required for normal development of fetal germ cells, and Rif1, a potential regulator of PGC pluripotency.

Sox2 and Pax6 maintain the proliferative and developmental potential of gliogenic neural stem cells In vitro.

Radial-glia-like neural stem (NS) cells may be derived from neural tissues or via differentiation of pluripotent embryonic stem (ES) cells. However, the mechanisms controlling NS cell propagation and differentiation are not yet fully understood. Here we investigated the roles of Sox2 and Pax6, transcription factors widely expressed in central nervous system (CNS) progenitors, in mouse NS cells. Conditional deletion of either Sox2 or Pax6 in forebrain-derived NS cells reduced their clonogenicity in a gene dosage-dependent manner. Cells heterozygous for either gene displayed moderate proliferative defects, which may relate to human pathologies attributed to SOX2 or PAX6 deficiencies. In the complete absence of Sox2, cells exited the cell cycle with concomitant downregulation of neural progenitor markers Nestin and Blbp. This occurred despite expression of the close relative Sox3. Ablation of Pax6 also caused major proliferative defects. However, a subpopulation of cells was able to expand continuously without Pax6. These Pax6-null cells retained progenitor markers but had altered morphology. They exhibited compromised differentiation into astrocytes and oligodendrocytes, highlighting that the role of Pax6 extends beyond neurogenic competence. Overall these findings indicate that Sox2 and Pax6 are both critical for self-renewal of differentiation-competent radial glia-like NS cells.

Mapping the Global Chromatin Connectivity Network for Sox2 Function in Neural Stem Cell Maintenance.

The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks. Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.

Neural progenitor cells orchestrate microglia migration and positioning into the developing cortex.

Microglia are observed in the early developing forebrain and contribute to the regulation of neurogenesis through still unravelled mechanisms. In the developing cerebral cortex, microglia cluster in the ventricular/subventricular zone (VZ/SVZ), a region containing Cxcl12-expressing basal progenitors (BPs). Here we show that the ablation of BP as well as genetic loss of Cxcl12 affect microglia recruitment into the SVZ. Ectopic Cxcl12 expression or pharmacological blockage of CxcR4 further supports that Cxcl12/CxcR4 signalling is involved in microglial recruitment during cortical development. Furthermore, we found that cell death in the developing forebrain triggers microglial proliferation and that this is mediated by the release of macrophage migration inhibitory factor (MIF). Finally, we show that the depletion of microglia in mice lacking receptor for colony-stimulating factor-1 (Csf-1R) reduces BPs into the cerebral cortex.

Sox2 is required to maintain cancer stem cells in a mouse model of high-grade oligodendroglioma.

The stem cell-determining transcription factor Sox2 is required for the maintenance of normal neural stem cells. In this study, we investigated the requirement for Sox2 in neural cancer stem-like cells using a conditional genetic deletion mutant in a mouse model of platelet-derived growth factor-induced malignant oligodendroglioma. Transplanting wild-type oligodendroglioma cells into the brain generated lethal tumors, but mice transplanted with Sox2-deleted cells remained free of tumors. Loss of the tumor-initiating ability of Sox2-deleted cells was reversed by lentiviral-mediated expression of Sox2. In cell culture, Sox2-deleted tumor cells were highly sensitive to differentiation stimuli, displaying impaired proliferation, increased cell death, and aberrant differentiation. Gene expression analysis revealed an early transcriptional response to Sox2 loss. The observed requirement of oligodendroglioma stem cells for Sox2 suggested its relevance as a target for therapy. In support of this possibility, an immunotherapeutic approach based on immunization of mice with SOX2 peptides delayed tumor development and prolonged survival. Taken together, our results showed that Sox2 is essential for tumor initiation by mouse oligodendroglioma cells, and they illustrated a Sox2-directed strategy of immunotherapy to eradicate tumor-initiating cells.

Sox2 requirement in sonic hedgehog-associated medulloblastoma.

The transcription factor Sox2 has been shown to play essential roles during embryonic development as well as in cancer. To more precisely understand tumor biology and to identify potential therapeutical targets, we thoroughly investigated the expression and function of Sox2 in medulloblastoma, a malignant embryonic brain tumor that initiates in the posterior fossa and eventually spreads throughout the entire cerebrospinal axis. We examined a large series of tumor samples (n = 188) to show that SOX2 is specifically expressed in Sonic hedgehog (SHH)-associated medulloblastoma with an interesting preponderance in adolescent and adult cases. We further show that cerebellar granule neuron precursors (CGNP), which are believed to serve as the cell of origin for this medulloblastoma subgroup, express Sox2 in early stages. Also, Shh-associated medulloblastoma can be initiated from such Sox2-positive CGNPs in mice. Independent of their endogenous Sox2 expression, constitutive activation of Shh signaling in CGNPs resulted in significantly enhanced proliferation and ectopic expression of Sox2 in vitro and Sox2-positive medulloblastoma in vivo. Genetic ablation of Sox2 from murine medulloblastoma did not affect survival, most likely due to a compensatory overexpression of Sox3. However, acute deletion of Sox2 from primary cultures of CGNPs with constitutive Shh signaling significantly decreased proliferation, whereas overexpression of Sox2 enhanced proliferation of murine medulloblastoma cells. We conclude that Sox2 is a marker for Shh-dependent medulloblastomas where it is required and sufficient to drive tumor cell proliferation.

DNA damage in mammalian neural stem cells leads to astrocytic differentiation mediated by BMP2 signaling through JAK-STAT.

The consequences of DNA damage generation in mammalian somatic stem cells, including neural stem cells (NSCs), are poorly understood despite their potential relevance for tissue homeostasis. Here, we show that, following ionizing radiation-induced DNA damage, NSCs enter irreversible proliferative arrest with features of cellular senescence. This is characterized by increased cytokine secretion, loss of stem cell markers, and astrocytic differentiation. We demonstrate that BMP2 is necessary to induce expression of the astrocyte marker GFAP in irradiated NSCs via a noncanonical signaling pathway engaging JAK-STAT. This is promoted by ATM and antagonized by p53. Using a SOX2-Cre reporter mouse model for cell-lineage tracing, we demonstrate irradiation-induced NSC differentiation in vivo. Furthermore, glioblastoma assays reveal that irradiation therapy affects the tumorigenic potential of cancer stem cells by ablating self-renewal and inducing astroglial differentiation.

R26R-GR: a Cre-activable dual fluorescent protein reporter mouse.

Green fluorescent protein (GFP) and its derivatives are the most widely used molecular reporters for live cell imagining. The development of organelle-specific fusion fluorescent proteins improves the labeling resolution to a higher level. Here we generate a R26 dual fluorescent protein reporter mouse, activated by Cre-mediated DNA recombination, labeling target cells with a chromatin-specific enhanced green fluorescence protein (EGFP) and a plasma membrane-anchored monomeric cherry fluorescent protein (mCherry). This dual labeling allows the visualization of mitotic events, cell shapes and intracellular vesicle behaviors. We expect this reporter mouse to have a wide application in developmental biology studies, transplantation experiments as well as cancer/stem cell lineage tracing.

Hippocampal development and neural stem cell maintenance require Sox2-dependent regulation of Shh.

Neural stem cells (NSCs) are controlled by diffusible factors. The transcription factor Sox2 is expressed by NSCs and Sox2 mutations in humans cause defects in the brain and, in particular, in the hippocampus. We deleted Sox2 in the mouse embryonic brain. At birth, the mice showed minor brain defects; shortly afterwards, however, NSCs and neurogenesis were completely lost in the hippocampus, leading to dentate gyrus hypoplasia. Deletion of Sox2 in adult mice also caused hippocampal neurogenesis loss. The hippocampal developmental defect resembles that caused by late sonic hedgehog (Shh) loss. In mutant mice, Shh and Wnt3a were absent from the hippocampal primordium. A SHH pharmacological agonist partially rescued the hippocampal defect. Chromatin immunoprecipitation identified Shh as a Sox2 target. Sox2-deleted NSCs did not express Shh in vitro and were rapidly lost. Their replication was partially rescued by the addition of SHH and was almost fully rescued by conditioned medium from normal cells. Thus, NSCs control their status, at least partly, through Sox2-dependent autocrine mechanisms.

Impaired generation of mature neurons by neural stem cells from hypomorphic Sox2 mutants.

The transcription factor Sox2 is active in neural stem cells, and Sox2 'knockdown' mice show defects in neural stem/progenitor cells in the hippocampus and eye, and possibly some neurons. In humans, heterozygous Sox2 deficiency is associated with eye abnormalities, hippocampal malformation and epilepsy. To better understand the role of Sox2, we performed in vitro differentiation studies on neural stem cells cultured from embryonic and adult brains of 'knockdown' mutants. Sox2 expression is high in undifferentiated cells, and declines with differentiation, but remains visible in at least some of the mature neurons. In mutant cells, neuronal, but not astroglial, differentiation was profoundly affected. beta-Tubulin-positive cells were abundant, but most failed to progress to more mature neurons, and showed morphological abnormalities. Overexpression of Sox2 in neural cells at early, but not late, stages of differentiation, rescued the neuronal maturation defect. In addition, it suppressed GFAP expression in glial cells. Our results show an in vitro requirement for Sox2 in early differentiating neuronal lineage cells, for maturation and for suppression of alternative lineage markers. Finally, we examined newly generated neurons from Sox2 ;knockdown' newborn and adult mice. GABAergic neurons were greatly diminished in number in newborn mouse cortex and in the adult olfactory bulb, and some showed abnormal morphology and migration properties. GABA deficiency represents a plausible explanation for the epilepsy observed in some of the knockdown mice, as well as in SOX2-deficient individuals.

Polynucleotide phosphorylase-deficient mutants of Pseudomonas putida.

In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved. In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature. We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity. In addition, we found that the transcription pattern of pnp upon cold shock in P. putida was markedly different from that in Escherichia coli. It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species.

Ternary complex formation between MADS-box transcription factors and the histone fold protein NF-YB.

MADS-box proteins are transcription factors present in different eukaryotic kingdoms. In contrast to plants, for mammalian and yeast MADS-box proteins ternary complex formation with unrelated transcription factors was reported. We show here the first identification of such ternary interaction in plants. A rice seed-specific NF-YB was identified as partner of OsMADS18 by two-hybrid screening. NF-YB contains a histone fold motif, HFM,(1) and is part of the trimeric CCAAT-binding NF-Y complex. OsMADS18, alone or in combination with a natural partner, interacts with OsNF-YB1 through the MADS and I regions. The mouse NF-YB also associates with OsMADS18 in vivo and in vitro as a NF-YB-NF-YC dimer. Other rice MADS-box proteins do not interact in these assays, indicating specificity for the interaction. OsNF-YB1 is capable of heterodimerizing with NF-YC, but not trimerizing with NF-YA, thus precluding CCAAT binding. Mutation of the variant Asp at position 99 of the HFM alpha2-helix into a conserved serine recovers the capacity to interact with NF-YA, but not with DNA. This is the first indication that members of the NF-YB family work through mechanisms independent of the CCAAT box.