Joint analysis of WES and RNA-Seq identify signature genes related to metastasis in prostate cancer.

Prostate cancer (PCa) has a certain degree of heritability, and metastasis occurs as cancer progresses. However, its underlying mechanism remains largely unknown. We sequenced four cases of cancer without metastasis, four metastatic cancer, and four benign hyperplasia tissues as controls. A total of 1839 damaging mutations were identified. Pathway analysis, gene clustering, and weighted gene co-expression network analysis were employed to find characteristics associated with metastasis. Chr19 had the most mutation density and 1p36 had the highest mutation frequency across the genome. These mutations occurred in 1630 genes, including the most frequently mutated genes TTN and PLEC, and dozens of metastasis-related genes, such as FOXA1, NCOA1, CD34, and BRCA2. Ras signalling and arachidonic acid metabolism were uniquely enriched in metastatic cancer. Gene programmes 10 and 11 showed the signatures indicating the occurrence of metastasis better. A module (135 genes) was specifically associated with metastasis. Of them, 67.41% reoccurred in program 10, with 26 genes further retained as the signature genes related to PCa metastasis, including AGR3, RAPH1, SOX14, DPEP1, and UBL4A. Our study provides new molecular perspectives on PCa metastasis. The signature genes and pathways could be served as potential therapeutic targets for metastasis or cancer progression.

Novel Candidate Regulators and Developmental Trajectory of Pituitary Thyrotropes.

The pituitary gland regulates growth, metabolism, reproduction, the stress response, uterine contractions, lactation, and water retention. It secretes hormones in response to hypothalamic input, end organ feedback, and diurnal cues. The mechanisms by which pituitary stem cells are recruited to proliferate, maintain quiescence, or differentiate into specific cell types, especially thyrotropes, are not well understood. We used single-cell RNA sequencing in juvenile P7 mouse pituitary cells to identify novel factors in pituitary cell populations, with a focus on thyrotropes and rare subtypes. We first observed cells coexpressing markers of both thyrotropes and gonadotropes, such as Pou1f1 and Nr5a1. This was validated in vivo by both immunohistochemistry and lineage tracing of thyrotropes derived from Nr5a1-Cre; mTmG mice and demonstrates that Nr5a1-progenitors give rise to a proportion of thyrotropes during development. Our data set also identifies novel factors expressed in pars distalis and pars tuberalis thyrotropes, including the Shox2b isoform in all thyrotropes and Sox14 specifically in Pou1f1-negative pars tuberalis thyrotropes. We have therefore used single-cell transcriptomics to determine a novel developmental trajectory for thyrotropes and potential novel regulators of thyrotrope populations.

Evolutionary Landscape of SOX Genes to Inform Genotype-to-Phenotype Relationships.

The SOX transcription factor family is pivotal in controlling aspects of development. To identify genotype-phenotype relationships of SOX proteins, we performed a non-biased study of SOX using 1890 open-reading frame and 6667 amino acid sequences in combination with structural dynamics to interpret 3999 gnomAD, 485 ClinVar, 1174 Geno2MP, and 4313 COSMIC human variants. We identified, within the HMG (High Mobility Group)- box, twenty-seven amino acids with changes in multiple SOX proteins annotated to clinical pathologies. These sites were screened through Geno2MP medical phenotypes, revealing novel SOX15 R104G associated with musculature abnormality and SOX8 R159G with intellectual disability. Within gnomAD, SOX18 E137K (rs201931544), found within the HMG box of ~0.8% of Latinx individuals, is associated with seizures and neurological complications, potentially through blood-brain barrier alterations. A total of 56 highly conserved variants were found at sites outside the HMG-box, including several within the SOX2 HMG-box-flanking region with neurological associations, several in the SOX9 dimerization region associated with Campomelic Dysplasia, SOX14 K88R (rs199932938) flanking the HMG box associated with cardiovascular complications within European populations, and SOX7 A379V (rs143587868) within an SOXF conserved far C-terminal domain heterozygous in 0.716% of African individuals with associated eye phenotypes. This SOX data compilation builds a robust genotype-to-phenotype association for a gene family through more robust ortholog data integration.

Sox14 is essential for initiation of neuronal differentiation in the chick spinal cord.

BACKGROUND: The neural tube comprises several different types of progenitors and postmitotic neurons that co-ordinately act with each other to play integrated functions. Its development consists of two phases: proliferation of progenitor cells and differentiation into postmitotic neurons. How progenitor cells differentiate into each corresponding neuron is an important question for understanding the mechanisms of neuronal development. RESULTS: Here we introduce one of the Sox transcription factors, Sox14, which plays an essential role in the promotion of neuronal differentiation. Sox14 belongs to the SoxB2 subclass and its expression starts in the progenitor regions before neuronal differentiation is initiated at the trunk level of the neural tube. After neuronal differentiation is initiated, Sox14 expression gradually becomes confined to the V2a region of the neural tube, where Chx10 is co-expressed. Overexpression of Sox14 restricts progenitor cell proliferation. Conversely, the blockade of Sox14 expression by the RNAi strategy inhibits V2a neuron differentiation and causes expansion of the progenitor domain. We further found that Sox14 acted as a transcriptional activator. CONCLUSIONS: Sox14 acts as a modulator of cell proliferation and is essential for initiation of neuronal differentiation in the chick neural tube.

SOX21 modulates SOX2-initiated differentiation of epithelial cells in the extrapulmonary airways.

SOX2 expression levels are crucial for the balance between maintenance and differentiation of airway progenitor cells during development and regeneration. Here, we describe patterning of the mouse proximal airway epithelium by SOX21, which coincides with high levels of SOX2 during development. Airway progenitor cells in this SOX2+/SOX21+ zone show differentiation to basal cells, specifying cells for the extrapulmonary airways. Loss of SOX21 showed an increased differentiation of SOX2+ progenitor cells to basal and ciliated cells during mouse lung development. We propose a mechanism where SOX21 inhibits differentiation of airway progenitors by antagonizing SOX2-induced expression of specific genes involved in airway differentiation. Additionally, in the adult tracheal epithelium, SOX21 inhibits basal to ciliated cell differentiation. This suppressing function of SOX21 on differentiation contrasts SOX2, which mainly drives differentiation of epithelial cells during development and regeneration after injury. Furthermore, using human fetal lung organoids and adult bronchial epithelial cells, we show that SOX2+/SOX21+ regionalization is conserved. Lastly, we show that the interplay between SOX2 and SOX21 is context and concentration dependent leading to regulation of differentiation of the airway epithelium.

Molecular Fingerprint and Developmental Regulation of the Tegmental GABAergic and Glutamatergic Neurons Derived from the Anterior Hindbrain.

Tegmental nuclei in the ventral midbrain and anterior hindbrain control motivated behavior, mood, memory, and movement. These nuclei contain inhibitory GABAergic and excitatory glutamatergic neurons, whose molecular diversity and development remain largely unraveled. Many tegmental neurons originate in the embryonic ventral rhombomere 1 (r1), where GABAergic fate is regulated by the transcription factor (TF) Tal1. We used single-cell mRNA sequencing of the mouse ventral r1 to characterize the Tal1-dependent and independent neuronal precursors. We describe gene expression dynamics during bifurcation of the GABAergic and glutamatergic lineages and show how active Notch signaling promotes GABAergic fate selection in post-mitotic precursors. We identify GABAergic precursor subtypes that give rise to distinct tegmental nuclei and demonstrate that Sox14 and Zfpm2, two TFs downstream of Tal1, are necessary for the differentiation of specific tegmental GABAergic neurons. Our results provide a framework for understanding the development of cellular diversity in the tegmental nuclei.

Silencing of long non-coding RNA SOX21-AS1 inhibits lung adenocarcinoma invasion and migration by impairing TSPAN8 via transcription factor GATA6.

Here, we revealed the novel role of long non-coding RNAs (lncRNAs) SOX21 antisense RNA 1 (SOX21-AS1)/TSPAN8/GATA6 in progression of lung adenocarcinoma. SOX21-AS1 expression was quantified in lung adenocarcinoma tissues and cells by RT-qPCR. Then, gain- and loss-of-function experiments were conducted in lung adenocarcinoma cells. Expression of GATA6, TSPAN8 and extracellular signal-regulated kinase (ERK) signaling pathway-related genes was determined in lung adenocarcinoma cells by western blot analysis. The interaction and relationship among SOX21-AS1, GATA6 and TSPAN8 were predicted and verified respectively by RNA pull down, RIP, ChIP, and dual-luciferase reporter assays. Next, lung adenocarcinoma cell proliferation, colony formation, invasion and migration were assessed by 5-ethynyl-2'-deoxyuridine staining, colony formation assay and Transwell assay. Xenograft tumors were established in nude mice and the tumor growth was observed and recorded. SOX21-AS1 was observed to be highly expressed in lung adenocarcinoma tissues. The overexpression of SOX21-AS1, GATA6 or TSPAN8 obviously enhanced cell biological functions in lung adenocarcinoma. Meanwhile, SOX21-AS1 interacted with GATA6 which bound to TSPAN8 promoter and promoted TSPAN8 expression, which further enhanced cell colony formation, proliferation and invasion, and also activated ERK signaling pathway. Silencing of SOX21-AS1 and inhibiting its binding to GATA6 downregulate TSPAN8 and thereby exert anti-oncogenic effects in lung adenocarcinoma.

Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair.

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. We found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, we found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.

Characterization of gonad differentially expressed SoxB2 genes in mud crab Scylla paramamosain.

Members of sox gene family play critical roles in development, and some of them have crucial functions in sexual dimorphism. To understand the role of two SoxB2 genes, Sox14b and Sox21 of mud crab Scylla paramamosain, the full-length 1939 bp SpSox14b cDNA sequence and 861 bp SpSox21 cDNA sequence were obtained from the crab's transcriptome database, which encode 397 and 259 amino acids respectively. The results of sq-PCR showed that SpSox14b was expressed in all tissues, while SpSox21 was only expressed in the testis and brain. qRT-PCR showed that the expression level of SpSox14b in ovary was significantly higher than that of testis, and during the gonad development its expression was the highest in O2 (previtellogenesis) stage. The expression level of SpSox21 in testis was much higher than in brain, and was significantly higher in T3 (the mature sperm stage) than in other stages of testis development. Meanwhile, in different stages of larval development, SpSox21 was low expressed in zoea, then increased significantly in megalopa. Therefore we speculated that SpSox14b and SpSox21 may play different roles in the gonad development of mud crab, especially SpSox21 may be involved in the development and maintenance of testis. The expression level of SpSox14b and SpSox21 during the eye-pigment formation was significantly higher than that in other embryonic development stages, the results of whole-mount in situ hybridization showed that SpSox14b and SpSox21 were mainly located near the head and the compound eyes in eye-pigment formation stage and hatching. It suggested that they may be involved in the formation of brain nerves and are related to the regulation of body segments, and play different roles in sexual dimorphism.

Sox100B Regulates Progenitor-Specific Gene Expression and Cell Differentiation in the Adult Drosophila Intestine.

Robust production of terminally differentiated cells from self-renewing resident stem cells is essential to maintain proper tissue architecture and physiological functions, especially in high-turnover tissues. However, the transcriptional networks that precisely regulate cell transition and differentiation are poorly understood in most tissues. Here, we identified Sox100B, a Drosophila Sox E family transcription factor, as a critical regulator of adult intestinal stem cell differentiation. Sox100B is expressed in stem and progenitor cells and required for differentiation of enteroblast progenitors into absorptive enterocytes. Mechanistically, Sox100B regulates the expression of another critical stem cell differentiation factor, Sox21a. Supporting a direct control of Sox21a by Sox100B, we identified a Sox21a intronic enhancer that is active in all intestinal progenitors and directly regulated by Sox100B. Taken together, our results demonstrate that the activity and regulation of two Sox transcription factors are essential to coordinate stem cell differentiation and proliferation and maintain intestinal tissue homeostasis.

SOX21 Ensures Rostral Forebrain Identity by Suppression of WNT8B during Neural Regionalization of Human Embryonic Stem Cells.

The generation of brain region-specific progenitors from human embryonic stem cells (hESCs) is critical for their application. However, transcriptional regulation of neural regionalization in humans is poorly understood. Here, we applied a rostrocaudal patterning system from hESCs to dissect global transcriptional networks controlling early neural regionalization. We found that SOX21 is required for rostral forebrain fate specification. SOX21 knockout led to activation of Wnt signaling, resulting in caudalization of regional identity of rostral forebrain neural progenitor cells. Moreover, we identified WNT8B as a SOX21 direct target. Deletion of WNT8B or inhibition of Wnt signaling in SOX21 knockout neural progenitor cells restored rostral forebrain identity. Furthermore, SOX21 interacted with ß-catenin, interfering with the binding of TCF4/ß-catenin complex to the WNT8B enhancer. Collectively, these results unveil the unknown role of SOX21 and shed light on how a transcriptional factor modulates early neural regionalization through crosstalk with a key component of Wnt signaling.

SOX14 activates the p53 signaling pathway and induces apoptosis in a cervical carcinoma cell line.

SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hypermethylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma.

SOX5/6/21 Prevent Oncogene-Driven Transformation of Brain Stem Cells.

Molecular mechanisms preventing self-renewing brain stem cells from oncogenic transformation are poorly defined. We show that the expression levels of SOX5, SOX6, and SOX21 (SOX5/6/21) transcription factors increase in stem cells of the subventricular zone (SVZ) upon oncogenic stress, whereas their expression in human glioma decreases during malignant progression. Elevated levels of SOX5/6/21 promoted SVZ cells to exit the cell cycle, whereas genetic ablation of SOX5/6/21 dramatically increased the capacity of these cells to form glioma-like tumors in an oncogene-driven mouse brain tumor model. Loss-of-function experiments revealed that SOX5/6/21 prevent detrimental hyperproliferation of oncogene expressing SVZ cells by facilitating an antiproliferative expression profile. Consistently, restoring high levels of SOX5/6/21 in human primary glioblastoma cells enabled expression of CDK inhibitors and decreased p53 protein turnover, which blocked their tumorigenic capacity through cellular senescence and apoptosis. Altogether, these results provide evidence that SOX5/6/21 play a central role in driving a tumor suppressor response in brain stem cells upon oncogenic insult. Cancer Res; 77(18); 4985-97. ©2017 AACR.

Nucleocytoplasmic shuttling of SOX14A and SOX14B transcription factors.

The nucleocytoplasmic shuttling of SOX transcription factors play a crucial role in the regulation of SOX protein functions during development. In this study, we have demonstrated two nuclear localization signals in the HMG box of Eriocheir sinensis SOX14A and SOX14B. These two conserved nuclear localization signals mediate nuclear transport. The N-termini nuclear localization signal mediates the calmodulin-dependent pathway and the C-termini nuclear localization signal interacts with the importin-ß pathway. The targeted deletion of nuclear localization signals of SOX14A/B dramatically inhibits the nuclear accumulation. We have first time revealed a non-classic nuclear export signal in the HMG box of E. sinensis SOX14A/B proteins is responds to leptomycin B. E. sinensis SOX14A/B is transported from the nucleus to the cytoplasm via a CRM1-dependent nuclear export pathway. And E. sinensis SOX14A/B are not belong to the subgroup E SOX proteins. Furthermore, these findings could shed a light on the mechanisms involved in the nuclear export of SOX proteins. The imperfect nuclear export signal on other SOX proteins, rather than just those of the SOXE group, may also be functional for nuclear export.

An Evolutionarily Conserved SoxB-Hdac2 Crosstalk Regulates Neurogenesis in a Cnidarian.

SoxB transcription factors and histone deacetylases (HDACs) are each major players in the regulation of neurogenesis, but a functional link between them has not been previously demonstrated. Here, we show that SoxB2 and Hdac2 act together to regulate neurogenesis in the cnidarian Hydractinia echinata during tissue homeostasis and head regeneration. We find that misexpression of SoxB genes modifies the number of neural cells in all life stages and interferes with head regeneration. Hdac2 was co-expressed with SoxB2, and its downregulation phenocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other's transcript levels, but Hdac2 counteracts this amplification cycle by deacetylating and destabilizing SoxB2 protein. Finally, we present evidence for conservation of these interactions in human neural progenitors. We hypothesize that crosstalk between SoxB transcription factors and Hdac2 is an ancient feature of metazoan neurogenesis and functions to stabilize the correct levels of these multifunctional proteins.

Tectal-derived interneurons contribute to phasic and tonic inhibition in the visual thalamus.

The release of GABA from local interneurons in the dorsal lateral geniculate nucleus (dLGN-INs) provides inhibitory control during visual processing within the thalamus. It is commonly assumed that this important class of interneurons originates from within the thalamic complex, but we now show that during early postnatal development Sox14/Otx2-expressing precursor cells migrate from the dorsal midbrain to generate dLGN-INs. The unexpected extra-diencephalic origin of dLGN-INs sets them apart from GABAergic neurons of the reticular thalamic nucleus. Using optogenetics we show that at increased firing rates tectal-derived dLGN-INs generate a powerful form of tonic inhibition that regulates the gain of thalamic relay neurons through recruitment of extrasynaptic high-affinity GABAA receptors. Therefore, by revising the conventional view of thalamic interneuron ontogeny we demonstrate how a previously unappreciated mesencephalic population controls thalamic relay neuron excitability.

A novel SoxB2 gene is required for maturation of sperm nucleus during spermiogenesis in the Chinese mitten crab, Eriocheir sinensis.

SRY-related HMG box (Sox) genes are characterized by the presence of a DNA-binding HMG domain and involved in a diverse range of developmental processes. In this study, we identified a novel Sox gene, designated as EsSoxB2-1, from the Chinese mitten crab Eriocheir sinensis. The EsSoxB2-1 encodes a protein of 259 amino acids, sharing the highest identity with the beetle Tribolium castaneum SOX21b. Unlike insect Sox21b, however, EsSoxB2-1 is intronless and exhibits a gonad-specific expression pattern at both mRNA and protein level. Two core promoters in 5' flanking region were demonstrated to be essential for inducing transcriptional regulatory activity. The transcription of EsSoxB2-1 mRNA begins in spermatogonia stage, while the translation of EsSOXB2-1 protein initiates at spermiogenesis stage. Interestingly, EsSOXB2-1 protein was exclusively localized in the nucleus of spermatid and spermatozoa even at the end of acrosome reaction, and was bound to the uncondensed chromatin in nucleoplasm of mature spermatozoa. Knockdown of EsSoxB2-1 by RNAi leads to abnormal transformation of the nucleus during spermiogenesis. Together, these findings demonstrated the requirement of EsSoxB2-1 for the spermatozoa nucleus maturation and also suggested that EsSoxB2-1 would be delivered into fertilized eggs along with chromatins as a paternal transcription factor for regulating early embryonic development.

A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis.

Homeostatic renewal of many adult tissues requires balanced self-renewal and differentiation of local stem cells, but the underlying mechanisms are poorly understood. Here we identified a novel feedback mechanism in controlling intestinal regeneration and tumorigenesis in Drosophila. Sox21a, a group B Sox protein, is preferentially expressed in the committed progenitor named enteroblast (EB) to promote enterocyte differentiation. In Sox21a mutants, EBs do not divide, but cannot differentiate properly and have increased expression of mitogens, which then act as paracrine signals to promote intestinal stem cell (ISC) proliferation. This leads to a feedback amplification loop for rapid production of differentiation-defective EBs and tumorigenesis. Notably, in normal intestine following damage, Sox21a is temporally downregulated in EBs to allow the activation of the ISC-EB amplification loop for epithelial repair. We propose that executing a feedback amplification loop between stem cells and their progeny could be a common mechanism underlying tissue regeneration and tumorigenesis.

Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos.

The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.

Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis.

Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. Here we report that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. Our data highlight the tumorigenic properties of transit differentiating cells.