Intrinsic Regulator Research Articles

Identification of Osr2 Transcriptional Target Genes in Palate Development

Previous studies have identified the odd-skipped related 2 (Osr2) transcription factor as a key intrinsic regulator of palatal shelf growth and morphogenesis. However, little is known about the molecular program acting downstream of Osr2 in the regulation of palatogenesis. In this study, we isolated palatal mesenchyme cells from embryonic day 12.5 (E12.5) and E13.5 Osr2RFP/+ and Osr2RFP/- mutant mouse embryos and performed whole transcriptome RNA sequencing analyses. Differential expression analysis of the RNA sequencing datasets revealed that expression of 70 genes was upregulated and expression of 61 genes was downregulated by >1.5-fold at both E12.5 and E13.5 in the Osr2RFP/- palatal mesenchyme cells, in comparison with Osr2RFP/+ littermates. Gene ontology analysis revealed enrichment of signaling molecules and transcription factors crucial for skeletal development and osteoblast differentiation among those significantly upregulated in the Osr2 mutant palatal mesenchyme. Using quantitative real-time polymerase chain reaction (RT-PCR)and in situ hybridization assays, we validated that the Osr2-/- embryos exhibit significantly increased and expanded expression of many osteogenic pathway genes, including Bmp3, Bmp5, Bmp7, Mef2c, Sox6, and Sp7 in the developing palatal mesenchyme. Furthermore, we demonstrate that expression of Sema3a, Sema3d, and Sema3e, is ectopically activated in the developing palatal mesenchyme in Osr2-/- embryos. Through chromatin immunoprecipitation, followed by RT-PCR analysis, we demonstrate that endogenous Osr2 protein binds to the promoter regions of the Sema3a and Sema3d genes in the embryonic palatal mesenchyme. Moreover, Osr2 expression repressed the transcription from the Sema3a and Sema3d promoters in cotransfected cells. Since the Sema3 subfamily of signaling molecules plays diverse roles in the regulation of cell proliferation, migration, and differentiation, these data reveal a novel role for Osr2 in regulation of palatal morphogenesis through preventing aberrant activation of Sema3 signaling. Together, these data indicate that Osr2 controls multiple molecular pathways, including BMP and Sema3 signaling, in palate development.

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Distinct effects on the dendritic arbor occur by microbead versus bath administration of brain-derived neurotrophic factor

Proper communication among neurons depends on an appropriately formed dendritic arbor, and thus, aberrant changes to the arbor are implicated in many pathologies, ranging from cognitive disorders to neurodegenerative diseases. Due to the importance of dendritic shape to neuronal network function, the morphology of dendrites is tightly controlled and is influenced by both intrinsic and extrinsic factors. In this work, we examine how brain-derived neurotrophic factor(BDNF), one of the most well-studied extrinsic regulators of dendritic branching, affects the arbor when it is applied locally via microbeads to cultures of hippocampal neurons. We found that local application of BDNF increases both proximal and distal branching in a time-dependent manner and that local BDNF application attenuates pruning of dendrites that occurs with neuronal maturation. Additionally, we examined whether cytosolic PSD-95 interactor(cypin), an intrinsic regulator of dendritic branching, plays a role in these changes and found strong evidence for the involvement of cypin in BDNF-promoted increases in dendrites after 24 but not 48h of application. This current study extends our previous work in which we found that bath application of BDNF for 72h, but not shorter times, increases proximal dendrite branching and that this increase occurs through transcriptional regulation of cypin. Moreover, this current work illustrates how dendritic branching is regulated differently by the same growth factor depending on its spatial localization, suggesting a novel pathway for modulation of dendritic branching locally.

XIAP 3'-untranslated region serves as a competitor for HMGA2 by arresting endogenous let-7a-5p in human hepatocellular carcinoma.

X-linked inhibitor of apoptosis protein functions as an intrinsic regulator of apoptosis by inhibition of caspase activity and possesses a pivotal role in human cancer development and progression. A growing body of literature has demonstrated that microRNAs lead to the degradation or translational repression of messenger RNAs by binding to the non-coding region of messenger RNA at the 3'-untranslated region. Here, we revealed that the expression of HMGA2 is upregulated with X-linked inhibitor of apoptosis protein after transfection of X-linked inhibitor of apoptosis protein 3'-untranslated region in hepatocellular carcinoma cells, suggesting that X-linked inhibitor of apoptosis protein 3'-untranslated region serves as a competitor for microRNAs and prevent the co-targeted messenger RNA, HMGA2, from being suppressed. We further identified that let-7a-5p could bind to both the X-linked inhibitor of apoptosis protein 3'-untranslated region and HMGA2 3'-untranslated region. Moreover, we demonstrated that the forced expression of X-linked inhibitor of apoptosis protein 3'-untranslated region increases the oncogenicity of hepatocellular carcinoma cells in vitro. Cell functional analyses were performed to examine the association of HMGA2 status and X-linked inhibitor of apoptosis protein 3'-untranslated region. We have also measured the functional readout of let-7a-5p and HMGA2, an assay often employed to provide substantial evidence for the effects of X-linked inhibitor of apoptosis protein 3'-untranslated region on hepatocellular carcinoma cells. In general, our findings suggest that X-linked inhibitor of apoptosis protein 3'-untranslated region serves as a competitive endogenous RNA for HMGA2 to activate hepatocellular carcinoma progression by arresting endogenous let-7a-5p.

Abstract 1667: Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach

Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer in the United States and affects 600,000 people each year worldwide. With a five-year survival rate of only 50% and a recent rise in HPV-associated HNSCCs, improved treatment protocols are urgently needed. Evidence of immunosuppression is often reported in HNSCC, making immunotherapy an attractive strategy for the management of this disease. The immune checkpoint inhibitor Pembrolizumab was recently approved for the treatment of metastatic and recurrent HNSCC, but only 18% of initial participants in a trial of Pembrolizumab responded, and it remains difficult to predict patients likely to experience benefit. To increase the number of patients that respond to immune checkpoint inhibition, we sought to identify targetable genetic factors modulating PD-L1, a molecule that serves to dampen the anti-tumor immune response. We show that several HNSCC models exhibit mild (2-4 fold) induction of PD-L1 expression following treatment with interferon-γ (adaptive expression), and recent reports suggest that common oncogenic pathways, including the EGFR and PI3K pathways, may provide a novel strategy to regulate PD-L1 expression. To this end, we developed a high throughput screen to identify targetable pathways that may be used to regulate PD-L1 expression in patients with specific genetic lesions. We have employed a Genome-scale CRISPR Knock-Out (GeCKO) screening technique in HNSCC cell lines and selected for genetic knockouts exhibiting altered PD-L1 expression. Stable knockout pools with representation of approximately 300 gRNAs per target gene (>20,000 target genes in the library) were expanded and serially sorted to create stable sub-populations with enhanced PD-L1 expression. These sub-populations were sequenced to identify gRNAs whose knockout causes a change in PD-L1 expression (e.g. genes that repress or enhance PD-L1 expression). We expect that these large-scale screens, when performed over multiple HNSCC cell lines with diverse genetic lesions, will identify patterns of targetable regulators that may ultimately be manipulated in combination with PD-1/PD-L1 inhibitors. Citation Format: Jacqueline E. Mann, Megan L. Ludwig, Rebecca C. Hoesli, Aditi Kulkarni, Simmy Patel, Judy Kafelghazal, Alexey Nesvizhskii, J Chad Brenner. Identifying intrinsic regulators of PD-L1 expression in cancer cells: A genome-scale CRISPR knock-out approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2017-1667

A systematic analysis of intrinsic regulators for HIV-1 R5 to X4 phenotypic switch

Human immunodeficiency virus isolates most often use chemokine receptor CCR5 or CXCR4 as a co-receptor to enter target cells. During early stages of HIV-1 infection, CCR5-tropic viruses are the predominant species. The CXCR4-tropic viruses may emerge late in infection. Recognition of factors influencing this phenotypic switch may give some hints on the antiviral strategies like anti-HIV/AIDS drugs, gene therapy and vaccines. To investigate the mechanism that triggers R5 to X4 phenotypic switch, we performed a systematic sensitivity analysis based on a five-dimensional model with time-varying parameters. We studied the sensitivity of each factor to the CCR5-to-CXCR4 tropism switch and acquired some interesting outcomes beyond expectation. The death rate of free virus (dV), rate that uninfected CD4+ Tcells arise from precursors (s) and proliferate as stimulated by antigens (r), and in vivo viral burst size (N) are four robust factors which are constantly observed to have a strong correlation with the evolution of viral phenotype for most patients longitudinally. Crucial factors, which are essential to phenotypic switch and disease progression, are almost the same for different patients at different time points, including the production of both virus and CD4+ Tcells and the decay of virion. It is also worth mentioning that although the sequence of factors sorted by the influence varies between patients, the trends of influences engendered by most factors as disease progresses are similar inter-patients.

Intrinsic regulation of enteroendocrine fate by Numb.

How terminal cell fates are specified in dynamically renewing adult tissues is not well understood. Here we explore terminal cell fate establishment during homeostasis using the enteroendocrine cells (EEs) of the adult Drosophila midgut as a paradigm. Our data argue against the existence of local feedback signals, and we identify Numb as an intrinsic regulator of EE fate. Our data further indicate that Numb, with alpha-adaptin, acts upstream or in parallel of known regulators of EE fate to limit Notch signaling, thereby facilitating EE fate acquisition. We find that Numb is regulated in part through its asymmetric and symmetric distribution during stem cell divisions; however, its de novo synthesis is also required during the differentiation of the EE cell. Thus, this work identifies Numb as a crucial factor for cell fate choice in the adult Drosophila intestine. Furthermore, our findings demonstrate that cell-intrinsic control mechanisms of terminal cell fate acquisition can result in a balanced tissue-wide production of terminally differentiated cell types.

Loop Diuretics Diminish Hemolysis Induced by α-Hemolysin from Escherichia coli.

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca2+-influx and activation of Ca2+-sensitive K+ and Cl- channels (KCa3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1-/-) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1-/- mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.

Netrin-1 Preserves Blood-Brain Barrier Integrity Through Deleted in Colorectal Cancer/Focal Adhesion Kinase/RhoA Signaling Pathway Following Subarachnoid Hemorrhage in Rats.

BackgroundNetrin‐1 (NTN‐1) has been established to be a novel intrinsic regulator of blood‐brain barrier (BBB) maintenance. This study was carried out to investigate the potential roles of exogenous NTN‐1 in preserving BBB integrity after experimental subarachnoid hemorrhage (SAH) as well as the underlying mechanisms of its protective effects.Methods and ResultsA total of 309 male Sprague‐Dawley rats were subjected to an endovascular perforation model of SAH. Recombinant NTN‐1 was administered intravenously 1 hour after SAH induction. NTN‐1 small interfering RNA or Deleted in Colorectal Cancer small interfering RNA was administered intracerebroventricular at 48 hours before SAH. Focal adhesion kinase inhibitor was administered by intraperitoneal injection at 1 hour prior to SAH. Neurological scores, brain water content, BBB permeability, RhoA activity, Western blot, and immunofluorescence staining were evaluated. The expression of endogenous NTN‐1 and its receptor Deleted in Colorectal Cancer were increased after SAH. Administration of exogenous NTN‐1 significantly reduced brain water content and BBB permeability and ameliorated neurological deficits at 24 and 72 hours after SAH. Exogenous NTN‐1 treatment significantly promoted phosphorylated focal adhesion kinase activation and inhibited RhoA activity, as well as upregulated the expression of ZO‐1 and Occludin. Conversely, depletion of endogenous NTN‐1 aggravated BBB breakdown and neurological impairments at 24 hours after SAH. The protective effects of NTN‐1 at 24 hours after SAH were also abolished by pretreatment with Deleted in Colorectal Cancer small interfering RNA and focal adhesion kinase inhibitor.Conclusions NTN‐1 treatment preserved BBB integrity and improved neurological functions through a Deleted in Colorectal Cancer/focal adhesion kinase/RhoA signaling pathway after SAH. Thus, NTN‐1 may serve as a promising treatment to alleviate early brain injury following SAH.

Control of Absence Seizures by the Thalamic Feed-Forward Inhibition.

As a subtype of idiopathic generalized epilepsies, absence epilepsy is believed to be caused by pathological interactions within the corticothalamic (CT) system. Using a biophysical mean-field model of the CT system, we demonstrate here that the feed-forward inhibition (FFI) in thalamus, i.e., the pathway from the cerebral cortex (Ctx) to the thalamic reticular nucleus (TRN) and then to the specific relay nuclei (SRN) of thalamus that are also directly driven by the Ctx, may participate in controlling absence seizures. In particular, we show that increasing the excitatory Ctx-TRN coupling strength can significantly suppress typical electrical activities during absence seizures. Further, investigation demonstrates that the GABAA- and GABAB-mediated inhibitions in the TRN-SRN pathway perform combination roles in the regulation of absence seizures. Overall, these results may provide an insightful mechanistic understanding of how the thalamic FFI serves as an intrinsic regulator contributing to the control of absence seizures.

C-Myb Regulates the T-Bet-Dependent Differentiation Program in B Cells to Coordinate Antibody Responses

Humoral immune responses are tailored to the invading pathogen through regulation of key transcription factors and their networks. This is criticalto establishing effective antibody-mediated responses, yet it is unknown how B cells integrate pathogen-induced signals to drive or suppress transcriptional programs specialized for each class of pathogen. Here, we detail the key role of the transcription factor c-Myb in regulating the T-bet-mediated anti-viral program. Deletion of c-Myb in mature B cells significantly increased serum IgG2c and CXCR3 expression by upregulating T-bet, normally suppressed during Th2-cell-mediated responses. Enhanced expression of T-bet resulted in aberrant plasma cell differentiation within the germinal center, mediated by CXCR3 expression. These findings identify a dual role for c-Myb in limiting inappropriate effector responses while coordinating plasma cell differentiation with germinal center egress. Identifying such intrinsic regulators of specialized antibody responses can assist in vaccine design and therapeutic intervention in B-cell-mediated immune disorders.

Characterization of a new allelic mutant of DWARF3 in rice and analysing its function and stability in the presence of strigolactone

Strigolactones (SLs) are important intrinsic growth regulators that control plant architecture by coordinating shoot and root development. Recent studies demonstrate that SL signals act via targeting the degradation protein DWARF53 (D53) family of chaperonin-like proteins. This process requires DWARF14 (D14) as strigolactones signal receptor and DWARF3 (D3) forming Skp-Cullin-F-box (SCF) complex as ubiquitin E3 ligase. Although the interactions of these signal components can be expected, where and how the SLs signalling occur within cells in a tissue-specific manner is still uncertain. In this study, we characterize a rice high-tillering dwarf mutant, ext.-M1B, displaying resistance to synthetic strigolactone mixture rac-GR24. Through genetic analysis, we find that ext.-M1B is a new allelic mutant of D3 with a nucleotide mutation resulting in a truncated protein of wide-type D3. We demonstrate that the mutation affects neither gene expression level nor the protein sub-cellular localization, whereas it disrupts the perception of SLs signal in ext.-M1B mutant. Moreover, we find that overexpression of D3 in wild type background causes no significant phenotype, but suppression of D3 by RNA interfering results in a clear phenocopy of SL mutants. By expressing fluorescent D3 fusion protein in rice, we first show that D3 is stable consistently in the nucleus with or without strigolactone treatment. Taken together, our data indicates that D3 encoding an F-box protein in nucleus, as a stable signal component response to strigolactone regulating rice shoot architecture.

Cyclin F/FBXO1 Interacts with HIV-1 Viral Infectivity Factor (Vif) and Restricts Progeny Virion Infectivity by Ubiquitination and Proteasomal Degradation of Vif Protein through SCFcyclin F E3 Ligase Machinery

Cyclin F protein, also known as FBXO1, is the largest among all cyclins and oscillates in the cell cycle like other cyclins. Apart from being a G2/M cyclin, cyclin F functions as the substrate-binding subunit of SCFcyclin F E3 ubiquitin ligase. In a gene expression analysis performed to identify novel gene modulations associated with cell cycle dysregulation during HIV-1 infection in CD4+ T cells, we observed down-regulation of the cyclin F gene (CCNF). Later, using gene overexpression and knockdown studies, we identified cyclin F as negatively influencing HIV-1 viral infectivity without any significant impact on virus production. Subsequently, we found that cyclin F negatively regulates the expression of viral protein Vif (viral infectivity factor) at the protein level. We also identified a novel host-pathogen interaction between cyclin F and Vif protein in T cells during HIV-1 infection. Mutational analysis of a cyclin F-specific amino acid motif in the C-terminal region of Vif indicated rescue of the protein from cyclin F-mediated down-regulation. Subsequently, we showed that Vif is a novel substrate of the SCFcyclin F E3 ligase, where cyclin F mediates the ubiquitination and proteasomal degradation of Vif through physical interaction. Finally, we showed that cyclin F augments APOBEC3G expression through degradation of Vif to regulate infectivity of progeny virions. Taken together, our results demonstrate that cyclin F is a novel F-box protein that functions as an intrinsic cellular regulator of HIV-1 Vif and has a negative regulatory effect on the maintenance of viral infectivity by restoring APOBEC3G expression.

EFA6 regulates selective polarised transport and axon regeneration from the axon initial segment.

ABSTRACTCentral nervous system (CNS) axons lose their intrinsic ability to regenerate upon maturity, whereas peripheral nervous system (PNS) axons do not. A key difference between these neuronal types is their ability to transport integrins into axons. Integrins can mediate PNS regeneration, but are excluded from adult CNS axons along with their Rab11 carriers. We reasoned that exclusion of the contents of Rab11 vesicles including integrins might contribute to the intrinsic inability of CNS neurons to regenerate, and investigated this by performing laser axotomy. We identify a novel regulator of selective axon transport and regeneration, the ARF6 guanine-nucleotide-exchange factor (GEF) EFA6 (also known as PSD). EFA6 exerts its effects from a location within the axon initial segment (AIS). EFA6 does not localise at the AIS in dorsal root ganglion (DRG) axons, and in these neurons, ARF6 activation is counteracted by an ARF GTPase-activating protein (GAP), which is absent from the CNS, ACAP1. Depleting EFA6 from cortical neurons permits endosomal integrin transport and enhances regeneration, whereas overexpressing EFA6 prevents DRG regeneration. Our results demonstrate that ARF6 is an intrinsic regulator of regenerative capacity, implicating EFA6 as a focal molecule linking the AIS, signalling and transport.This article has an associated First Person interview with the first author of the paper.

A Quiescent Bcl11b High Stem Cell Population Is Required for Maintenance of the Mammary Gland

Stem cells in many tissues sustain themselves by entering a quiescent state to avoid genomic insults and to prevent exhaustion caused by excessive proliferation. In the mammary gland, the identity and characteristics of quiescent epithelial stem cells are not clear. Here, we identify a quiescent mammary epithelial cell population expressing high levels of Bcl11b and located at the interface between luminal and basal cells. Bcl11bhigh cells are enriched for cells that can regenerate mammary glands in secondary transplants. Loss of Bcl11b leads to a Cdkn2a-dependent exhaustion of ductal epithelium and loss of epithelial cell regenerative capacity. Gain- and loss-of-function studies show that Bcl11b induces cells to enter the G0 phase of the cell cycle and become quiescent. Taken together, these results suggest that Bcl11b acts as acentral intrinsic regulator of mammary epithelial stem cell quiescence and exhaustion and is necessary for long-term maintenance of the mammary gland.

A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin

Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear. In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells. Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-β-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression. Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.

Impaired Planar Germ Cell Division in the Testis, Caused by Dissociation of RHAMM from the Spindle, Results in Hypofertility and Seminoma

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM-a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382-95. ©2016 AACR.

The influence of FKBP5 genotype on expression of FKBP5 and other glucocorticoid-regulated genes, dependent on trauma exposure.

The FK506 binding protein 51 (FKBP5), an intrinsic regulator of the glucocorticoid receptor, has been associated with pathological behaviors particularly in the context of childhood trauma (CT), via a putatively regulatory polymorphism, rs1360780. However, trans- and cis-acting effects of this locus and its interaction with CT are incompletely understood. To study its effects on the expression of glucocorticoid-regulated genes including FKBP5, we used lymphoblastoid cell lines (LCLs) derived from 16 CT-exposed patients with greater than two substance dependence/suicidal behavior diagnoses (casesCT+) and 13 non-CT-exposed controls (controlsCT-). This study in LCLs measures long-term trait-like differences attributable to genotype or lasting epigenetic modification. Through analysis of differential allelic expression (DAE) using an FKBP5 3'-UTR reporter single nucleotide polymorphism (SNP), rs3800373, that is in strong linkage disequilibrium with rs1360780, we confirmed that the rs1360780 risk allele (A) (or conceivably that of a linked SNP) leads to higher FKBP5 expression in controlsCT-. Intriguingly, casesCT+ did not show DAE, perhaps because of a genotype-predicted difference in FKBP5 DNA methylation restricted to casesCT+. Furthermore, through correlation analyses on FKBP5 expression at baseline and after induction by dexamethasone, we observed that casesCT+ had lower induction of FKBP5 expression, indicating that overall they may have strong ultra-short negative-feedback. Only casesCT+ showed an effect of rs1360780 genotype on expression of FKBP5 and other glucocorticoid-regulated genes. Together, these results confirm that the rs1360780 locus alters FKBP5 expression and further that in trans-fashion this locus affects the expression of other glucocorticoid-regulated genes after a glucocorticoid challenge. The CT exposure appears to be essential for trans-effects of rs1360780 on glucocorticoid-regulated genes.

Orphan nuclear receptor SHP regulates iron metabolism through inhibition of BMP6-mediated hepcidin expression.

Small heterodimer partner (SHP) is a transcriptional corepressor regulating diverse metabolic processes. Here, we show that SHP acts as an intrinsic negative regulator of iron homeostasis. SHP-deficient mice maintained on a high-iron diet showed increased serum hepcidin levels, decreased expression of the iron exporter ferroportin as well as iron accumulation compared to WT mice. Conversely, overexpression of either SHP or AMP-activated protein kinase (AMPK), a metabolic sensor inducing SHP expression, suppressed BMP6-induced hepcidin expression. In addition, an inhibitory effect of AMPK activators metformin and AICAR on BMP6-mediated hepcidin gene expression was significantly attenuated by ablation of SHP expression. Interestingly, SHP physically interacted with SMAD1 and suppressed BMP6-mediated recruitment of the SMAD complex to the hepcidin gene promoter by inhibiting the formation of SMAD1 and SMAD4 complex. Finally, overexpression of SHP and metformin treatment of BMP6 stimulated mice substantially restored hepcidin expression and serum iron to baseline levels. These results reveal a previously unrecognized role for SHP in the transcriptional control of iron homeostasis.

Multiple intrinsic factors act in concert with Lhx2 to direct retinal gliogenesis.

Müller glia (MG) are the principal glial cell type in the vertebrate retina. Recent work has identified the LIM homeodomain factor encoding gene Lhx2 as necessary for both Notch signaling and MG differentiation in late-stage retinal progenitor cells (RPCs). However, the extent to which Lhx2 interacts with other intrinsic regulators of MG differentiation is unclear. We investigated this question by investigating the effects of overexpression of multiple transcriptional regulators that are either known or hypothesized to control MG formation, in both wildtype and Lhx2-deficient RPCs. We observe that constitutively elevated Notch signaling, induced by N1ICD electroporation, inhibited gliogenesis in wildtype animals, but rescued MG development in Lhx2-deficient retinas. Electroporation of Nfia promoted the formation of cells with MG-like radial morphology, but did not drive expression of MG molecular markers. Plagl1 and Sox9 did not induce gliogenesis in wildtype animals, but nonetheless activated expression of the Müller marker P27Kip1 in Lhx2-deficient cells. Finally, Sox2, Sox8, and Sox9 promoted amacrine cell formation in Lhx2-deficient cells, but not in wildtype retinas. These findings demonstrate that overexpression of individual gliogenic factors typically regulates only a subset of characteristic MG markers, and that these effects are differentially modulated by Lhx2.