Distinct Subsets Of Genes Research Articles

Abstract 4561: Mechanism of histone H3K27me3 demethylase therapy for restoring cisplatin sensitivity in refractory testicular cancer

Abstract Testicular germ cell tumors (TGCTs) can be cured at a high rate with standard cisplatin-based chemotherapy, but 15% of metastatic patients are cisplatin resistant/refractory and die from progressive disease. In prior work, we used de novo pharmacogenetic approaches leveraging a series of unique cisplatin resistant cell models that uncovered an important role for polycomb and histone H3K27me3 methylation in mediating TGCT sensitivity and resistance to cisplatin. Importantly, we demonstrated that pharmacological inhibition of H3K27 methylation with the EZH2 inhibitor GSK-126 conferred cisplatin resistance to parental cells while induction of H3K27 methylation with the histone lysine demethylase inhibitor GSK-J4 resulted in a high degree of cisplatin sensitization of TGCT tumors including cisplatin resistant TGCT cells. In order to elucidate mechanisms that account for the remarkable cisplatin sensitization effects of GSKJ4 in TGCTs, we used two distinct cisplatin resistant TGCT cell lines 2102EP-C1 and NT2D1-A4. Cells were pretreated for three days with either vehicle control or 1µM GSK-J4 to modulate H3K27me3 followed by treatment with vehicle control or 0.5µM cisplatin for 6 hours. RNA was extracted from cells after 24 hours of cisplatin removal. This treatment protocol was designed to capture “early” transcriptional responses to GSKJ4 sensitization before the onset of cell death. RNA-seq transcriptomic profiling and differential gene expression analysis revealed as expected a severely attenuated transcriptional response to cisplatin in cisplatin refractory cells that was restored with GSKJ4 pretreatment. Interestingly GSKJ4 alone resulted in a robust transcriptional response with a large overlap with gene changed with cisplatin alone, despite the fact that GSKJ4 alone did not affect cell proliferation or survival. However, GSKJ4 uniquely regulated a subset of genes and uniquely restored cisplatin response to a subset of genes which are candidates for driving the remarkable cisplatin sensitizing effects of GSKJ4 in TGCTs. Further enrichment and biological classification analysis of these distinct gene subsets are ongoing and will be presented. In summary, our data suggests mechanisms to account for why directly targeting H3K27 methylation with GSKJ4 appear highly effective in treating cisplatin resistant/refractory TGCTs and may provide biomarkers for future clinical investigation of this strategy. Citation Format: Doha Naguib Ahmed Mohamed Shokry, Ratnakar Singh, Mehwish Khan, Zeeshan Fazal, Raya Boyd, Christine Powell, Sarah J. Freemantle, Michael J. Spinella. Mechanism of histone H3K27me3 demethylase therapy for restoring cisplatin sensitivity in refractory testicular cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4561.

Decoupling of mRNA and Protein Expression in Aging Brains Reveals the Age-Dependent Adaptation of Specific Gene Subsets.

During aging, changes in gene expression are associated with a decline in physical and cognitive abilities. Here, we investigate the connection between changes in mRNA and protein expression in the brain by comparing the transcriptome and proteome of the mouse cortex during aging. Our transcriptomic analysis revealed that aging mainly triggers gene activation in the cortex. We showed that an increase in mRNA expression correlates with protein expression, specifically in the anterior cingulate cortex, where we also observed an increase in cortical thickness during aging. Genes exhibiting an aging-dependent increase of mRNA and protein levels are involved in sensory perception and immune functions. Our proteomic analysis also identified changes in protein abundance in the aging cortex and highlighted a subset of proteins that were differentially enriched but exhibited stable mRNA levels during aging, implying the contribution of aging-related post- transcriptional and post-translational mechanisms. These specific genes were associated with general biological processes such as translation, ribosome assembly and protein degradation, and also important brain functions related to neuroplasticity. By decoupling mRNA and protein expression, we have thus characterized distinct subsets of genes that differentially adjust to cellular aging in the cerebral cortex.

Mechanisms encoding STAT functional diversity for context-specific inflammatory responses.

Cells integrate complex cytokine cues and other inflammatory stimuli through activation of the signal transducers and activators of transcription (STAT) family of transcription factors to drive the appropriate anti-microbial, inflammatory, and resolving functions. Here, we discuss recent progress in our understanding of mechanisms supporting STAT functional diversity. Signaling component availability and the strength of receptor and STAT interactions emerge as important determinants of immune function. The resultant dynamics of STAT activation, together with stimulus-specific variation in STAT post-translationally modified states, will impact downstream binding partners to support transcription of distinct gene subsets. Understanding how context-dependent STAT function is encoded to dictate cytokine specificity, crosstalk, and control of inflammation will guide therapeutic efforts to selectively perturb STAT-regulated responses.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism.

The type VII protein secretion system (T7SS) has been characterized in members of the phyla Actinobacteria and Firmicutes. In mycobacteria the T7SS is intimately linked with pathogenesis and intracellular survival, while in Firmicutes there is mounting evidence that the system plays a key role in interbacterial competition. A conserved membrane-bound ATPase protein, termed EssC in Staphylococcus aureus, is a critical component of the T7SS and is the primary receptor for substrate proteins. Genetic diversity in the essC gene of S. aureus has previously been reported, resulting in four protein variants that are linked to specific subsets of substrates. Here we have analysed the genetic diversity of the T7SS-encoding genes and substrate proteins across Listeria monocytogenes genome sequences. We find that there are seven EssC variants across the species that differ in their C-terminal region; each variant is correlated with a distinct subset of genes for likely substrate and accessory proteins. EssC1 is most common and is exclusively linked with polymorphic toxins harbouring a YeeF domain, whereas EssC5, EssC6 and EssC7 variants all code for an LXG domain protein adjacent to essC. Some essC1 variant strains encode an additional, truncated essC at their T7 gene cluster. The truncated EssC, comprising only the C-terminal half of the protein, matches the sequence of either EssC2, EssC3 or EssC4. In each case the truncated gene directly precedes a cluster of substrate/accessory protein genes acquired from the corresponding strain. Across L. monocytogenes strains we identified 40 LXG domain proteins, most of which are encoded at conserved genomic loci. These loci also harbour genes encoding immunity proteins and sometimes additional toxin fragments. Collectively our findings strongly suggest that the T7SS plays an important role in bacterial antagonism in this species.

Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.

Interferon Regulatory Factors IRF1 and IRF7 Directly Regulate Gene Expression in Bats in Response to Viral Infection

Bat cells and tissue have elevated basal expression levels of antiviral genes commonly associated with interferon alpha (IFNα) signaling. Here, we show Interferon Regulatory Factor 1 (IRF1), 3, and 7 levels are elevated in most bat tissues and that, basally, IRFs contribute to the expression of type I IFN ligands and high expression of interferon regulated genes (IRGs). CRISPR knockout (KO) of IRF 1/3/7 in cells reveals distinct subsets of genes affected by each IRF in an IFN-ligand signaling-dependent and largely independent manner. As the master regulators of innate immunity, the IRFs control the kinetics and maintenance of the IRG response and play essential roles in response to influenza A virus (IAV), herpes simplex virus 1 (HSV-1), Melaka virus/Pteropine orthoreovirus 3 Melaka (PRV3M), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) infection. With its differential expression in bats compared to that in humans, this highlights a critical role for basal IRF expression in viral responses and potentially immune cell development in bats with relevance for IRF function in human biology.

Coordinated Regulation of Pre-mRNA Splicing by the SFPS-RRC1 Complex to Promote Photomorphogenesis.

Light signals perceived by the phytochrome (phy) family of photoreceptors control gene expression at both transcriptional and posttranscriptional levels to promote photomorphogenesis. Recently, we identified a factor called SPLICING FACTOR FOR PHYTOCHROME SIGNALING (SFPS) that directly interacts with the photoreceptor phyB and regulates pre-mRNA splicing in Arabidopsis (Arabidopsis thaliana). To identify SFPS-interacting proteins, we performed an immunoprecipitation followed by a mass spectrometry and identified the Ser/Arg-like protein REDUCED RED-LIGHT RESPONSES IN CRY1CRY2 BACKGROUND1 (RRC1). Genetic analyses revealed that the sfps-2 rrc1-3 phenotypes are similar to those of the single mutants, suggesting that RRC1 and SFPS might function together. RNA sequence analyses of rrc1-3 identified a large number of genes whose pre-mRNA splicing is altered under dark and light conditions. Comparison of the sequence data revealed a subset of common genes coregulated by SFPS and RRC1 under dark and light conditions. Similar to SFPS, RRC1 also interacts with phyB, colocalizes in nuclear photobodies, and regulates light-dependent pre-mRNA splicing of a subset of genes. Taken together, these data suggest that although SFPS and RRC1 can regulate distinct subsets of genes, they also form a complex and coordinately control pre-mRNA splicing of a subset of genes involved in light signaling and circadian clock pathways to promote photomorphogenesis.

Resting cells rely on the DNA helicase component MCM2 to build cilia.

Minichromosome maintenance (MCM) proteins facilitate replication by licensing origins and unwinding the DNA double strand. Interestingly, the number of MCM hexamers greatly exceeds the number of firing origins suggesting additional roles of MCMs. Here we show a hitherto unanticipated function of MCM2 in cilia formation in human cells and zebrafish that is uncoupled from replication. Zebrafish depleted of MCM2 develop ciliopathy-phenotypes including microcephaly and aberrant heart looping due to malformed cilia. In non-cycling human fibroblasts, loss of MCM2 promotes transcription of a subset of genes, which cause cilia shortening and centriole overduplication. Chromatin immunoprecipitation experiments show that MCM2 binds to transcription start sites of cilia inhibiting genes. We propose that such binding may block RNA polymerase II-mediated transcription. Depletion of a second MCM (MCM7), which functions in complex with MCM2 during its canonical functions, reveals an overlapping cilia-deficiency phenotype likely unconnected to replication, although MCM7 appears to regulate a distinct subset of genes and pathways. Our data suggests that MCM2 and 7 exert a role in ciliogenesis in post-mitotic tissues.

Abstract B031: CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer

Abstract Many genes aberrantly expressed in prostate cancer are involved in normal basal to luminal cell differentiation. We previously demonstrated that transient ING4 expression is required for luminal cell differentiation and is downregulated in ~60% of primary prostate tumors. We further demonstrated in a primary prostate cancer model overexpressing ERG, MYC, and shPTEN (EMP) that loss of PTEN was responsible for ING4 loss. Furthermore, half of the human tumor samples that lose ING4 have also lost PTEN. However, we did not know how PTEN loss inhibits ING4 expression. Utilizing our in vitro differentiation model, whereby prostate basal epithelial cells (iPrEC) treated with KGF and androgen induce a suprabasal layer of luminal-like cells, and RNA-seq we identified transcriptional nodes required for luminal differentiation. Differentially expressed genes were analyzed by GeneGo to identify enriched transcription-factor signatures. Of the ~600 differentially regulated genes during differentiation, the largest signature (29% of genes) was CREB/ATF targets. Induction of Blimp1, Claudin1, and Plk2 and inhibition of Chek1 were further validated by qRT-PCR and immunoblotting. CREB/ATF bind constitutively to open chromatin CRE elements in the promoters of genes and are activated through signaling-induced phosphorylation at Ser133 by kinases, including AKT. We found that both CREB1 and ATF1 are inducibly phosphorylated midway through luminal differentiation, with ATF1 preceding CREB1. Knockdown of CREB1 with shRNA increased ING4, accelerated differentiation, and induced premature luminal cell death. Conversely, knockdown of ATF1 blocked ING4 induction and prevented suprabasal layer formation. CREB1/ATF1 ChIP was enriched at the ING4 promoter at mid-differentiation, when ING4 expression peaks. Additionally, CREB1/ATF1 was constitutively bound to the promoter of JFK, an E3-ligase that targets ING4 and whose mRNA levels increase during differentiation. Thus, we propose that ATF1 is required to induce ING4 transcription, while CREB1 suppresses ING4 and simultaneously activates its E3-ligase to tightly control the timing of ING4 expression. We compared the gene signature of luminal cells to that of the tumorigenic EMP cells and, surprisingly, found that 30% of the differentially expressed genes were also CREB/ATF targets. However, there is less than 10% overlap in these targets, indicating that CREB/ATF control distinct subsets of genes in differentiated luminal cells versus cancer cells. Some of EMP-specific CREB/ATF targets included GATA2, TWIST1, Necdin, and PPM1F, which were further validated by qRT-PCR and immunoblotting. CREB1 and ATF1 were highly phosphorylated in EMP cells and knockdown of CREB1 restored ING4 expression and suprabasal formation. Our working model is that AKT activation upon PTEN loss in transiently differentiating luminal cells results in premature and constitutive activation of CREB1/ATF1 bound to genes prior to induction of the ING4 chromatin switch. This prevents ING4 induction and the chromatin rearrangements required for terminal differentiation. In normal PrECs, CREB/ATF1 activation is tightly controlled by as yet undetermined factors and is only permitted when the proper CRE binding sites are exposed. This model helps to explain how loss of PTEN disrupts luminal cell terminal differentiation to promote prostate cancer oncogenesis. Citation Format: McLane Watson, Penny Berger, Sander Frank, Mary Winn, Cindy Miranti. CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B031.

RNA Polymerase II Activity of Type 3 Pol III Promoters

In eukaryotes, three RNA polymerases (Pol I, II, and III) are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters (7SK, U6, and H1) mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1 ≫ U6 > 7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nt upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters, and they offer additional flexibility for biotechnology applications of these genetic elements.

697 Epidermal tissue differentiation requires the uncharacterized protein RED1

The human genome encodes >4,000 uncharacterized open reading frames (ORFs) with undiscovered biological functions. To identify potentially novel mediators of epidermal differentiation from this candidate pool, RNA-Seq of primary human keratinocytes during calcium-induced differentiation was used to screen for uncharacterized ORFs whose transcript levels are increased during this process. The top up-regulated ORF encodes a 50 kD protein that we have termed Regulator of Epidermal Differentiation (RED) 1. An orthogonal screen also identified RED1 as highly down-regulated in primary human squamous cell carcinomas compared to patient-matched normal skin. As malignant neoplasms frequently demonstrate loss of differentiation, we hypothesized that RED1 is required for epidermal differentiation. To test this possibility, we undertook gain- and loss-of-function experiments in organotypic human epidermal tissue. Enforced expression of RED1 accelerated differentiation in regenerated human skin while CRISPR/Cas9-mediated depletion attenuated this process. In each case, whole transcriptome profiling to assess the global impact of altering RED1 expression identified gene groups that control skin differentiation, development, and function. Proximity-dependent biotin ligation assays in organotypic human epidermal tissue identified validated RED1 interactors including envoplakin (EVPL) and periplakin (PPL), genes known to be essential to skin differentiation, as well as genes not previously implicated in skin homeostasis, such as calcyclin binding protein (CACYBP). Gene set enrichment analysis suggests RED1 and its interacting proteins co-control distinct subsets of genes that induce epidermal differentiation and drive neoplastic transformation. Efforts are underway to study RED1s impacts on epidermal neoplasia. RED1 therefore represents a new regulator of epidermal differentiation with potential roles in skin carcinogenesis.

High-Resolution Mapping and Dynamics of the Transcriptome, Transcription Factors, and Transcription Co-Factor Networks in Classically and Alternatively Activated Macrophages.

Macrophages are the prime innate immune cells of the inflammatory response, and the combination of multiple signaling inputs derived from the recognition of host factors [e.g., interferon-g (IFN-γ)] and invading pathogen products (e.g., toll-like receptors (TLRs) agonists) are required to maintain essential macrophage function. The profound effects on biological outcomes of inflammation associated with IFN-γ pretreatment (“priming”) and TLR4 ligand bacterial lipopolysaccharide (LPS)-induced macrophage activation (M1 or classical activation) have long been recognized, but the underlying mechanisms are not well defined. Therefore, we analyzed gene expression profiles of macrophages and identified genes, transcription factors (TFs), and transcription co-factors (TcoFs) that are uniquely or highly expressed in IFN-γ-mediated TLR4 ligand LPS-inducible versus only TLR4 ligand LPS-inducible primary macrophages. This macrophage gene expression has not been observed in macrophage cell lines. We also showed that interleukin (IL)-4 and IL-13 (M2 or alternative activation) elicited the induction of a distinct subset of genes related to M2 macrophage polarization. Importantly, this macrophage gene expression was also associated with promoter conservation. In particular, our approach revealed novel roles for the TFs and TcoFs in response to inflammation. We believe that the systematic approach presented herein is an important framework to better understand the transcriptional machinery of different macrophage subtypes.

SUMOylation Regulates Transcription by the Progesterone Receptor A Isoform in a Target Gene Selective Manner.

Luminal breast cancers express estrogen (ER) and progesterone (PR) receptors, and respond to endocrine therapies. However, some ER+PR+ tumors display intrinsic or acquired resistance, possibly related to PR. Two PR isoforms, PR-A and PR-B, regulate distinct gene subsets that may differentially influence tumor fate. A high PR-A:PR-B ratio is associated with poor prognosis and tamoxifen resistance. We speculate that excessive PR-A marks tumors that will relapse early. Here we address mechanisms by which PR-A regulate transcription, focusing on SUMOylation. We use receptor mutants and synthetic promoter/reporters to show that SUMOylation deficiency or the deSUMOylase SENP1 enhance transcription by PR-A, independent of the receptors’ dimerization interface or DNA binding domain. De-SUMOylation exposes the agonist properties of the antiprogestin RU486. Thus, on synthetic promoters, SUMOylation functions as an independent brake on transcription by PR-A. What about PR-A SUMOylation of endogenous human breast cancer genes? To study these, we used gene expression profiling. Surprisingly, PR-A SUMOylation influences progestin target genes differentially, with some upregulated, others down-regulated, and others unaffected. Hormone-independent gene regulation is also PR-A SUMOylation dependent. Several SUMOylated genes were analyzed in clinical breast cancer database. In sum, we show that SUMOylation does not simply repress PR-A. Rather it regulates PR-A activity in a target selective manner including genes associated with poor prognosis, shortened survival, and metastasis.

A genomic atlas of human adrenal and gonad development.

In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis determination, the onset of steroidogenesis and primordial germ cell development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical stages of human embryonic and fetal development. RNA was extracted from 53 tissue samples between 6-10 wpc (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A mathematical model was constructed to investigate time-series changes across the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry. Using this approach, we have identified novel components of adrenal development (e.g. ASB4, NPR3) and confirmed the role of SRY as the main human testis-determining gene. By mathematical modelling time-series data we have found new genes up-regulated with SOX9 in the testis (e.g. CITED1), which may represent components of the testis development pathway. We have shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis (e.g. MGARP, FOXO4, MAP3K15, GRAMD1B, RMND2), as well as testis biomarkers (e.g. SCUBE1). We have also shown that the developing human ovary expresses distinct subsets of genes (e.g. OR10G9, OR4D5), but enrichment for established biological pathways is limited. This genomic atlas is revealing important novel aspects of human development and new candidate genes for adrenal and reproductive disorders.

A genomic atlas of human adrenal and gonad development

Background: In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis determination, the onset of steroidogenesis and primordial germ cell development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical stages of human embryonic and fetal development. Methods: RNA was extracted from 53 tissue samples between 6-10 wpc (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A mathematical model was constructed to investigate time-series changes across the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry. Results: Using this approach, we have identified novel components of adrenal development (e.g. ASB4, NPR3) and confirmed the role of SRY as the main human testis-determining gene. By mathematical modelling time-series data we have found new genes up-regulated with SOX9 in the testis (e.g. CITED1), which may represent components of the testis development pathway. We have shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis (e.g. MGARP, FOXO4, MAP3K15, GRAMD1B, RMND2), as well as testis biomarkers (e.g. SCUBE1). We have also shown that the developing human ovary expresses distinct subsets of genes (e.g. OR10G9, OR4D5), but enrichment for established biological pathways is limited. Conclusion: This genomic atlas is revealing important novel aspects of human development and new candidate genes for adrenal and reproductive disorders.

Corrigendum: The histone H2B-specific ubiquitin ligase RNF20/hBRE1 acts as a putative tumor suppressor through selective regulation of gene expression.

Histone monoubiquitylation is implicated in critical regulatory processes. We explored the roles of histone H2B ubiquitylation in human cells by reducing the expression of hBRE1/RNF20, the major H2B-specific E3 ubiquitin ligase. While H2B ubiquitylation is broadly associated with transcribed genes, only a subset of genes was transcriptionally affected by RNF20 depletion and abrogation of H2B ubiquitylation. Gene expression dependent on RNF20 includes histones H2A and H2B and the p53 tumor suppressor. In contrast, RNF20 suppresses the expression of several proto-oncogenes, which reside preferentially in closed chromatin and are modestly transcribed despite bearing marks usually associated with high transcription rates. Remarkably, RNF20 depletion augmented the transcriptional effects of epidermal growth factor (EGF), increased cell migration, and elicited transformation and tumorigenesis. Furthermore, frequent RNF20 promoter hypermethylation was observed in tumors. RNF20 may thus be a putative tumor suppressor, acting through selective regulation of a distinct subset of genes.

Biotechnological Potential of Cold Adapted Pseudoalteromonas spp. Isolated from 'Deep Sea' Sponges.

The marine genus Pseudoalteromonas is known for its versatile biotechnological potential with respect to the production of antimicrobials and enzymes of industrial interest. We have sequenced the genomes of three Pseudoalteromonas sp. strains isolated from different deep sea sponges on the Illumina MiSeq platform. The isolates have been screened for various industrially important enzymes and comparative genomics has been applied to investigate potential relationships between the isolates and their host organisms, while comparing them to free-living Pseudoalteromonas spp. from shallow and deep sea environments. The genomes of the sponge associated Pseudoalteromonas strains contained much lower levels of potential eukaryotic-like proteins which are known to be enriched in symbiotic sponge associated microorganisms, than might be expected for true sponge symbionts. While all the Pseudoalteromonas shared a large distinct subset of genes, nonetheless the number of unique and accessory genes is quite large and defines the pan-genome as open. Enzymatic screens indicate that a vast array of enzyme activities is expressed by the isolates, including β-galactosidase, β-glucosidase, and protease activities. A β-glucosidase gene from one of the Pseudoalteromonas isolates, strain EB27 was heterologously expressed in Escherichia coli and, following biochemical characterization, the recombinant enzyme was found to be cold-adapted, thermolabile, halotolerant, and alkaline active.

A genomic atlas of human adrenal and gonad development

Background: In humans, the adrenal glands and gonads undergo distinct biological events between 6-10 weeks post conception (wpc), such as testis determination, the onset of steroidogenesis and primordial germ cell development. However, relatively little is currently known about the genetic mechanisms underlying these processes. We therefore aimed to generate a detailed genomic atlas of adrenal and gonad development across these critical stages of human embryonic and fetal development.Methods: RNA was extracted from 53 tissue samples between 6-10 wpc (adrenal, testis, ovary and control). Affymetrix array analysis was performed and differential gene expression was analysed using Bioconductor. A mathematical model was constructed to investigate time-series changes across the dataset. Pathway analysis was performed using ClueGo and cellular localisation of novel factors confirmed using immunohistochemistry.Results: Using this approach, we have identified novel components of adrenal development (e.g.ASB4,NPR3) and confirmed the role ofSRYas the main human testis-determining gene. By mathematical modelling time-series data we have found new genes up-regulated withSOX9in the testis (e.g.CITED1), which may represent components of the testis development pathway. We have shown that testicular steroidogenesis has a distinct onset at around 8 wpc and identified potential novel components in adrenal and testicular steroidogenesis (e.g.MGARP,FOXO4,MAP3K15,GRAMD1B,RMND2), as well as testis biomarkers (e.g.SCUBE1). We have also shown that the developing human ovary expresses distinct subsets of genes (e.g.OR10G9,OR4D5), but enrichment for established biological pathways is limited.Conclusion: This genomic atlas is revealing important novel aspects of human development and new candidate genes for adrenal and reproductive disorders.

Decreased expression of indoleamine 2, 3-dioxygenase in villous stromal endothelial cells of placentas with preeclampsia and/or fetal growth restriction

Hypoxia inducible factor (HIF)-1 and HIF-2 are transcription factors that mediate the cellular response to hypoxia. Although HIF-1 and HIF-2 share the same target genes, both proteins activate a distinct subset of genes. To identify the target genes preferentially activated by HIF-2 in endothelial cells, DNA microarray analysis was performed to human umbilical vein endothelial cells (HUVECs) with forced expression of either HIF-1α or HIF-2α. In the present study, which is the first comparative study of target genes induced by either HIF-1 or HIF-2 in HUVECs, HIF-1 (and not HIF-2) stimulated mainly glycolytic, hexose metabolic and alcohol metabolic gene expression. However, HIF-2 (but not HIF-1) induced developmental gene expressions such as Fms-like tyrosine kinase 1 (Flt-1) and angiopoietin 2 (Angpt2). Furthermore, CD82 was up-regulated by HIF-2, but not by HIF-1, in response to hypoxia. HIF-2 regulated CD82 gene expression by binding to its HRE consensus sequence located within its first intron. Assessing the function of CD82 in HUVECs forced its expression. This result revealed that CD82 negatively regulates the HUVECs cell migration. The induction of CD82 gene expression in endothelial cells provided new insights into a specific function of HIF-2.