Disease-gene discovery by integration of 3D gene expression and transcription factor binding affinities

Abstract Motivation: The computational evaluation of candidate genes for hereditary disorders is a non-trivial task. Several excellent methods for disease-gene prediction have been developed in the past 2 decades, exploiting widely differing data sources to infer disease-relevant functional relationships between candidate genes and disorders. We have shown recently that spatially mapped, i.e. 3D, gene expression data from the mouse brain can be successfully used to prioritize candidate genes for human Mendelian disorders of the central nervous system. Results: We improved our previous work 2-fold: (i) we demonstrate that condition-independent transcription factor binding affinities of the candidate genes' promoters are relevant for disease-gene prediction and can be integrated with our previous approach to significantly enhance its predictive power; and (ii) we define a novel similarity measure—termed Relative Intensity Overlap—for both 3D gene expression patterns and binding affinity profiles that better exploits their disease-relevant information content. Finally, we present novel disease-gene predictions for eight loci associated with different syndromes of unknown molecular basis that are characterized by mental retardation. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Bis-(3 ',5 ') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K-d similar to 2 mu M). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence

Functional effects of variation in transcription factor binding highlight long-range gene regulation by epromoters.

Identifying DNA cis-regulatory modules (CRMs) that control the expression of specific genes is crucial for deciphering the logic of transcriptional control. Natural genetic variation can point to the possible gene regulatory function of specific sequences through their allelic associations with gene expression. However, comprehensive identification of causal regulatory sequences in brute-force association testing without incorporating prior knowledge is challenging due to limited statistical power and effects of linkage disequilibrium. Sequence variants affecting transcription factor (TF) binding at CRMs have a strong potential to influence gene regulatory function, which provides a motivation for prioritizing such variants in association testing. Here, we generate an atlas of CRMs showing predicted allelic variation in TF binding affinity in human lymphoblastoid cell lines and test their association with the expression of their putative target genes inferred from Promoter Capture Hi-C and immediate linear proximity. We reveal >1300 CRM TF-binding variants associated with target gene expression, the majority of them undetected with standard association testing. A large proportion of CRMs showing associations with the expression of genes they contact in 3D localize to the promoter regions of other genes, supporting the notion of 'epromoters': dual-action CRMs with promoter and distal enhancer activity

BosR (BB0647) Controls the RpoN-RpoS Regulatory Pathway and Virulence Expression in Borrelia burgdorferi by a Novel DNA-Binding Mechanism

In Borrelia burgdorferi (Bb), the Lyme disease spirochete, the alternative σ factor σ54 (RpoN) directly activates transcription of another alternative σ factor, σS (RpoS) which, in turn, controls the expression of virulence-associated membrane lipoproteins. As is customary in σ54-dependent gene control, a putative NtrC-like enhancer-binding protein, Rrp2, is required to activate the RpoN-RpoS pathway. However, recently it was found that rpoS transcription in Bb also requires another regulator, BosR, which was previously designated as a Fur or PerR homolog. Given this unexpected requirement for a second activator to promote σ54-dependent gene transcription, and the fact that regulatory mechanisms among similar species of pathogenic bacteria can be strain-specific, we sought to confirm the regulatory role of BosR in a second virulent strain (strain 297) of Bb. Indeed, BosR displayed the same influence over lipoprotein expression and mammalian infectivity for strain Bb 297 that were previously noted for Bb strain B31. We subsequently found that recombinant BosR (rBosR) bound to the rpoS gene at three distinct sites, and that binding occurred despite the absence of consensus Fur or Per boxes. This led to the identification of a novel direct repeat sequence (TAAATTAAAT) critical for rBosR binding in vitro. Mutations in the repeat sequence markedly inhibited or abolished rBosR binding. Taken together, our studies provide new mechanistic insights into how BosR likely acts directly on rpoS as a positive transcriptional activator. Additional novelty is engendered by the facts that, although BosR is a Fur or PerR homolog and it contains zinc (like Fur and PerR), it has other unique features that clearly set it apart from these other regulators. Our findings also have broader implications regarding a previously unappreciated layer of control that can be involved in σ54–dependent gene regulation in bacteria

Integrative modeling of Transcription Factor cooperativity and its effects on phenotypic variability

The regulation of biological processes relies on a complex nucleotide code embedded in our DNA. Its decoding and interpretation is the main task of Transcription Factors (TFs), which altogether enable the recognition and modulation of gene expression. Whenever factors bind to DNA, a set of additional criteria and conditions need to be satisfied, such as TF concentration, DNA openness, and cooperativity with other binding factors. Such combinations of DNA-bound TFs, as well as their structural and functional cooperativity, allow a more fine-grained control of gene expression due to subtle changes in specificity in both DNA recognition and functional outcomes. This thesis explores the prediction of structural TF cooperativity and its biological consequences. Additionally, examples of functional cooperativity are presented and discussed in the context of neuronal activity and reprogramming. Altogether, this dissertation provides an extensive set of insights to better understand the complex interplay between TFs cooperativity and phenotypes

Drug Discovery Using Chemical Systems Biology: Identification of the Protein-Ligand Binding Network To Explain the Side Effects of CETP Inhibitors

Systematic identification of protein-drug interaction networks is crucial to correlate complex modes of drug action to clinical indications. We introduce a novel computational strategy to identify protein-ligand binding profiles on a genome-wide scale and apply it to elucidating the molecular mechanisms associated with the adverse drug effects of Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors are a new class of preventive therapies for the treatment of cardiovascular disease. However, clinical studies indicated that one CETP inhibitor, Torcetrapib, has deadly off-target effects as a result of hypertension, and hence it has been withdrawn from phase III clinical trials. We have identified a panel of off-targets for Torcetrapib and other CETP inhibitors from the human structural genome and map those targets to biological pathways via the literature. The predicted protein-ligand network is consistent with experimental results from multiple sources and reveals that the side-effect of CETP inhibitors is modulated through the combinatorial control of multiple interconnected pathways. Given that combinatorial control is a common phenomenon observed in many biological processes, our findings suggest that adverse drug effects might be minimized by fine-tuning multiple off-target interactions using single or multiple therapies. This work extends the scope of chemogenomics approaches and exemplifies the role that systems biology has in the future of drug discovery

T cell-mediated cytotoxicity against patient-derived cervical cancer organoids

Cervical cancer is one of the most common cancers affecting women, and the mortality-to incidence ratio (MIR) of cervical cancer varies between developed and developing regions, North America had the lowest MIR (0.36), followed by MIR in Europe (0.40), while Africa had the highest MIR (0.68). Infection with human high-risk papillomaviruses (HR-HPV) is the most important driver of cervical cancer, but after persistent HR-HPV infection, infected cervix cells still need many years to transform into cancer. Since 90% of failures in current treatments are related to drug resistance, the development of more effective therapies is urgently required. However, many new therapies have to be terminated because of the unsatisfactory drug efficacy at clinical phases, which reflects the current flaws of preclinical drug models. In this context, establishing precise in vitro models to screen out the therapeutic targets and to assess the new drug efficacy against cervical cancer is vital. In this study, based on patient-derived cervical organoids, multi-omic analysis including RNA sequencing and HLA-restricted peptidome sequencing was used to investigate dysregulated genes, biology processes, and HLA-presented epitopes in HPV-transformed and cancerous cervical organoids. Totally 6,515 HLA class I and 719 HLA class II restricted peptides were yielded, thereafter the immunogenicity of the epitopes was predicted with public machine learning-based tools. Interestingly, a large number of upregulated genes and highly immunogenic epitopes in HPV+ and cancer samples were associated with DNA repair. Thus, the role DNA repair-related molecules appear to play in cervical cancer therapies and prognosis was further explored. I first constructed a DNA repair-related gene score (DRGscore) with the public cervical cancer dataset of the Cancer Genome Atlas Program (TCGA-CESE). In the following, I observed a negative correlation between DRGscore and the prognosis of cervical cancer patients. The tumor microenvironment of patients with lower DRGscore contained more immune effectors and less immune suppressors, which suggested the negative correlation between DRGscore and sensitivity to immunotherapy. This finding could provide clues for patient medication guidance: high DRGscore patients might benefit from DNA repair inhibitors to reduce chemoradiotherapy resistance and gain a better prognosis. In contrast, low DRGscore patients rather might be sensitive to different immunotherapy including immune checkpoint inhibitors, and adoptive T cell therapy. Due to HPV infection and viral genome integration, HPV+ and cervical cancer cells are generally under stress conditions, which induces the intracellular accumulation of phosphoantigens (pAgs) that can be recognized by Vγ9Vδ2 T cells in an HLA-independent manner. The DNA repair activities in host cells are exploited by HPV for replication, which may provide some potential ligands recognized by Vγ9Vδ2 T cell receptors. To figure out our hypotheses, I investigated the Vγ9Vδ2 T cell cytotoxic effect against HPV-transformed and cervical cancer cells based on our cervical organoid model. Importantly, the cytotoxic effect was significantly enhanced against HPV+ and cancer organoids compared to healthy organoids in the presence or the absence of bromohydrin pyrophosphate (BrHPP), a synthetic phosphoantigen which activates Vγ9Vδ2 T. Moreover, the BrHPP dependent cytotoxic effect was found strikingly to be reduced to the level in non-activated Vγ9Vδ2 T group with the blockade of butyrophilin-subfamily members (BTN3A, BTN2A1). Besides, CD107a over expression was also associated with BrHPP stimulation. Both pieces of evidence indicated the BTN-subfamily members were mainly responsible for the cytotoxic effect with BrHPP activation and the self-active Vγ9Vδ2 T cells induced indiscriminate killing against HPV+, cancer cells but also healthy cells via degranulation including granzyme B (GzmB), platelet rich fibrin (Prf), interferon gamma (IFNγ) secretion. While the mechanism of the specific cytotoxicity against HPV+ and cancer organoids in the BrHPP absence condition still remained vague. I concluded based on the bioinformatic analysis of multi-omic data that the differential recognition of non-healthy cells by Vγ9Vδ2 T cells depends on multiple-ligand interactions, which should be further explored in the future. Overall, within my thesis project, stable patient-derived cervical organoid lines were established, which acted as a promising in vitro model for screening cervical cancer associated epitopes and for exploring T cell-induced cytotoxic effects. Thus, the organoid-T cell coculture model that I established could prove useful for testing additional modulators (e.g. small molecules, antibodies, tumor sensitizers, etc.). Consequently, my investigations on the Vγ9Vδ2 T-mediated killing effect against HPV-transformed cervical cancer cells have shed light on the potential future utilization of Vγ9Vδ2 T in clinical settings

The International Human Epigenome Consortium: A Blueprint for Scientific Collaboration and Discovery

The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease

Structure-based mutagenesis of the integrase-LEDGF/p75 interface uncouples a strict correlation between in vitro protein binding and HIV-1 fitness

AbstractLEDGF/p75 binding-defective IN mutant viruses were previously characterized as replication-defective, yet RNAi did not reveal an essential role for the host factor in HIV-1 replication. Correlative analyses of protein binding and viral fitness were expanded here by targeting 12 residues at the IN-LEDGF/p75 binding interface. Whereas many of the resultant viruses were defective, the majority of the INs displayed wild-type in vitro integration activities. Though an overall trend of parallel loss of LEDGF/p75 binding and HIV-1 infectivity was observed, a strict correlation was not. His-tagged INA128Q, derived from a phenotypically wild-type virus, failed to pull-down LEDGF/p75, but INA128Q was effectively recovered in a reciprocal GST pull-down assay. Under these conditions, INH171A, also derived from a phenotypically wild-type virus, interacted less efficiently than a previously described interaction-defective mutant, INQ168A. Thus, the relative affinity of the in vitro IN-LEDGF/p75 interaction is not a universal predictor of IN mutant viral fitness

Mechanism Of Nucleosome Targeting By Pioneer Transcription Factors

Transcription factors (TFs) forage the genome to instruct cell plasticity, identity, and differentiation. These developmental processes are elicited through TF engagement with chromatin. Yet, how and which TFs can engage with chromatin and thus, nucleosomes, remains largely unexplored. Pioneer TFs are TF that display a high affinity for nucleosomes. Extensive genetic and biochemical studies on the pioneer TF FOXA, a driver of fibroblast to hepatocyte reprogramming, revealed its nucleosome binding ability and chromatin targeting lead to chromatin accessibility and subsequent cooperative binding of TFs. Similarly, a number of reprogramming TFs have been suggested to have pioneering activity due to their ability to target compact chromatin and increase accessibility and enhancer formation in vivo. But whether these factors directly interact with nucleosomes remains to be assessed. Here we test the nucleosome binding ability of the cell reprogramming TFs, Oct4, Sox2, Klf4 and cMyc, that are required for the generation of induced pluripotent stem cells. In addition, we also test neuronal and macrophage reprogramming TFs. Our study shows that reprogramming TFs bind nucleosomes with a range of nucleosome binding affinities, indicating that although specific cocktails of TFs are required for reprogramming, mechanistically these TFs show differential nucleosome interacting behaviors. These results allowed us to assess differential features between TFs nucleosome binding ability and to correlate their binding with reprogramming potential. To determine how general is nucleosome binding we extended our analysis to screen 593 of the 2,000 predicted human TFs in the genome for potential nucleosome binding and validated their binding in solution. Based on 3D structural analysis, we proposed that strong nucleosome binders anchor DNA through short -helixes and have a flexible and adaptable DNA binding domain while weak nucleosome binders use -sheets or unstructured regions and have a higher rigidity within their DNA binding domain. Through the experiments presented in this dissertation we present the first study revealing the shared structural features contributing to nucleosome binding potential of pioneer TFs and thus allow for predication of novel pioneer TFs with cell reprogramming potential