Deregulation upon DNA damage revealed by joint analysis of context-specific perturbation data

<p>Abstract</p> <p>Background</p> <p>Deregulation between two different cell populations manifests itself in changing gene expression patterns and changing regulatory interactions. Accumulating knowledge about biological networks creates an opportunity to study these changes in their cellular context.</p> <p>Results</p> <p>We analyze re-wiring of regulatory networks based on cell population-specific perturbation data and knowledge about signaling pathways and their target genes. We quantify deregulation by merging regulatory signal from the two cell populations into one score. This joint approach, called JODA, proves advantageous over separate analysis of the cell populations and analysis without incorporation of knowledge. JODA is implemented and freely available in a Bioconductor package 'joda'.</p> <p>Conclusions</p> <p>Using JODA, we show wide-spread re-wiring of gene regulatory networks upon neocarzinostatin-induced DNA damage in Human cells. We recover 645 deregulated genes in thirteen functional clusters performing the rich program of response to damage. We find that the clusters contain many previously characterized neocarzinostatin target genes. We investigate connectivity between those genes, explaining their cooperation in performing the common functions. We review genes with the most extreme deregulation scores, reporting their involvement in response to DNA damage. Finally, we investigate the indirect impact of the ATM pathway on the deregulated genes, and build a hypothetical hierarchy of direct regulation. These results prove that JODA is a step forward to a systems level, mechanistic understanding of changes in gene regulation between different cell populations.</p

Deregulation of embryonic transcription factors in human epithelial cancers: new perspectives in breast and liver tumors

Carcinogenesis is commonly referred to as a multi-step process in which normal cells develop progressively into hyperplasia, carcinoma in situ, invasive cancer and metastasis. Several evidences indicate that transcription factors, which act as master regulators of embryonic development, may play a central role in this pathologic process. Indeed, growing evidence suggests that cancer cells often reactivate latent developmental programs in order to efficiently execute the multi-step process of tumorigenesis. Reminiscent of their function during development, embryonic transcription factors regulate changes in gene expression that promote tumor cell growth, cell survival and motility, as well as a morphogenetic process called epithelial-mesenchymal transition (EMT), which is implicated in both metastasis and tumor recurrence. Because of their pivotal roles in tumor progression, these factors represent valuable new biomarkers for cancer detection as well as promising new targets for alternative anti-cancer therapies. The present doctoral work explores the role of embryonic transcription factors deregulation in epithelial cancers and their therapeutic implications in the frontiers of precision oncology. More specifically, the first project identified MDM2 as a specific synthetic lethal partner of GATA3, an embryonic master regulator of the mammary gland often mutated in estrogen receptor-positive breast cancers. The second project identified the homeobox transcription factor HOXA13 as a novel oncogene, whose overexpression results in hepatocarcinogenesis in mice through the induction of chromosomal instability

Modeling signal transduction pathways and their transcriptional response

This thesis is concerned with revealing regulation of gene expression. The basic motivation behind our work is that gene regulation can be better resolved when analyzed in a cellular context of the upstream signaling pathway and known regulatory targets. Our source of data are perturbation experiments, which are performed on pathway components and induce changes in gene expression. In such a way, they connect the signaling pathway to its downstream target genes. This chapter starts with an introduction to the cellular con- text considered in the thesis (section 1.1) and the principles of perturbation experiments (section 1.2). We end with a concise summary of three approaches that comprise this thesis. The approaches tackle various problems in the process of revealing context-speci c regulatory networks (section 1.3). We deal with di erential expression analysis of the per- turbation data, enhanced with known transcription factor targets serving as examples of di erential genes (chapter 2), pathway model-based planning of informative perturbation experiments (chapter 3), and nally, with deregulation analysis, i.e., comparing changes in gene regulation between two di erent cell populations (chapter 4)

Phosphorylation by CK2 regulates MUS81/EME1 in mitosis and after replication stress

The MUS81 complex is crucial for preserving genome stability through the resolution of branched DNA intermediates in mitosis. However, untimely activation of the MUS81 complex in S-phase is dangerous. Little is known about the regulation of the human MUS81 complex and how deregulated activation affects chromosome integrity. Here, we show that the CK2 kinase phosphorylates MUS81 at Serine 87 in late-G2/mitosis, and upon mild replication stress. Phosphorylated MUS81 interacts with SLX4, and this association promotes the function of the MUS81 complex. In line with a role in mitosis, phosphorylation at Serine 87 is suppressed in S-phase and is mainly detected in the MUS81 molecules associated with EME1. Loss of CK2-dependent MUS81 phosphorylation contributes modestly to chromosome integrity, however, expression of the phosphomimic form induces DSBs accumulation in S-phase, because of unscheduled targeting of HJ-like DNA intermediates, and generates a wide chromosome instability phenotype. Collectively, our findings describe a novel regulatory mechanism controlling the MUS81 complex function in human cells. Furthermore, they indicate that, genome stability depends mainly on the ability of cells to counteract targeting of branched intermediates by the MUS81/EME1 complex in S-phase, rather than on a correct MUS81 function in mitosis

Understanding the molecular pathogenesis of HIV-associated Burkitt Lymphoma – the impact of HIV-1 protein Tat on lymphoma driver genes

Burkitt Lymphoma (BL) is a B cell non-Hodgkin lymphoma that occurs as three distinct subtypes, namely: endemic, sporadic, and immunodeficiency/HIV-associated. This cancer represents a frequent cause of mortality among HIV+ people in Southern Africa which has the highest incidence of HIV/AIDS worldwide. Recent reports associate a direct oncogenic function of HIV in BL development. However, the molecular mechanisms underlying this HIV-associated malignancy are not well understood. This study explores the oncogenic potential of HIV-1 protein Tat in BL via its ability to manipulate the expression of c-MYC and activation-induced cytidine deaminase (AID), two key drivers of BL progression. Using dual-luciferase reporter assays, HIV-1 Tat was shown to enhance the activity of the cMYC promoter (-2324 bp - +537 bp), which corresponded with elevated c-MYC protein levels in BL cells (Ramos) expressing HIV-1 Tat. By generating sequential promoter deletions, the minimal promoter region mediating HIV-1 Tat induced activation was identified. Site-directed mutagenesis indicated that this response was mediated by AP-1 binding elements, and coimmunoprecipitation assays revealed that HIV-1 Tat and the AP-1 factor JunB interacted within the same complex. Chromatin immunoprecipitation assays confirmed that JunB bound the c-MYC promoter in vivo under the influence of HIV-1 Tat. The effect of HIV-1 Tat on the expression of the DNA editing enzyme AID was also investigated. Dual-luciferase assays revealed that HIV-1 Tat could enhance the activity of the three regulatory regions of the AICDA gene, namely R1, R2 and R4. This translated into elevated AID protein expression in Ramos cells expressing HIV-1 Tat, which was also reflected in an increase in genomic instability as shown by enhanced phosphorylated H2AX expression. Sequential promoter deletions of the R1 promoter did not lead to a loss in HIV-1 Tat-mediated activation, pointing to potential post-transcriptional regulation. Indeed, HIV-1 Tat was found to downregulate the expression of hsa-miRNA-181b-5p, a known repressor of the murine Aicda gene. Furthermore, using reporter assays, we show that an hsa-miRNA-181b5p mimic could repress AICDA via the full-length 3'UTR which contains three putative binding elements for the miRNA. To date, this study reveals that HIV-1 Tat induced c-MYC promoter activation is mediated by two AP-1 binding sites. HIV-1 Tat was shown to couple with JunB and bind to both AP-1 sites on the c-MYC promoter inducing promoter activation. Furthermore, this study reveals a novel mechanism of AID deregulation via HIV-1 Tat-mediated miRNA perturbation. Lastly, we show that HIV-1 Tat interferes with hsa-miRNA-181b-5p expression in B cells, alleviating AICDA 3'UTR repression

Study of the human SOX17 locus and its genetic determinants in definitive endoderm

Embryonic development and organogenesis depend on the precise spatiotemporal expression of specific sets of genes. Precisely controlled gene expression ensures cell state transitions, especially in the early stages of development, as gastrulation. These complex multi-layered cellular processes are orchestrated by the interfacing of the epigenome, 3-dimensional (3D) nuclear organization, cis-regulatory elements (CREs) with transcription factors (TF), and long non-coding RNAs (lncRNAs). In the gastrulating embryo, definitive endoderm is specified from the pluripotent epiblast following a series of regulatory events, including the activation of SOX17, a key TF of that particular germ layer. Although SOX17 has been extensively studied in early embryonic development, the precise control of its activation, the locus, and the epigenetic rules governing its genetic regulatory network (GRN) remains poorly investigated. In my thesis, I in-depth characterized the human SOX17 locus, exploring the relevance and regulatory impact of 3D nuclear organization, its distal CREs, and their activity. I applied a series of loss of function (LOF) and transgenic experiments to dissect the locus at a satisfactory resolution. In particular, I showed SOX17 among a subset of developmental regulators topologically isolated within CTCF-CTCF loop domains and highlighted the importance of gene control in 3D within this type of domain. I pinpointed the relevance of SOX17’s distal CREs and their definitive endoderm-specific interaction and showed this interaction to be highly dependent on CTCF-CTCF loop-formation to guarantee proper gene control. I found CRE-dependent SOX17 gene deregulation associated with poor definitive endoderm differentiation outcome and a stalled “mesendodermal-like” phenotype. Assessing the genetic identity of different CREs, I divulged the presence of a novel lncRNA within the locus, namely LNCSOX17. I fully characterized LNCSOX17 and established its identity as a bona fide lncRNA through a series of genetic perturbations. I demonstrated the importance of LNCSOX17 for forming definitive endoderm and the lack of participation in SOX17 cis-acting gene control. I associated the loss of LNCSOX17 RNA but not its active transcription at the locus with an aberrant endodermal transcriptome, a lack of epithelial-to-mesenchymal transition (EMT), and the hyperactivity of the detrimental definitive endoderm JNK/JUN/AP1 signaling pathway. I found definitive endoderm lacking LNCSOX17 to be functionally impeded in the generation of pancreatic progenitor populations. The studies within this thesis serve as valuable examples to support the functional relevance of 3D nuclear organization and its importance for developmental gene control in cis via CTCF-CTCF loop domain-mediated CRE-promoter contact facilitation. They associate developmental gene expression levels with various phenotypes, identify a so far unknown developmental lncRNA molecule, and imply its relevance for the formation of definitive endoderm. The outlined results advance our knowledge of developmental TF gene-control and its importance for the development of human definitive endoderm

Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models

Drug-induced liver injury (DILI) is a patient-specific, temporal, multifactorial pathophysiological process that cannot yet be recapitulated in a single in vitro model. Current preclinical testing regimes for the detection of human DILI thus remain inadequate. A systematic and concerted research effort is required to address the deficiencies in current models and to present a defined approach towards the development of new or adapted model systems for DILI prediction. This Perspective defines the current status of available models and the mechanistic understanding of DILI, and proposes our vision of a roadmap for the development of predictive preclinical models of human DILI

Proteomics Reveals Global Regulation of Protein SUMOylation by ATM and ATR Kinases during Replication Stress

Summary: The mechanisms that protect eukaryotic DNA during the cumbersome task of replication depend on the precise coordination of several post-translational modification (PTM)-based signaling networks. Phosphorylation is a well-known regulator of the replication stress response, and recently an essential role for SUMOs (small ubiquitin-like modifiers) has also been established. Here, we investigate the global interplay between phosphorylation and SUMOylation in response to replication stress. Using SUMO and phosphoproteomic technologies, we identify thousands of regulated modification sites. We find co-regulation of central DNA damage and replication stress responders, of which the ATR-activating factor TOPBP1 is the most highly regulated. Using pharmacological inhibition of the DNA damage response kinases ATR and ATM, we find that these factors regulate global protein SUMOylation in the protein networks that protect DNA upon replication stress and fork breakage, pointing to integration between phosphorylation and SUMOylation in the cellular systems that protect DNA integrity. : Munk et al. use mass spectrometry-based proteomics to analyze the interplay between SUMOylation and phosphorylation in replication stress. They analyze changes in the SUMO and phosphoproteome after MMC and hydroxyurea treatments and find that the DNA damage response kinases ATR and ATM globally regulate SUMOylation upon replication stress and fork breakage. Keywords: Replication stress, quantitative proteomics, phosphoproteomics, SUMO, ATR, ATM, TOPBP1, MMC, kinase inhibitors, hydroxyure