B-cell lymphoma gene regulatory networks: biological consistency among inference methods

Despite the development of numerous gene regulatory network (GRN) inference methods in the last years, their application, usage and the biological significance of the resulting GRN remains unclear for our general understanding of large-scale gene expression data in routine practice. In our study, we conduct a structural and a functional analysis of B-cell lymphoma GRNs that were inferred using 3 mutual information-based GRN inference methods: C3Net, BC3Net and Aracne. From a comparative analysis on the global level, we find that the inferred B-cell lymphoma GRNs show major differences. However, on the edge-level and the functional-level - that are more important for our biological understanding - the B-cell lymphoma GRNs were highly similar among each other. Also, the ranks of the degree centrality values and major hub genes in the inferred networks are highly conserved as well. Interestingly, the major hub genes of all GRNs are associated with the G-protein-coupled receptor pathway, cell-cell signaling and cell cycle. This implies that hub genes of the GRNs can be highly consistently inferred with C3Net, BC3Net and Aracne, representing prominent targets for signaling pathways. Finally, we describe the functional and structural relationship between C3Net, BC3Net and Aracne gene regulatory networks. Our study shows that these GRNs that are inferred from large-scale gene expression data are promising for the identification of novel candidate interactions and pathways that play a key role in the underlying mechanisms driving cancer hallmarks. Overall, our comparative analysis reveals that these GRNs inferred with considerably different inference methods contain large amounts of consistent, method independent, biological information

Beyond transcription : a post-transcriptional role of 3D chromatin crosstalk in oncogene regulation

This thesis explores how stochastic chromatin fibre interactions, chromatin organization in the 3D nuclear architecture, and environmental signals collaborate to regulate MYC oncogene expression in human colon cancer cells. In Paper I, we employ the ultra-sensitive Nodewalk technique to uncover the dynamic and stochastic nature of chromatin networks impinging on MYC. The analyses revealed that the MYC interactome mainly consists of stochastic pairwise interactions between MYC and its flanking enhancers in two neighbouring topologically associated domains (TADs), which are insulated self-interacting genomic domains. The limits of Nodewalk were also pushed to enable the detection of interactions in very small cell populations, corresponding to the genomic content of ~7 cells. Comparing the frequency of interactions detected in such small input samples with ensemble interactomes of large cell populations uncovered that the enhancer hubs of the ensemble interactomes that appear to simultaneously interact with MYC likely represent virtual events, which are not present in reality at the single cell level. These data support a model where MYC interacts with its enhancers in a mutually exclusive way, with MYC screening for enhancer contacts, rather than the other way around. Paper II provides a detailed understanding of a novel post-transcriptional mechanism of enhancer action on MYC expression. We have thus uncovered that the cancer-specific recruitment of the MYC gene to nuclear pores and ensuing rapid nuclear export of MYC transcripts - a process that increases MYC expression by enabling the escape of MYC mRNAs from rapid decay in the nucleus - require a CTCF binding site positioned within the colorectal oncogenic super-enhancer. Genetic editing by CRISPR-Cas9 was thus commissioned to establish two clones of human colon cancer cells with a mutated sequence in the OSE-specific CTCFBS. Comparing the mutant cells to the parental cell line, we uncovered that the WNT-dependent increase in the nuclear export rate of MYC transcripts was abrogated in the CTCFBS mutant clones, providing the first genetic evidence of super- enhancer-mediated gene gating in human cells. In line with this finding, the OSE-specific CTCFBS thus conferred a significant growth advantage to the parental colon cancer cells, compared to the mutant clones. Moreover, we found that WNT-dependent CCAT1 eRNA transcription is mediated by the OSE-specific CTCFBS that is required for recruitment of AHCTF1 to the OSE to mediate the positioning of the OSE to the nuclear periphery, enabling the subsequent facilitation of MYC mRNA export. A multistep molecular process including WNT signalling and the OSE-specific CTCFBS thus underlies the gene gating of MYC in human colon cancer cells, and could potentially be targeted for diagnostic or therapeutic uses. In summary, this thesis explores the dynamics of the stochastic interactomes impinging on the MYC oncogene, and provides new insights on the role of 3D chromatin orchestration in the transcriptional regulation of MYC. Our analyses uncovered the molecular factors involved in the gene gating of MYC, and thus increase our understanding of tumour development. These findings could potentially be beneficial for future diagnostic approaches, or for targeted therapeutic strategies in the treatment of cancer

Herpesviruses and Intermediate Filaments: Close Encounters with the Third Type

Intermediate filaments (IF) are essential to maintain cellular and nuclear integrity and shape, to manage organelle distribution and motility, to control the trafficking and pH of intracellular vesicles, to prevent stress-induced cell death, and to support the correct distribution of specific proteins. Because of this, IF are likely to be targeted by a variety of pathogens, and may act in favor or against infection progress. As many IF functions remain to be identified, however, little is currently known about these interactions. Herpesviruses can infect a wide variety of cell types, and are thus bound to encounter the different types of IF expressed in each tissue. The analysis of these interrelationships can yield precious insights into how IF proteins work, and into how viruses have evolved to exploit these functions. These interactions, either known or potential, will be the focus of this review

3D genome organization during lymphocyte development and activation

Chromosomes have a complex three-dimensional (3D) architecture comprising A/B compartments, topologically associating domains and promoter-enhancer interactions. At all these levels, the 3D genome has functional consequences for gene transcription and therefore for cellular identity. The development and activation of lymphocytes involves strict control of gene expression by transcription factors (TFs) operating in a three-dimensionally organized chromatin landscape. As lymphocytes are indispensable for tissue homeostasis and pathogen defense, and aberrant lymphocyte activity is involved in a wide range of human morbidities, acquiring an in-depth understanding of the molecular mechanisms that control lymphocyte identity is highly relevant. Here we review current knowledge of the interplay between 3D genome organization and transcriptional control during B and T lymphocyte development and antigen-dependent activation, placing special emphasis on the role of TFs

IL-36 gamma promotes anti-tumor immunity through therapeutic induction of tumor-associated tertiary lymphoid structures

The last decade has advanced our understanding of the composition of the tumor-immune microenvironment and its role as a potential target for therapeutic intervention. Recently, tertiary lymphoid structures have been observed to develop in the tumor microenvironment and serve as a positive prognostic marker in many types of solid tumors. The major signals that control tertiary lymphoid organogenesis are the same as those that direct the development of secondary lymphoid organs. Tertiary lymphoid structures are classically characterized by high endothelial venules, which serve to recruit T cells, dendritic cells, and B cells to sites of persistent inflammation and locally prime T cells against tumor-derived antigens. Our group has long been interested in understanding whether tertiary lymphoid structures can be therapeutically induced to form within the tumor microenvironment and induce a protective anti-tumor immune response. In previous studies, we characterized dendritic cells engineered to overexpress the Type-1 transactivator Tbet (i.e. DC.Tbet) and showed that they are able to delay tumor progression following intratumoral injection in a murine model of sarcoma. In this work, I show that the effector molecule responsible for the therapeutic efficacy of DC.Tbet is IL-36 gamma. Dendritic cells engineered to ectopically overexpress IL-36 gamma and injected intratumorally into the murine MC38 model of colorectal carcinoma can direct the same magnitude of immune response as DC.Tbet therapy, even in the absence of Tbet expression by the injected cells. IL-36 gamma drives intratumoral expression of lymphotoxins and chemokines that direct tertiary lymphoid organogenesis, and promotes an intratumoral Type-1 immune response in conjunction with delayed tumor progression. Finally, I evaluated the expression pattern of IL-36 gamma in human colorectal cancer. I show that within the immune compartment, expression of IL-36 gamma by M1 macrophages is positively correlated with a high CD4+ central memory T cell infiltrate into those tumors; and that IL-36 gamma expression on the tumor vasculature is associated with an increased density of B cells within tumor-associated tertiary lymphoid structures. Together, these data support IL-36 gamma as a novel mediator of anti-tumor immunity and its further investigation as a therapeutic agent to enhance protective Type-1 immune responses in the tumor microenvironment