Transcriptional regulation in mouse macrophages : the role of enhancers in macrophage activation and infection

The results presented in Chapter 3 of this thesis were published as (the Authors retain copyright): Denisenko, E., Guler, R., Mhlanga, M. M., Suzuki, H., Brombacher, F., & Schmeier, S. (2017). Genome-wide profiling of transcribed enhancers during macrophage activation. Epigenetics & Chromatin, 10(1), 50. doi: 10.1186/s13072-017-0158-9Macrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli. Of those, M(IFN-γ) and M(IL-4/IL-13) macrophage activation states are well known for their opposing pro- and anti-inflammatory roles. Imbalance in these populations of macrophages has been implicated in progression of various diseases. Macrophages also comprise the first line of an organism’s defence against Mycobacterium tuberculosis, the causative agent of tuberculosis; interactions between the bacteria and host macrophages define the infection outcome. The area of mammalian transcriptional regulation progressed remarkably with recent advances in high-throughput technologies. Enhancers emerged as crucial regulatory DNA elements capable of activating transcription of target genes at distance in an orientation-independent manner. A recent discovery revealed that enhancers can be transcribed themselves into enhancer RNAs, or eRNAs. Enhancers were shown to be pervasive, yet the associated regulatory patterns remain largely unknown and require further research. In this thesis, we investigated in silico transcribed enhancers in mouse tissues and cell lines, with a particular focus on macrophages. We have performed a large-scale study to identify transcribed enhancers across multiple tissues and to characterise their properties. In macrophages, we have established the most accurate, to our knowledge, genome-wide catalogue of transcribed enhancers and enhancer-gene regulatory interactions. We have inferred enhancers that might drive transcriptional responses of protein-coding genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation, and demonstrated stimuli specificity of regulatory associations. We have conducted the first to our knowledge study of the role of transcribed enhancers in macrophage response to Mycobacterium tuberculosis infection. Taken together, the present work provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage protein-coding genes in different conditions. Given the increasing promise for enhancer and chromatin-directed therapy, this work paves the way for further studies towards hostdirected therapies and novel treatments for tuberculosis and immune diseases associated with macrophage dysfunction

Genome-wide profiling of transcribed enhancers during macrophage activation

Background: Macrophages are sentinel cells essential for tissue homeostasis and host defence. Owing to their plasticity, macrophages acquire a range of functional phenotypes in response to microenvironmental stimuli, of which M(IFN-γ) and M(IL-4/IL-13) are well known for their opposing pro- and anti-inflammatory roles. Enhancers have emerged as regulatory DNA elements crucial for transcriptional activation of gene expression. Results: Using cap analysis of gene expression and epigenetic data, we identify on large-scale transcribed enhancers in bone marrow-derived mouse macrophages, their time kinetics, and target protein-coding genes. We observe an increase in target gene expression, concomitant with increasing numbers of associated enhancers, and find that genes associated with many enhancers show a shift towards stronger enrichment for macrophage-specific biological processes. We infer enhancers that drive transcriptional responses of genes upon M(IFN-γ) and M(IL-4/IL-13) macrophage activation and demonstrate stimuli specificity of regulatory associations. Finally, we show that enhancer regions are enriched for binding sites of inflammation-related transcription factors, suggesting a link between stimuli response and enhancer transcriptional control. Conclusions: Our study provides new insights into genome-wide enhancer-mediated transcriptional control of macrophage genes, including those implicated in macrophage activation, and offers a detailed genome-wide catalogue of transcribed enhancers in bone marrow-derived mouse macrophages

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified