Browsing by Author "Mhlanga, Musa"

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    Open Access
    LncRNA discovery in the Listeria monocytogenes infection model
    (2015) Magagula, Loretta Q; Mhlanga, Musa; Brombacher, Frank
    A growing body of evidence indicates that long noncoding RNAs (lncRNAs), the most abundant noncoding RNA (ncRNA) species of the pervasively transcribed mammalain genome, have functional roles in the gene regulation of an array of cellular processes. These observations have since discredited the long standing central dogma formulated by Franscis Crick in 1958, which states that genetic output is entirely conducted by protein. Recent studies collectively indicate that lncRNAs play important functional roles in the transcriptional regulation of a wide array of cellular processes. In the last year alone, a handful of studies have identified lncRNAs linc-Cox2, Lethe, PACER and THRIL as central players in host cell innate immune response against microbial infection. These discoveries and the vast numbers of uncharacterized lncRNAs identified by high-throughput nextgeneration transcriptome sequencing technologies, set a precedence for further investigation and characterization of lncRNAs in infection biology. Importantly, lncRNAs may serve as important diagnostic markers of infection as well as therapeutic targets. These aspect of lncRNAs field although extensively being explored in cancer research, have been neglected in infection biology, particularly in microbial infection. In this study, next-generation technologies were used to identify subtle vairations in transcriptional activity, with particular emphasis to lncRNA differential expression, and uncover their physiological relevance during Listeria monotocytogenes infection. To this end, an RNA-Seq dataset of Listeria-infected HeLa cells was subjected to several variations of data analysis lncRNA discovery pipelines. Potential lncRNA functioning was hypothesized using the Rinn & Chang "guilt by association" approach in which lncRNA functioning was hypothesized based on the known functions of tightly co-expressed protein coding mRNAs. "Guilty" lncRNAs were then knocked down in the HeLa cells using transcription activator-like nucleases (TALENs) to validate their candidacy as infection-regulating lncRNAs. Preliminary investigations conducted in this study have revealed potential Listeria infectionregulating lncRNA candidates. Furthermore, we explored the use of the physiologically relevant cellular model of iPSC-MDMs to validate identified lncRNA candidates. This work provides a framework for lncRNA discovery from RNA-Seq data by iterative and intergrative analysis.
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    Open Access
    The chromatin landscape of colorectal cancer cells
    (2020) Magagula, Loretta Qinisile; Mhlanga, Musa; Skok, Jane
    Chromatin organization is at the heart of deciphering gene regulation as it is instructive to transcription. Current technological advances in next-generation sequencing approaches have offered unprecedented opportunities to interrogate the genomic landscape in multiple pathological and clinical presentations. Historically, mutations and alterations at the genomic loci of protein-coding genes were thought to be exclusively causal to many human diseases. However, the non-coding genome has emerged as the master regulator of chromatin dynamics and transcriptional activity. With cancer increasingly becoming the greatest health epidemic of our time, the comprehensive genomic characterization of tumor genotypes has become central to current therapeutic approaches. Functioning as the basic unit of chromatin organisation, chromatin loops and topologically associating domains (TADs) compartmentalize genomic loci and their corresponding molecular transcriptional elements in three-dimensional space. Transcription of the human genome is proximity-dependent requiring the cooperative engagement of non-coding elements and epigenetic modifiers to create permissive topological chromatin contacts and structures. The repertoire of chromatin contacts at any given time is regulated by the threedimensional structure and organization of the chromatin. TAD structures are formed and maintained by chromatin insulating proteins such as CTCF (CCCTC-binding factor) and multiprotein complex, cohesin. The dysfunction of which, through mutational and epigenetic aberrations, directly impacts a plethora of chromatin contacts and the resultant transcriptional profiles within each cell. Loops and TADs are formed by the binding of CTCF on the conserved 19 bp CTCF binding motif as the chromatin is protruded through the "ring-like" multi-protein complex, cohesin. When two convergently oriented and CTCF enriched CTCF-binding sites (CBSs) come into contact within the ring, cohesin is thought to "hand-cuff" the chromatin resulting in the formation a chromatin loop. These loop structures then serve to compartmentalize and restrict the chromatin contacts and their frequency within each loop. Promoter-resident CBSs can also function as "docking sites” for tissue- and context-specific enhancers. The dysregulation of CTCF binding has been repeatedly demonstrated to directly alter chromatin contacts in a vast array of cellular contexts including cancer. Fundamentally, CTCF functions as a potent regulator of chromatin contacts, which directly instruct transcriptional status. Thus, CTCF binding has become an attractive regulatory target for manipulating the topological and transcriptional activity of chromatin. In this study, we sought to identify CBS swith differential, specifically abrogated CTCF enrichment that may be hijacked by oncogenes in an attempt to modify transcriptional programmes to favour cancer progression. To this end, we developed an integrated bioinformatic pipeline to identify promoter-associated lower-CTCF enrichment sites (PA-LCes) in colorectal cancer (CRC) cell lines as compared to primary colonic tissue from CTCF ChIP-Seq data. With ever-growing catalogues of nextgeneration sequencing datasets, including ChIP-Seq, in the public domain, the use of ENCODE datasets proved to be an economical option and added layer of standardization in our analysis. Briefly the pipeline developed in this study takes ENCODE ChIP-Seq FASTQ files from the NCBI SRA using fastqdump as input files. The FASTQ files undergo a quality control and dataset filtration with FASTQC. The filtered datasets are then aligned to the hg38 human genome and fed back into FASTQC to ensure aligned reads pass quality control metrics. The mapped reads are then processed using samtools and duplicate reads are marked with the picard markduplicates argument. Narrow peaks are then called from processed reads using MACS2 and processed using bedtools. Called peaks then undergo a final quality control step using ChIPQCr and are visualized using IGV before undergoing differential enrichment analysis. Differential CTCF enrichment analysis between the peaks in primary sigmoidal colon cells and CRC cell lines is then conducted using DeSeq2 within DiffBind. Lower CTCF enrichment peaks are then used for the discovery of the canonical CTCF MA00139.1 motif using homer and compared to similar annotations in the primary consensus peakset. The resultant lower CTCF enrichment peaks are then annotated using homer and ChiPpeakAnno to determine their genomic locations and extract LCes located proximal (<1kb) to annotated TSS or promoter regions i.e. PA-LCes. The PA-LCe discovery pipeline developed in this study is highly robust, resulting in some previously validated CBSs implicated in oncogenesis. Intriguingly, the PA-LCe sites identified in this study emanate from bidirectional promoters at oncogenes with differential methylation and transcriptional patterns in cancer. Additionally these PA-LCes transcribe antisense lncRNAs such as the tumor-suppressive aslncRNA ZNF582-AS1. This data adds to the recent body of evidence that suggests that disruption of promoter-associated CBSs leads to fluctuations in promoter activity. Recent studies have implicated the requirement of CTCFlncRNA complexes at promoter regions in facilitating and regulating CTCF docking on chromatin which subsequently influences transcriptional activity. In accordance with this, our data suggests that the lncRNAs at PA-LCe loci may be molecular targets for the regulation ofCTCF binding and transcriptional activity in CRC. Perturbation of CTCF enrichment at PALCes in CRC result in differential chromatin contacts, epigenetic context and, the transcriptional activity of the promoters in which they reside. As CTCF binding at CBSs sites is highly modular, the use of targeted CRISPR-mediated gene-editing and DNA methylation at PA-LCe CBSs may represent viable and druggable oncogenic targets.
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    Open Access
    Transcriptionally induced enhancers in the macrophage immune response to Mycobacterium tuberculosis infection
    (BioMed Central, 2019-01-22) Denisenko, Elena; Guler, Reto; Mhlanga, Musa; Suzuki, Harukazu; Brombacher, Frank; Schmeier, Sebastian
    Background Tuberculosis is a life-threatening infectious disease caused by Mycobacterium tuberculosis (M.tb). M.tb subverts host immune responses to build a favourable niche and survive inside of host macrophages. Macrophages can control or eliminate the infection, if acquire appropriate functional phenotypes. Transcriptional regulation is a key process that governs the activation and maintenance of these phenotypes. Among the factors orchestrating transcriptional regulation during M.tb infection, transcriptional enhancers still remain unexplored. Results We analysed transcribed enhancers in M.tb-infected mouse bone marrow-derived macrophages. We established a link between known M.tb-responsive transcription factors and transcriptional activation of enhancers and their target genes. Our data suggest that enhancers might drive macrophage response via transcriptional activation of key immune genes, such as Tnf, Tnfrsf1b, Irg1, Hilpda, Ccl3, and Ccl4. We report enhancers acquiring transcription de novo upon infection. Finally, we link highly transcriptionally induced enhancers to activation of genes with previously unappreciated roles in M.tb infection, such as Fbxl3, Tapt1, Edn1, and Hivep1. Conclusions Our findings suggest the importance of macrophage host transcriptional enhancers during M.tb infection. Our study extends current knowledge of the regulation of macrophage responses to M.tb infection and provides a basis for future functional studies on enhancer-gene interactions in this process.
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    Uncovering the hidden mechanisms governing the transcriptional regulation of inflammation
    (2020) Fok, Ezio T; Mhlanga, Musa; Fanucchi, Stephanie
    Inflammation provides broad immunological protection that is essential for our survival. This cellular response is characterised by a biphasic cycle consisting of an initial acute pro-inflammatory phase and a subsequent resolving anti-inflammatory phase. Underlying each of these phases are changes in the expression of hundreds of immune genes, which encode for inflammatory mediators called cytokines. Importantly, the biphasic nature of inflammation requires cytokine expression to be highly regulated and coordinated to different timescales during each phase of inflammation. For the initial proinflammatory response, cytokine expression needs to be rapid and robust to efficiently initiate host defence mechanisms and provide effective immunological protection. In contrast, the expression of anti-inflammatory cytokines is temporally delayed to ensure that anti-inflammation always follows pro-inflammation. In order to choreograph the expression of these cytokines during inflammation, numerous mechanisms within the cell serve to regulate and coordinate cytokine transcription. Within the eukaryotic nucleus, multiple modes of transcriptional regulation function cooperatively to provide the regulatory capacity that is required for complex transcription patterns to emerge. These include the organisation of the genome, which confine cognate chromosomal contacts that are causal to transcription, and long-non coding RNAs (lncRNAs) that function to discretely fine tune transcriptional activity. Although many of the mechanisms that regulate transcription have been well described, their role in cytokine expression during inflammation remains largely unknown. In particular, the mechanisms that facilitate rapid and robust cytokine expression during proinflammation and the regulatory networks that coordinate the biphasic regulation of inflammation are unresolved. In this work, two novel lncRNAs were discovered to transcriptionally regulate these key features of cytokine expression during inflammation. The first, UMLILO (Upstream Master LncRNA of the Inflammatory chemokine LOcus), was found to emanate from the ELR+ CXCL chemokine TAD and regulate the transcriptional activation of the pro-inflammatory ELR+ CXCL chemokines (IL-8, CXCL1, CXCL2 and CXCL3). By exploiting the pre-formed local 3D topology, UMLILO is able to epigenetically prime the chemokines for transcriptional activation. This involves the discrete deposition of H3K4me3 onto the promoters of the chemokines, which allows for the pre-loading of transcriptional machinery prior to their signal-dependent activation. This reveals a fundamental mechanism for the epigenetic priming and rapid activation of pro-inflammatory cytokine genes. The second lncRNA, called AMANZI (A MAster Non-coding RNA antagoniZing Inflammation), was found to coordinate the transcription of two functionally opposed cytokines: the master pro-inflammatory IL-1β and the broad antiinflammatory IL-37. AMANZI is encoded in the promoter of IL-1β, which results in its concomitant expression when IL-1β is transcriptionally active. Functionally, AMANZI mediates the formation of a dynamic chromosomal contact between IL-1β and IL-37. This leads to the delayed transcriptional activation of IL-37 ensuring that the pro-inflammatory function of IL-1β precedes IL-37 mediated anti-inflammation. This revealed a novel biphasic circuit that coordinated the expression of IL-1β and IL-37, through the activity of AMANZI, to regulate the two functionally opposed states of inflammation. Clinical observations in healthy individuals revealed that a polymorphism occurring in AMANZI (rs16944) was able to augment the state of this genetic circuit and shift the relative levels of IL-1β and IL-37 to influence an individual's inflammatory capacity. This affected the establishment of innate immunological memory, which is involved in the progression of many inflammatory conditions and the efficacy of certain vaccines. The work described here uncovers novel mechanisms that transcriptionally regulate key features of the inflammatory response. Importantly, this work implicates the role of two novel lncRNAs in inflammation, essentially contributing to the functional annotation to the genome and providing novel targets for the modulation of pathogenic inflammation.