1,721,011 research outputs found
Low levels of methylation are targeted to aberrantly methylated CGIs in human colorectal cancer cells
Get PDFDisruption of DNA methylation patterns is a primary hallmark of cancer. One
main disruption is CpG islands (CGIs) aberrant hypermethylation which is
associated with transcriptional repression of tumour suppressor genes such as
BRCA1, MLH1, and CDKN2A (p16/ARF). However, the mechanism(s)
underpinning this process are still unknown. One hypothesis is that high de novo
methylation activity is targeted to CGIs that get aberrantly methylated in cancer.
To investigate this, I used human colorectal cancer cell line (HCT116) as a
model. Firstly, I have ectopically integrated representative aberrantly methylated
CGIs randomly into the genome and assessed their methylation at ectopic loci. I
show that integrated CGIs rarely gained methylation at ectopic locations. This
suggested that the sequence doesn’t program CGIs aberrant methylation at
ectopic loci in HCT116 and that low de novo methylation is targeted to
aberrantly methylated CGIs. Next, I wanted to confirm this result by integrating
CGIs at a chosen genomic loci in order to exclude any effects from the CGI
position on its methylation status. This was performed by recombinase mediated
cassette exchange (RMCE). Successful targeting of a RMCE cassette to the
desired chromosomal locus was achieved. However, isolating clonal cell lines
with integrated CGIs was technically infeasible due to difficulty in using the
thymidine kinase selection in HCT116. In order to investigate which CGIs have
higher de novo methylation in HCT116 on a genome wide level, I restored
DNMT3B expression in DNMTA1/DNMT3B double knockout (DKO) of HCT116.
In agreement with previous reports, DNMT3B showed higher de novo
methylation activity at CGIs overlapping H3K36me3. Most importantly,
aberrantly methylated CGIs showed ~ 78.5% less methylation gain compared to
normally methylated CGIs. To confirm results from this experiment, I treated
HCT116 with 5-Aza-2′-deoxycytidine and then allowed cells to recover
methylation. The rate of methylation recovery of CGIs was assessed. This
showed that aberrantly methylated CGIs recover slower than normally
methylated CGIs, confirming that lower de novo methylation is targeted to
aberrantly methylated CGIs compared to normally methylated ones. The work
carried out during the course of this thesis provides novel insights into the
process of de novo methylating CGIs in cancer on a genome wide level. It
suggests that low levels, rather than high levels, of methylation are targeted to
aberrantly methylated CGIs in colorectal tumours
Uncovering key transcriptional and epigenetic changes underpinning the emergence of the glioma stem cell state
Get PDFGlioblastoma Multiforme (GBM) is the most aggressive and common adult primary brain tumour, with an awful prognosis of 14 months median survival. GBMs can also occur in children, as paediatric high-grade glioma (pHGG), or diffuse intrinsic pontine glioma (DIPG), and like adult GBMs are incurable. GBM tumour growth and relapse after therapy is thought to be driven by the glioma stem cell (GSC). GSCs display similarities to neural stem and progenitor cells and likely arise from these cells. However, we currently have a poor understanding of the transcriptional and epigenetic reconfiguration which follows neural stem cell (NSC) to GSC transformation. In particular, further work is required to understand how common driver mutations contribute to immune evasion in adult GBM, and how the common paediatric mutation H3.3-G34R drives pHGG oncogenesis in a forebrain specific context.
In the first instance using a novel set of engineered mouse NSC lines harbouring common adult glioma-associated driver mutations (RTK/PI3K/MAPK/P53 pathways) and transcriptional and epigenetic profiling, we explored mechanisms of adult GBM immune evasion as tumour-initiating GSCs were serially transplanted into C57BL/6J (BL6) immunocompetent mice. Second, to model the paediatric disease, an additional set of cortex and hindbrain human foetal NSC lines were engineered with paediatric glioma-associated mutations (H3.3-G34R mutations and RTK/P53 pathways). We used multi-omics data and bioinformatics approaches to characterise these adult and paediatric models of GBM, alongside adult and paediatric GSCs, to understand the transcriptional and epigenetic alterations imposed by these driver mutations.
We found that triple mutants (Nf1 and Pten knock-out (KO); EGFRvIII overexpression), herein termed NPE cells, demonstrated unconstrained proliferation and a differentiation block through transcriptional downregulation and epigenetic silencing of Bmpr1b. Furthermore, we found an acquired capacity for immune evasion in NPE derivatives (in the absence of any genetic selection), suggesting a form of epigenetic immunoediting. In particular we found, upregulation of myeloid associated transcription factors (e.g. Irf8), interferon (IFN) response genes, and myeloid suppressor cell chemo-attractants such as Ccl9 which drove a myeloid-derived suppressive cell (MDSC) weighted tumour micro-environment. In human we demonstrated that GSCs could be defined by a single axis of IFN-gamma, immune response gene activation, associated with the ‘mesenchymal’ (MES) transcriptional subtype. We have therefore uncovered a key IFN driven immune evasion signal present within MES like GBM, imposed by the tumour microenvironment, which likely supports immune evasion.
In a parallel analysis of foetal engineered NSC transcriptomes (H3.3-G34R, PDGFRA overexpression, and TP53 KO) obtained from cortex and hindbrain regions, we demonstrated that in a forebrain context the H3.3-G34R mutation reinforces pre-existing forebrain progenitor transcriptional circuits, whilst in the hindbrain it drives a cytostatic response. The G34R mutation does not cause widespread epigenetic changes, but instead maintains highly expressed forebrain genes through reduced binding of transcriptional regulator ZMYND11. A key mechanistic role for the oncohistone mutation H3.3-G34R in pHGG is therefore to reinforce forebrain NSC identity, explaining the mutations incompatibility within the hindbrain
Investigating chromosome cohesion loss in mammalian oocytes
Get PDFThe most significant risk factor associated with decreasing female fertility is advanced
maternal age. Oocytes from older women are frequently aneuploid, contributing to the
increased incidence of miscarriages and genetic disorders such as Down’s syndrome in their
offspring. Cohesin proteins that hold sister chromatids together may be subject to gradual
degradation in ageing oocytes, accounting for some of the chromosome mis-segregation
observed. So far, mature mouse oocytes have provided evidence for an age-related decline
in chromosome cohesion and the chromosome-associated REC8 cohesin protein. However,
there has been limited and contradictory evidence for these changes in dictyate-arrested
oocytes, especially in human oocytes.
Preliminary work in the Adams Lab has provided evidence for an age-related separation of
chromatids in dictyate oocytes from older mice. To determine the separation of chromatids
and infer chromosome cohesion levels in dictyate human oocytes, I used Fluorescence in
situ Hybridisation (FISH) to fluorescently label specific chromosomal regions. Using human
ovary sections from fertility preservation patients and Caesarian section patients, I
investigated if the age-related separation of chromatids was conserved in humans. I
measured the distances between each FISH signal to quantify inter-chromatid separation in
each oocyte. My analysis revealed an increase in inter-chromatid distances at telomereadjacent regions of chromosome 13, 16 and 21 with increasing maternal age, which are amongst the most common chromosome aneuploidies in human genetic disease and
miscarriages. Next, I assessed whether changes in chromosome-associated cohesin may
relate to this separation. Whilst previous work in human oocytes have not reliably shown
any changes in cohesin protein levels with maternal age, it is possible that these changes are
occurring in specific cohesin subunits yet to be identified. In mitotic cells, the acetylation of
SMC3 stabilises chromosome-bound cohesin involved in sister chromatid cohesion, thus
marking a subpopulation of ‘cohesive’ cohesin. Additionally, in Tex19.1⁻/⁻ oocytes, cohesion
loss was accompanied by a decrease in AcSMC3 but not REC8, suggesting that AcSMC3 may
be more representative of cohesion changes. Indeed, I found a drastic decline in nuclear
AcSMC3 in dictyate oocytes from older women. For the first time in human oocytes, my data
shows an age-related decrease in inter-chromatid cohesion, corresponding to changes in the
cohesive cohesin, AcSMC3.
Alongside age-dependent changes in cohesion during dictyate arrest, additional pathways
have been implicated in further cohesion loss in the post-dictyate stages of oogenesis.
Enhancing cohesion levels in these later stages has the potential to improve aneuploidy
outcomes in oocytes, so I took both a genetic and pharmacological approach to attempt to
manipulate cohesion levels. TEX19.1 is a protein that regulates AcSMC3 levels and maintains
cohesion levels in oocytes, likely by negatively regulating its partner UBR2, an E3 ubiquitin
ligase. However, I found that neither Ubr2⁻/⁻ nor Ubr2 separation of function mutations
affect chromosome cohesion in mouse oocytes. It is likely that the regulation of cohesion by
a TEX19.1-UBR2 pathway in post-dictyate oocytes is more complex than in somatic tissues,
possibly due to the activity of other UBR proteins in oocytes.
In old mouse oocytes with depleted cohesin levels, sufficient cohesion remains to keep
bivalents intact. However, once the bivalent is pulled by the spindle in prometaphase,
chromosomes begin to separate prematurely. This suggests that an alternative pathway
may contribute to chromosome cohesion loss in oocytes during prometaphase. To
determine if the attachment of spindle fibres that applies tension across the bivalent
contributes to cohesion loss, I treated mouse oocytes with various spindle drugs to assess
the effects on chromosome cohesion. In the absence of a bipolar spindle, oocytes exhibited
more proximally positioned chiasma in metaphase I with higher levels of AcSMC3 on their
chromosomes, suggesting that spindle drug treatment prevents chromosomes from ‘ageing’
and instead resemble chromosomes from a ‘younger’ oocyte. Further investigation into the
molecular players involved in a spindle-induced cohesion loss pathway may provide a
therapeutic route to enhance cohesion and improve oocyte quality during fertility
treatment
Patient-led functional genomics identifies novel drivers of intrahepatic cholangiocarcinoma
Get PDFIntrahepatic cholangiocarcinoma (iCCA) is a rare and universally lethal
malignancy arising in the liver. Incidence has steadily been increasing globally yet
effective therapeutics are lacking. Currently, gold standard treatment is complete
resection for which a minority of patients are suitable and recurrence rates post
resection are high. For the majority of patients unsuitable for surgery, standard
chemotherapeutic options extend life by only 3-6 months on average. As such there
is a pressing need to elucidate the genetic and molecular features of this
malignancy so as to inform the development of more effective therapies. To date, a
small but robust set of driver genes have been discovered and for some targeted
therapies in development, with mixed results in clinical trials.
This work aimed to extend the set of known driver genes by exploring the long tail
of infrequently mutated genes in patient sequencing data. Through computational
prediction and in vivo screening, a set of driver genes were uncovered which
cooperate with the more commonly encountered oncogenic RASG12 isoforms of
KRAS and NRAS. Validation studies in vivo confirmed that the membrane
cytoskeleton adaptor protein Merlin, encoded by NF2, and the canonical
transmembrane semaphorin receptor PLXNB2 encoded by PLXNB2 are tumour
suppressors occurring at low frequency in the patient population. Mutation of
these genes significantly accelerated disease progression and propagated
aggressive, invasive tumour phenotypes
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Deciphering the role of DNA sequence features in human gene expression using factorial design of experiments
Get PDFGene expression is regulated at multiple levels, including the coding sequence itself. One key factor is guanine and cytosine (GC) content, which has been shown to enhance gene expression through various mechanisms. In the human genome, regions with distinct GC content, known as isochores, define functional domains with unique properties. However, the strong correlation between GC content (nucleotide level), CpG dinucleotide frequency, and codon optimality (trinucleotide level) makes it challenging to disentangle their individual contributions to gene expression. To address this, I used a synthetic library of synonymous Green Fluorescent Protein (GFP) variants generated via a full factorial design of experiments. Over 150 GFP variants were systematically generated by varying GC content, CpG dinucleotide frequency, and codon optimality at three levels (low, medium, and high). Transient overexpression experiments in two human cell lines revealed that GC content primarily determined steady-state mRNA levels, whereas codon optimality had a stronger influence on protein levels. Interestingly, CpG frequency had the least impact, largely due to its intrinsic negative correlation with codon optimality. Taken together, these sequence features accounted for the majority of the differences in gene expression observed. I also investigated the role of the Zinc-Finger Antiviral Protein (ZAP) and its cofactor TRIM25 in regulating GFP expression. While CpG-rich viral RNA is known to be suppressed by ZAP during the interferon response, depletion of endogenous ZAP and TRIM25 in human cells did not significantly affect CpG-rich GFP variants. Additionally, increased GFP expression imposed a cellular burden on cells, suggesting an interplay between sequence composition and cellular stress. To extend these findings, I constructed and validated a stable HeLa cell pool expressing GFP variants tagged with unique 20-nucleotide barcodes in the 3′ UTR. Using flow-seq, I analysed protein expression and the effects of gene silencing over prolonged passaging, confirming codon optimality as the most critical factor. Moreover, differences in gene silencing were dependent on the combination of coding sequence features. Overall, these findings highlight how different sequence features influence gene expression in human cells, with important implications for biotechnology, synthetic biology, and gene regulation
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
DNA methylation & its regulation in colorectal tumours
Get PDFColorectal cancer (CRC) is the fourth most common form of cancer in the UK and is associated with approximately 17,000 deaths each year. DNA methylation represents a critical epigenetic mechanism by which gene expression is controlled in cells, with aberrant DNA hyper-methylation often reported in CRC. Significant DNA hyper-methylation has been reported in a subset of CRCs and is referred to as the CpG Island Methylator Phenotype (CIMP). The precise mechanisms underlying CIMP and how DNA hyper-methylation contributes to colorectal tumorigenesis remains incompletely understood, therefore this thesis seeks to address both of these questions.
Firstly, Genome-Wide Association Study (GWAS) meta-analysis data revealed an association between the 4q24 locus of chromosome 4 and CRC predisposition in Europeans. Subsequent conditional analysis, fine-mapping and in silico functional annotation identified a list of candidate causal polymorphisms with functional relevance in colonic tissues. Mendelian Randomisation and genotype-expression analysis identified down-regulation of Ten-Eleven Translocation 2 (TET2) as the candidate causal factor underpinning the GWAS association with CRC risk at this locus. The TET2 gene is involved in active DNA de-methylation, with pathogenic loss-of-function mutations thought to drive a hyper-methylated phenotype in acute myeloid leukaemia (AML). Similarly, pathogenic mutations in isocitrate dehydrogenase (IDH) 1 and 2 have also been reported in AML and present with a similar hyper-methylated phenotype. It has been hypothesised that 2-hydroxyglutarate produced by mutant IDH drives this hyper-methylation via the competitive inhibition of TET2. Mutations in TET2 and IDH are uncommon in CRC but it is possible that mutations in these genes may drive CIMP in such cancers.
Methylation array data of colorectal adenocarcinomas from The Cancer Genome Atlas identified elevated DNA methylation in TET2-mutant and IDH-mutant CRCs compared to their wild-type (WT) counterparts, as well as a significant correlation between IDH mutations and CIMP+ CRC. CIMP+ CRCs presented with an enrichment of significantly hyper-methylated probes in bivalent promoter regions, with TET2-mutant and IDH-mutant cancers possibly showing an even greater enrichment at these regions than their WT CIMP+ counterparts. A number of significantly hyper-methylated probes in TET2-mutant CIMP+ cancers were also hyper-methylated in IDH-mutant CIMP+ cancers, localising around the transcription start site of a number of candidate tumour suppressor genes – indicating TET2 and IDH mutations may both drive CIMP+ CRC via the aberrant hyper-methylation and transcriptional silencing of tumour suppressor genes.
5-methylcytosine (5-mC) regularly undergoes spontaneous deamination to thymine. The methyl-CpG binding domain 4 (MBD4) gene encodes a protein involved in catalysing the repair of this deamination by excising the thymine from the resultant T:G DNA mismatch. Germline inactivation of MBD4 has been suggested to predispose affected individuals to intestinal polyposis and some forms of cancer, including uveal melanoma. This predisposition is potentially driven by pathogenic C → T mutations at CpG sites arising within the protein-coding sequence of cancer driver genes as a consequence of spontaneous deaminations of 5-mC going unrepaired. Individuals with germline pathogenic mutations in MBD4 and subsequent somatic loss of heterozygosity were shown to have increased numbers of C → T mutations at CpG sites, including a number of pathogenic mutations in cancer driver genes that have been previously reported in the literature. It was also found that CpG sites that were highly-methylated and in later-replicating regions of the genome were at the greatest risk of spontaneous deamination, thereby describing MBD4 as a cancer predisposition gene and how DNA methylation is mechanistically associated with C → T mutagenesis at CpG sites.
In addition to unrepaired spontaneous deaminations, errors made by DNA polymerase epsilon (POL-ε) during DNA replication are an alternative mechanism by which C → T mutations at CpG sites may arise. CRCs with POL-ε exonuclease domain mutations (EDMs) and microsatellite unstable (MSI+ ) cancers presented with more C → T mutations at CpG sites than microsatellite stable (MSS) polymerase wild-type (POL-WT) CRCs. There was an excess of C → T mutations at CpG sites on the leading strand template in MSI+ CRCs and cancers with POL-ε EDMs, indicating that these mutations were likely the result of unrepaired DNA mismatches arising as a consequence of the erroneous incorporation of adenine opposite a template 5-mC by POL-ε propagating into C → T mutations in the next round of DNA replication. Similarly to spontaneous deaminations, C → T mutagenesis was more common at highly-methylated CpG sites, indicating that DNA methylation also influences the likelihood of replication errors. Therefore, methylation-induced DNA replication error may represent another mechanism by which DNA methylation may contribute to colorectal tumorigenesis.
In conclusion, this thesis provides an in-depth investigation into the role of DNA methylation in CRC pathogenesis and suggests that DNA hyper-methylation may drive colorectal tumorigenesis via multiple mechanisms, including the direct modification of the expression of CRC driver genes or by indirectly driving C → T mutagenesis at CpG sites
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