Biology subjects

Williams, M. M.

Publications and source records attributed to Williams, M. M..

3 recordsLinked to original sources

bioRxiv2020

A single N6-methyladenosine site in lncRNA HOTAIR regulates its function in breast cancer cells

N6-methyladenosine (m6A) modification of RNA plays important roles in normal and cancer biology, but knowledge of its function on long noncoding RNAs (lncRNAs) remains limited. Here, we investigate whether m6A regulates the function of the human HOTAIR lncRNA, which contributes to multiple pro-tumor phenotypes in triple-negative breast cancer (TNBC) cells. We identify at least 8 individual m6A sites within HOTAIR, with a single site (A783) consistently methylated. Mutation of A783 impairs cellular proliferation and invasion in HOTAIR-overexpressing TNBC cells. m6A at A783 regulates HOTAIRs ability to localize to chromatin and induce gene pathways that affect tumor progression. In contrast, A783U mutant HOTAIR demonstrates loss-of-function and antimorph behaviors by impairing gene expression changes induced by WT HOTAIR and, in some cases, inducing opposite changes in gene expression. HOTAIR interacts with nuclear m6A reader YTHDC1 and high HOTAIR is significantly associated with shorter overall patient survival, particularly in the context of high YTHDC1. At the molecular level, YTHDC1-HOTAIR interactions are required for chromatin localization and regulation of gene repression. Our work demonstrates how modification of one base in a lncRNA can elicit a distinct gene regulation mechanism and drive disease-associated phenotypic changes such as proliferation and invasion.

molecular biology↗

bioRxiv2020

Estrogen receptor alpha mutations in breast cancer cells cause gene expression changes through constant activity and through secondary effects

While breast cancer patients with tumors that express estrogen receptor (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele-specific and were not observed upon estrogen treatment of wildtype cells. These mutant-specific genes showed consistent differential expression across ER mutant lines developed in other laboratories. Wildtype cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity.

cancer biology↗

bioRxiv2020

Duplications drive diversity in Bordetella pertussis on an underestimated scale.

Bacterial genetic diversity is often described using solely base pair changes despite a wide variety of other mutation types likely being major contributors. Tandem duplications of genomic loci are thought to be widespread among bacteria but due to their often intractable size and instability, comprehensive studies of the range and genome dynamics of these mutations are rare. We define a methodology to investigate duplications in bacterial genomes based on read depth of genome sequence data as a proxy for copy number. We demonstrate the approach with Bordetella pertussis, whose insertion sequence element-rich genome provides extensive scope for duplications to occur. Analysis of genome sequence data for 2430 B. pertussis isolates identified 272 putative duplications, of which 94% were located at 11 hotspot loci. We demonstrate limited phylogenetic connection for the occurrence of duplications, suggesting unstable and sporadic characteristics. Genome instability was further described in-vitro using long read sequencing via the Nanopore platform. Clonally derived laboratory cultures produced heterogenous populations containing multiple structural variants. Short read data was used to predict 272 duplications, whilst long reads generated on the Nanopore platform enabled the in-depth study of the genome dynamics of tandem duplications in B. pertussis. Our work reveals the unrecognised and dynamic genetic diversity of B. pertussis and, as the complexity of the B. pertussis genome is not unique, highlights the need for a holistic and fundamental understanding of bacterial genetics.

microbiology↗