Biology subjects

Knowles, M. R.

Publications and source records attributed to Knowles, M. R..

3 recordsLinked to original sources

bioRxiv2024

Long G4-rich enhancer physically interacts with EXOC3 promoter via a G4:G4 DNA-based mechanism

Enhancers are genomic sequences that function as regulatory elements capable of increasing the transcription of a given gene often located at a considerable distance. The broadly accepted model of enhancer activation involves bringing an enhancer-bound activator protein complex into close spatial proximity to its target promoter through chromatin looping. Equally relevant to the work described herein, roles for guanine (G) rich sequences in transcriptional regulation are now widely accepted. Non-coding G-rich sequences are commonly found in gene promoters and enhancers, and various studies have described specific instances where G-rich sequences regulate gene expression via their capacity to form G-quadruplex (G4) structures under physiological conditions. In light of this, our group previously performed a search for long human genomic stretches significantly enriched for minimal G4 motifs (referred to as LG4s herein) leading to the identification of 301 LG4 loci with a density of at least 80 GGG repeats / 1,000 basepairs (bp) and averaging 1,843 bp in length. Further, in agreement with previous reports indicating that minimal G4s are highly enriched in promoters and enhancers, we found 217/301 LG4 sequences overlap a GeneHancer annotated enhancer, and the gene promoters regulated by these LG4 enhancers were found to be similarly, markedly enriched with G4-capable sequences. Importantly, while the generally accepted model for enhancer:promoter specificity maintains that interactions are dictated by enhancer- and promoter-bound transcriptional activator proteins, the current study was designed to test an alternative hypothesis: that LG4 enhancers physically interact with their cognate promoters via a direct G4:G4 DNA-based mechanism. As such, this work employs a combination of informatic mining and locus-specific immunoprecipitation strategies to establish the spatial proximity of enhancer:promoter pairs within the nucleus then biochemically confirms the ability of individual LG4 ssDNAs to directly and specifically interact with DNA sequences found in their target promoters. In addition, we also identify four single nucleotide polymorphisms (SNPs), occurring within a LG4 enhancer on human chromosome 5, significantly associated with Cystic Fibrosis (CF) lung disease severity (avg. p value = 2.83E-9), presumably due to their effects on the expressions of CF-relevant genes directly regulated by this LG4 enhancer (e.g., EXOC3 and CEP72). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/577212v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1fee785org.highwire.dtl.DTLVardef@11f0a9org.highwire.dtl.DTLVardef@1cf198corg.highwire.dtl.DTLVardef@1963a55_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief: LG4 enhancers physically interact with gene promoters by forming composite G4 structures where both the LG4 and cognate promoter contribute half of the necessary sequence for G4 formation.

genetics↗

bioRxiv2021

Leveraging TOPMed Imputation Server and Constructing a Cohort-Specific Imputation Reference Panel to Enhance Genotype Imputation among Cystic Fibrosis Patients

Cystic fibrosis (CF) is a severe genetic disorder that can cause multiple comorbidities affecting the lungs, the pancreas, the luminal digestive system and beyond. In our previous genome-wide association studies (GWAS), we genotyped [~]8,000 CF samples using a mixture of different genotyping platforms. More recently, the Cystic Fibrosis Genome Project (CFGP) performed deep ([~]30x) whole genome sequencing (WGS) of 5,095 samples to better understand the genetic mechanisms underlying clinical heterogeneity among CF patients. For mixtures of GWAS array and WGS data, genotype imputation has proven effective in increasing effective sample size. Therefore, we first performed imputation for the [~]8,000 CF samples with GWAS array genotype using the TOPMed freeze 8 reference panel. Our results demonstrate that TOPMed can provide high-quality imputation for CF patients, boosting genomic coverage from [~]0.3 - 4.2 million genotyped markers to [~]11 - 43 million well-imputed markers, and significantly improving Polygenic Risk Score (PRS) prediction accuracy. Furthermore, we built a CF-specific CFGP reference panel based on WGS data of CF patients. We demonstrate that despite having [~]3% the sample size of TOPMed, our CFGP reference panel can still outperform TOPMed when imputing some CF disease-causing variants, likely due to allele and haplotype differences between CF patients and general populations. We anticipate our imputed data for 4,656 samples without WGS data will benefit our subsequent genetic association studies, and the CFGP reference panel built from CF WGS samples will benefit other investigators studying CF.

genetics↗

bioRxiv2021

Robust W1282X-CFTR rescue by a small molecule GSPT1 degrader

With the approval of elexacaftor/tezacaftor/ivacaftor (trade name Trikafta), the vast majority of people with cystic fibrosis (CF) are eligible for CF transmembrane conductance regulator (CFTR) modulator therapy. Remaining individuals have premature termination codons or rare CFTR variants with limited treatment options. Although clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, these cells can be expanded by overexpression of mouse Bmi-1 and human TERT (hTERT). We therefore used this approach to develop two non-CF and three CF (F508del/F508del, F508del/S492F, W1282X/W1282X) nasal cell lines and two W1282X/W1282X bronchial cell lines. Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The F508del/F508del and F508del/S492F cell lines robustly responded to Trikafta, which was mirrored in the parent primary cells and the cell donors clinical response. CC-90009, a novel cereblon E3 ligase modulator targeting the GSPT1 protein, rescued ~20% of wildtype CFTR function in our panel of W1282X/W1282X cell lines and primary cells. Intriguingly, CC-90009 also diminished epithelial sodium channel function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirror primary cell responses to CFTR modulators and illustrate novel therapeutic approaches for the W1282X CFTR variant.

pathology↗