Biology subjects

Furlong, E. E.

Publications and source records attributed to Furlong, E. E..

3 recordsLinked to original sources

bioRxiv2024

Contextualising transcription factor binding during embryogenesis using natural sequence variation

Understanding how genetic variation impacts transcription factor (TF) binding remains a major challenge, limiting our ability to model disease-associated variants. Here, we used a highly controlled system of F1 crosses with extensive genetic diversity to profile allele-specific binding of four TFs at several embryonic time-points, using Drosophila as a model. Using a combined haplotype test, we identified 9-18% of TF bound regions impacted by genetic variation. By expanding WASP (a tool for allele-specific read mapping) to examine INDELs, we increased detection of allele imbalanced (AI) peaks by 30-50%. This fine-grained mutagenesis could reconstruct functionalized binding motifs of all factors. To prioritise potential causal variants, we trained a convolutional neural network (Basenji) to predict TF binding from DNA sequence. The model could accurately predict experimental AI for strong effect variants, providing a mechanistic interpretation for how genetic variation impacted TF binding. This revealed unexpected relationships between TFs, including potential cooperative pairs, and mechanisms of tissue specific recruitment of the ubiquitous factor CTCF.

genetics↗

bioRxiv2024

A function of Spalt proteins in heterochromatin organization and maintenance of genomic DNA integrity

The phylogenetically conserved Spalt proteins regulate gene expression and participate in a variety of cell fate choices during multicellular development, generally acting as transcriptional repressors in different gene regulatory networks. Paradoxically, besides their roles as DNA sequence-specific transcription factors, Spalt proteins show a consistent localization to heterochromatic regions. They can act through interactions with the Nucleosome remodeling and deacetylase complex (NuRD) to promote closing of open chromatin domains, but their activities as epigenetic regulators also rely on interactions with DNA Methyltransferases or with the Lysine-specific histone demethylase LSD1, suggesting that they can participate in multiple regulatory mechanisms. Here we describe several major consequences of loss of spalt function in Drosophila cells, including changes in chromatin accessibility affecting mostly pericentromeric heterochromatin, the generation of DNA damage, alterations in the localization of chromosomes within the nucleus in polyploid cells of the salivary glands and miss-expression of transposable elements. We suggest that most of these effects are related to roles of Spalt proteins in the regulation of heterochromatin formation. We propose that Drosophila Spalt proteins have two complementary functions, acting as sequence-specific transcriptional repressors on specific target genes and regulating more global gene silencing through the generation or maintenance of heterochromatic domains.

developmental biology↗

bioRxiv2023

In vivo silencing of regulatory elements using a single AAV-CRISPRi vector

CRISPR-Cas9 based transcriptional repressors (CRISPRi) have emerged as specific and robust tools for functional epigenetic silencing of regulatory elements. Adeno-associated viruses (AAVs) are promising CRISPRi delivery vectors for cardiovascular research and therapy. However, compact vectors enabling codelivery of all CRISPRi components by a single AAV are needed for an enhanced and consistent performance. We engineered a 4.7kb all-in-one CRISPRi construct compatible with AAV-mediated delivery and produced cardiotropic AAVi 6 and 9 particles for in vitro and in vivo application, respectively. AAVi vectors targeting the Nppa promoter (AAViNppa) reduced gene expression in cultivated cardiomyocytes (HL-1 cells) in a dose-dependent manner. The maximum effect was a >95% reduction as measured by qPCR and RNA-seq. This effect was orchestrated by loss of chromatin accessibility (ATAC-seq) and establishment of heterochromatin (H3K9me3 ChIP-seq) specifically at the target promoter region. We confirmed the broad applicability of AAVi to different cardiomyocyte cell culture systems by silencing several genes in primary neonatal rat ventricular cardiomyocytes (NRVMs), human iPSC-derived cardioids and iPSC-CMs. To demonstrate the efficacy of AAVi in vivo we injected 8-week-old C57Bl/6 WT mice with a single dose of AAViNppa and implanted osmotic minipumps releasing Phenylephrine (50 mg/kg/d) and Angiotensin II (0.45 mg/kg/d) to induce Nppa transcription. AAViNppa silenced Nppa transcription as revealed by qPCR and single nuclei RNA-seq even under stress conditions. On the epigenome layer AAViNppa induced closed chromatin at the Nppa promoter site comparable to the in vitro effect. Here, we present an efficient AAV-based method for CRISPRi-mediated epigenetic silencing of gene expression in cardiac myocytes in vivo and in vitro. This functional epigenetic approach provides an efficient way to modulate gene expression in the heart and could become a standard method for cardiovascular disease modelling and translational research.

genomics↗