Dysregulation of alternative poly-adenylation as a potential player in Autism Spectrum Disorder

We present here the hypothesis that alternative poly-adenylation (APA) is dysregulated in the brains of individuals affected by Autism Spectrum Disorder (ASD), due to disruptions in the calcium signaling networks. APA, the process of selecting different poly-adenylation sites on the same gene, yielding transcripts with different-length 3′ untranslated regions (UTRs), has been documented in different tissues, stages of development and pathologic conditions. Differential use of poly-adenylation sites has been shown to regulate the function, stability, localization and translation efficiency of target RNAs. However, the role of APA remains rather unexplored in neurodevelopmental conditions. In the human brain, where transcripts have the longest 3′ UTRs and are thus likely to be under more complex post-transcriptional regulation, erratic APA could be particularly detrimental. In the context of ASD, a condition that affects individuals in markedly different ways and whose symptoms exhibit a spectrum of severity, APA dysregulation could be amplified or dampened depending on the individual and the extent of the effect on specific genes would likely vary with genetic and environmental factors. If this hypothesis is correct, dysregulated APA events might be responsible for certain aspects of the phenotypes associated with ASD. Evidence supporting our hypothesis is derived from standard RNA-seq transcriptomic data but we suggest that future experiments should focus on techniques that probe the actual poly-adenylation site (3′ sequencing). To address issues arising from the use of post-mortem tissue and low numbers of heterogeneous samples affected by confounding factors (such as the age, gender and health of the individuals), carefully controlled in vitro systems will be required to model the effect of calcium signaling dysregulation in the ASD brain

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U

Transcriptional and epigenetic regulation of lineage identity in endothelial cells by the transcription factor ERG via super-enhancers

Transcriptional programs establish and maintain cell identities. Regulation of gene expression is mediated by sequence-specific transcription factors (TFs) and cis-regulatory elements present in the genome. More recently, cell identity has been associated with lineage-defining super-enhancers: comprising dense TF platforms. Endothelial cells are key players of vascular integrity. The ETS TF ERG is constitutively expressed in endothelial cells and essential for endothelial lineage specification, vascular homeostasis and angiogenesis. However, the genomic programs that are regulated by ERG in endothelial cells are poorly understood. In this thesis, I show that ERG densely occupies super-enhancers in human umbilical vein endothelial cells (HUVEC), and its high occupancy can identify super-enhancers. I find that variants associated with cardiovascular disease are enriched in ERG-defined super-enhancers providing insight to the endothelial contribution to complex disease. Depletion of ERG causes profound modulation of the active enhancer mark H3K27ac genome-wide and in recruitment of the transcriptional co-activator Mediator complex. Loss of ERG leads to a decrease in 107 endothelial super-enhancers that have reduced co-occupancy of TFs GATA2 and AP-1. This indicates that ERG plays an essential role as a positive regulator of a core set of endothelial super-enhancers. Interestingly, aberrant ERG overexpression in prostate cancer via androgen-responsive TMPRSS2:ERG fusion proteins is oncogenic. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinct lineage-specific transcriptome and super-enhancer profiles. In endothelial cells, I show that ERG is required at promoters and enhancers yet assembles distinct TF complexes at these two regions. ERG also colocalises with structural chromatin regulator CTCF in HUVEC, implying a role for ERG in coordinating chromatin structural organisation. Finally, I adopt CRISPR-Cas9 gene editing technology to genetically dissect the super-enhancer of adhesion molecular VE-cadherin. The mechanistic exploration of ERG and endothelial super-enhancers provide insight into the regulation of the endothelial-specific gene expression program.Open Acces

The Transcription Factor ERG Regulates Super-Enhancers Associated with an Endothelial-Specific Gene Expression Program

Rationale: The ETS transcription factor (TF) ERG is essential for endothelial homeostasis, driving expression of lineage genes and repressing pro-inflammatory genes. Loss of ERG expression is associated with diseases including atherosclerosis. ERG’s homeostatic function is lineage-specific, since aberrant ERG expression in cancer is oncogenic. The molecular basis for ERG lineage-specific activity is unknown. Transcriptional regulation of lineage specificity is linked to enhancer clusters (super-enhancers). Objective: To investigate whether ERG regulates endothelial-specific gene expression via super-enhancers. Methods and Results: Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) in human umbilical vein endothelial cells (HUVEC) showed that ERG binds 93% of super-enhancers ranked according to H3K27ac, a mark of active chromatin. These were associated with endothelial genes such as DLL4, CLDN5, VWF and CDH5. Comparison between HUVEC and prostate cancer TMPRSS2:ERG fusion-positive VCaP cells revealed distinctive lineage-specific transcriptome and super-enhancer profiles. At a subset of endothelial super-enhancers (including DLL4 and CLDN5), loss of ERG results in significant reduction in gene expression which correlates with decreased enrichment of H3K27ac and Mediator subunit MED1, and reduced recruitment of acetyltransferase p300. At these super-enhancers, co-occupancy of GATA2 and AP-1 is significantly lower compared to super-enhancers that remained constant following ERG inhibition. These data suggest distinct mechanisms of super-enhancer regulation in EC and highlight the unique role of ERG in controlling a core subset of super-enhancers. Most disease-associated single nucleotide polymorphisms (SNPs) from genome-wide association studies (GWAS) lie within noncoding regions and perturb TF recognition sequences in relevant cell types. Analysis of GWAS data shows significant enrichment of risk variants for CVD and other diseases, at ERG endothelial enhancers and superenhancers. Conclusions: The TF ERG promotes endothelial homeostasis via regulation of lineage-specific enhancers and super-enhancers. Enrichment of CVD-associated SNPs at ERG super-enhancers suggests that ERGdependent transcription modulates disease risk.This work was funded by grants from the British Heart Foundation (RG/11/17/29256; RG/17/4/32662; FS/15/65/32036; PG/17/33/32990) and Cancer Research U