Biology subjects

Tam, T. W.

Publications and source records attributed to Tam, T. W..

3 recordsLinked to original sources

bioRxiv2024

CRISPR tiling deletion screens reveal functional enhancers of neuropsychiatric risk genes and allelic compensation effects (ACE) on transcription

Precise transcriptional regulation is critical for cellular function and development, yet the mechanism of this process remains poorly understood for many genes. To gain a deeper understanding of the regulation of neuropsychiatric disease risk genes, we identified a total of 39 functional enhancers for four dosage-sensitive genes, APP, FMR1, MECP2, and SIN3A, using CRISPR tiling deletion screening in human induced pluripotent stem cell (iPSC)-induced excitatory neurons. We found that enhancer annotation provides potential pathological insights into disease-associated copy number variants. More importantly, we discovered that allelic enhancer deletions at SIN3A could be compensated by increased transcriptional activities from the other intact allele. Such allelic compensation effects (ACE) on transcription is stably maintained during differentiation and, once established, cannot be reversed by ectopic SIN3A expression. Further, ACE at SIN3A occurs through dosage sensing by the promoter. Together, our findings unravel a regulatory compensation mechanism that ensures stable and precise transcriptional output for SIN3A, and potentially other dosage-sensitive genes.

genetics↗

bioRxiv2021

Parallel Characterization of cis-Regulatory Elements for Multiple Genes UsingCRISPRpath

Current pooled CRISPR screens for cis-regulatory elements (CREs) can only accommodate one gene based on its expression level. Here, we describe CRISPRpath, a scalable screening strategy for parallelly characterizing CREs of genes linked to the same biological pathway and converging phenotypes. We demonstrate the ability of CRISPRpath for simultaneously identifying functional enhancers of six genes in the 6-thioguanine-induced DNA mismatch repair pathway using both CRISPR interference (CRISPRi) and CRISPR nuclease (CRISPRn) approaches. 60% of the identified enhancers are known promoters with distinct epigenomic features compared to other active promoters, including increased chromatin accessibility and interactivity. Furthermore, by imposing different levels of selection pressure, CRISPRpath can distinguish enhancers exerting strong impact on gene expression from those exerting weak impact. Our results offer a nuanced view of cis-regulation and demonstrate that CRISPRpath can be leveraged for understanding the complex gene regulatory program beyond transcriptional output at scale.

genomics↗

bioRxiv2018

cis-Regulatory Chromatin Contacts in Neural Cells Reveal Contributions of Genetic Variants to Complex Neurological Disorders

Mutations in gene regulatory elements have been associated with a wide range of complex neurological disorders. However, due to their inherent cell type-specificity and difficulties in characterizing their regulatory targets, our ability to identify causal genetic variants has remained limited. To address these constraints, we perform integrative analysis of chromatin interactions using promoter capture Hi-C (pcHi-C), open chromatin regions using ATAC-seq, and transcriptomes using RNA-seq in four functionally distinct neural cell types: iPSC-induced excitatory neurons and lower motor neurons, iPSC-derived hippocampal dentate gyrus (DG)-like neurons, and primary astrocytes. We identify hundreds of thousands of long-range cis interactions between promoters and distal promoter-interacting regions (PIRs), enabling us to link regulatory elements to their target genes and reveal putative pathways that are dysregulated in disease. We validate several novel PIRs using CRISPR techniques in human excitatory neurons, demonstrating that CDK5RAP3, STRAP, and DRD2 are transcriptionally regulated by physically linked enhancers. Finally, we show that physical chromatin interactions mediate genetic interactions in autism spectrum disorder (ASD). Our study illustrates how characterizing the 3D epigenome elucidates novel regulatory relationships in the central nervous system (CNS), shedding light on previously unknown functions for noncoding variants in complex neurological disorders.

genomics↗