publications

2025

1. The PRC2.1 subcomplex opposes G1 progression through regulation of CCND1 and CCND2

   Adam D Longhurst, Kyle Wang, Harsha Garadi Suresh, and 12 more authors

   eLife, Feb 2025

   Abs DOI

   Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenetic approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued proliferation inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in antagonizing G1 progression in a diversity of cell linages, including chronic myeloid leukemia (CML), breast cancer, and immortalized cell lines.

2. Comparative characterization of human accelerated regions in neurons

   Xiekui Cui, Han Yang, Charles Cai, and 16 more authors

   Nature, Apr 2025

   DOI
3. 3D-epigenome of Glial Cell Types in Developing Human Cortex [Under Revision in Nature]

   Ian R. Jones, Li Wang, Michael Kosicki, and 15 more authors

   May 2025

   Abs

   Abstract The human cortex is complex and heterogeneous, undergoing extensive expansion during development. Our study of neurogenesis, including radial glia (RG), intermediate progenitor cells (IPCs), excitatory neurons (eNs), and interneurons (iN) demonstrated that chromatin looping underlies transcriptional regulation for lineage-specific genes, shedding insight on how non-coding genetic variants contribute to neuropsychiatric disorders via cell type-specific gene regulation. Radial glia play a crucial role in generating cellular diversity through both neurogenesis and gliogenesis and can be further classified into ventricular radial glia (vRG) and outer radial glia (oRG). Given their significance in cortical development, we conducted a comprehensive 3D epigenomic analysis of four major glial populations, including vRG, oRG, oligodendrocyte precursor cells (OPC), and microglia (MG), from the mid-gestational human neocortex. By integrating gene expression, chromatin accessibility, DNA methylation, and 3D chromatin interactions, we identified cell type-specific candidate cis-regulatory elements (cCREs) and validated their enhancer function using transgenic mouse embryos. Using machine learning, we prioritized 112 schizophrenia risk variants within glia cCREs and further confirmed the predicted vRG enhancer disruption by rs4449074 risk allele in vivo. Finally, oRG cCREs are enriched for human accelerated regions (HARs) compared to other cCREs and a subset of HARs have predicted activity differences compared to their chimpanzee orthologs that interact with genes involved in neuronal development. Our findings advance the understanding of human-specific gene regulation during corticogenesis.

4. Cell type- and temporal-specific 3D epigenome regulates RNA splicing during human cortical development [In Prep.]

   Jing Wang, Ian R. Jones, Li Wang, and 10 more authors

   May 2025

   Abs

   Abstract Genome organization and the transcriptome undergo substantial changes during human brain development. However, the precise developmental dynamics of the 3D epigenome and their role in regulating transcriptomic diversity and cellular function remain poorly understood. To fill this gap, we generated and integrated 3D epigenome and transcriptome data of excitatory neurons, inhibitory neurons, oligodendrocytes, microglia, and astrocytes across second-trimester, third-trimester, neonatal, and adolescent human cortical samples. As neurons matured, we observed strengthened long-range chromatin interactions coupled with the emergence of more complex gene co-expression networks, enriched for functions related to neuronal differentiation and maturation. Furthermore, by leveraging machine learning, we identified key features predictive of cell type-specific RNA splicing, including chromatin accessibility and 3D interaction, enabling the prediction of splicing events from single-cell multi-omics data of diverse brain samples and identification of potential disease-associated splicing events. Overall, our findings illuminate how the 3D epigenome is dynamically rewired to regulate gene expression and RNA splicing during cortical development.

2024

1. A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex

   Xiaoyi G. Liang, Kendy Hoang, Brandon L. Meyerink, and 15 more authors

   Proceedings of the National Academy of Sciences, May 2024

   Abs DOI

   During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1 , Egfr, and Olig2 . The increased Ascl1 expression and appearance of Egfr + and Olig2 + cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2 , a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

2023

1. Functional characterization of Alzheimer’s disease genetic variants in microglia

   Xiaoyu Yang, Jia Wen, Han Yang, and 24 more authors

   Nature Genetics, Oct 2023

   DOI
2. Functional characterization of gene regulatory elements and neuropsychiatric disease-associated risk loci in iPSCs and iPSC-derived neurons

   Xiaoyu Yang, Ian R. Jones, Poshen B. Chen, and 14 more authors

   Aug 2023

   Abs DOI

   Abstract Genome-wide association studies (GWAS) have identified thousands of non-coding variants that contribute to psychiatric disease risks, likely by perturbing cis -regulatory elements (CREs). However, our ability to interpret and explore their mechanisms of action is hampered by a lack of annotation of functional CREs (fCREs) in neural cell types. Here, through genome-scale CRISPR screens of 22,000 candidate CREs (cCREs) in human induced pluripotent stem cells (iPSCs) undergoing differentiation to excitatory neurons, we identify 2,847 and 5,540 fCREs essential for iPSC fitness and neuronal differentiation, respectively. These fCREs display dynamic epigenomic features and exhibit increased numbers and genomic spans of chromatin interactions following terminal neuronal differentiation. Furthermore, fCREs essential for neuronal differentiation show significantly greater enrichment of genetic heritability for neurodevelopmental diseases including schizophrenia (SCZ), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorders (ASD) than cCREs. Using high-throughput prime editing screens we experimentally confirm 45 SCZ risk variants that act by affecting the function of fCREs. The extensive and in-depth functional annotation of cCREs in neuronal types therefore provides a crucial resource for interpreting non-coding risk variants of neuropsychiatric disorders.

2022

1. High-throughput CRISPRi and CRISPRa technologies in 3D genome regulation for neuropsychiatric diseases

   Ian R Jones, Xingjie Ren, and Yin Shen

   Human Molecular Genetics, Oct 2022

   Abs DOI

   Abstract Advances in genomics have led to the identification of many risk loci with hundreds of genes and thousands of DNA variants associated with neuropsychiatric disorders. A significant barrier to understanding the genetic underpinnings of complex diseases is the lack of functional characterization of risk genes and variants in biological systems relevant to human health and connecting disease-associated variants to pathological phenotypes. Characterizing gene and DNA variant functions requires genetic perturbations followed by molecular and cellular assays of neurobiological phenotypes. However, generating null or mutant alleles is low throughput, making it impossible to characterize disease-associated variants in large quantities efficiently. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) screens can be leveraged to dissect the biological consequences of the tested genes and variants in their native context. Nevertheless, testing non-coding variants associated with complex diseases remains non-trivial. In this review, we first discuss the current challenges of interpreting the function of the non-coding genome and approaches to prioritizing disease-associated variants in the context of the 3D epigenome. Second, we provide a brief overview of high-throughput CRISPRi and CRISPRa screening strategies applicable for characterizing non-coding sequences in appropriate biological systems. Lastly, we discuss the promising prospects of using CRISPR-based technologies to dissect DNA sequences associated with neuropsychiatric diseases.

2021

1. Parallel characterization of cis-regulatory elements for multiple genes using CRISPRpath

   Xingjie Ren, Mengchi Wang, Bingkun Li, and 18 more authors

   Science Advances, Sep 2021

   Abs DOI

   A new CRISPR screening strategy allows characterizing enhancers for multiple genes associated with converging phenotypes. , Current pooled CRISPR screens for cis-regulatory elements (CREs), based on transcriptional output changes, are typically limited to characterizing CREs of only one gene. 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. Sixty percent 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.

2. HPRep: Quantifying Reproducibility in HiChIP and PLAC-Seq Datasets

   Jonathan D. Rosen, Yuchen Yang, Armen Abnousi, and 6 more authors

   Current Issues in Molecular Biology, Sep 2021

   Abs DOI

   HiChIP and PLAC-Seq are emerging technologies for studying genome-wide long-range chromatin interactions mediated by the protein of interest, enabling more sensitive and cost-efficient interrogation of protein-centric chromatin conformation. However, due to the unbalanced read distribution introduced by protein immunoprecipitation, existing reproducibility measures developed for Hi-C data are not appropriate for the analysis of HiChIP and PLAC-Seq data. Here, we present HPRep, a stratified and weighted correlation metric derived from normalized contact counts, to quantify reproducibility in HiChIP and PLAC-Seq data. We applied HPRep to multiple real datasets and demonstrate that HPRep outperforms existing reproducibility measures developed for Hi-C data. Specifically, we applied HPRep to H3K4me3 PLAC-Seq data from mouse embryonic stem cells and mouse brain tissues as well as H3K27ac HiChIP data from human lymphoblastoid cell line GM12878 and leukemia cell line K562, showing that HPRep can more clearly separate among pseudo-replicates, real replicates, and non-replicates. Furthermore, in an H3K4me3 PLAC-Seq dataset consisting of 11 samples from four human brain cell types, HPRep demonstrated the expected clustering of data that could not be achieved by existing methods developed for Hi-C data, highlighting the need for a reproducibility metric tailored to HiChIP and PLAC-Seq data.

3. Transcriptional network orchestrating regional patterning of cortical progenitors

   Athéna R. Ypsilanti, Kartik Pattabiraman, Rinaldo Catta-Preta, and 17 more authors

   Proceedings of the National Academy of Sciences, Dec 2021

   Abs DOI

   Significance Development of cortical areas begins in cortical stem cells through the action of morphogens controlling the graded expression of transcription factors (TFs). Here, we have systematically identified the TFs and gene regulatory elements (REs) that together control regional pattering of the cortical progenitor zone; these data have led us to propose a cortical regionalization TF network. To identify REs active in this network, we performed TF chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromatin-looping conformation experiments as well as assays for epigenomic marks and DNA accessibility in purified ventricular zone (VZ) progenitor cells in wild-type and patterning mutant mice. This integrated approach has laid the foundations to identify a TF network and cortical VZ REs involved in cortical regional patterning. , We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects ( Emx2, Nr2f1 , and Pax6 ), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

2020

1. Cell-type-specific 3D epigenomes in the developing human cortex

   Michael Song, Mark-Phillip Pebworth, Xiaoyu Yang, and 20 more authors

   Nature, Nov 2020

   DOI

2019

1. Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain

   Lihong Zhan, Grietje Krabbe, Fei Du, and 13 more authors

   PLOS Biology, Feb 2019

   DOI
2. Mapping cis-regulatory chromatin contacts in neural cells links neuropsychiatric disorder risk variants to target genes

   Michael Song, Xiaoyu Yang, Xingjie Ren, and 20 more authors

   Nature Genetics, Aug 2019

   DOI