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

Lineage-specific epigenomic changes during human corticogenesis have been difficult to study owing to challenges with sample availability and tissue heterogeneity. For example, previous studies using single-cell RNA sequencing identified at least 9 major cell types and up to 26 distinct subtypes in the dorsal cortex alone1,2. Here we characterize cell-type-specific cis-regulatory chromatin interactions, open chromatin peaks, and transcriptomes for radial glia, intermediate progenitor cells, excitatory neurons, and interneurons isolated from mid-gestational samples of the human cortex. We show that chromatin interactions underlie several aspects of gene regulation, with transposable elements and disease-associated variants enriched at distal interacting regions in a cell-type-specific manner. In addition, promoters with increased levels of chromatin interactivity-termed super-interactive promoters-are enriched for lineage-specific genes, suggesting that interactions at these loci contribute to the fine-tuning of transcription. Finally, we develop CRISPRview, a technique that integrates immunostaining, CRISPR interference, RNAscope, and image analysis to validate cell-type-specific cis-regulatory elements in heterogeneous populations of primary cells. Our findings provide insights into cell-type-specific gene expression patterns in the developing human cortex and advance our understanding of gene regulation and lineage specification during this crucial developmental window.

Sensory experience remodels genome architecture in neural circuit to drive motor learning.

Neuronal-activity-dependent transcription couples sensory experience to adaptive responses of the brain including learning and memory. Mechanisms of activity-dependent gene expression including alterations of the epigenome have been characterized1-8. However, the fundamental question of whether sensory experience remodels chromatin architecture in the adult brain in vivo to induce neural code transformations and learning and memory remains to be addressed. Here we use in vivo calcium imaging, optogenetics and pharmacological approaches to show that granule neuron activation in the anterior dorsal cerebellar vermis has a crucial role in a delay tactile startle learning paradigm in mice. Of note, using large-scale transcriptome and chromatin profiling, we show that activation of the motor-learning-linked granule neuron circuit reorganizes neuronal chromatin including through long-distance enhancer-promoter and transcriptionally active compartment interactions to orchestrate distinct granule neuron gene expression modules. Conditional CRISPR knockout of the chromatin architecture regulator cohesin in anterior dorsal cerebellar vermis granule neurons in adult mice disrupts enhancer-promoter interactions, activity-dependent transcription and motor learning. These findings define how sensory experience patterns chromatin architecture and neural circuit coding in the brain to drive motor learning.

Publisher Correction: Sensory experience remodels genome architecture in neural circuit to drive motor learning.

In this Letter, '≥' should be '≤' in the sentence: "Intra-chromosomal reads were further split into short-range reads (≥1 kb) and long-range reads (>1 kb)". This error has been corrected online.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circular ecDNA promotes accessible chromatin and high oncogene expression.

Oncogenes are commonly amplified on particles of extrachromosomal DNA (ecDNA) in cancer1,2, but our understanding of the structure of ecDNA and its effect on gene regulation is limited. Here, by integrating ultrastructural imaging, long-range optical mapping and computational analysis of whole-genome sequencing, we demonstrate the structure of circular ecDNA. Pan-cancer analyses reveal that oncogenes encoded on ecDNA are among the most highly expressed genes in the transcriptome of the tumours, linking increased copy number with high transcription levels. Quantitative assessment of the chromatin state reveals that although ecDNA is packaged into chromatin with intact domain structure, it lacks higher-order compaction that is typical of chromosomes and displays significantly enhanced chromatin accessibility. Furthermore, ecDNA is shown to have a significantly greater number of ultra-long-range interactions with active chromatin, which provides insight into how the structure of circular ecDNA affects oncogene function, and connects ecDNA biology with modern cancer genomics and epigenetics.

HiC-ACT: improved detection of chromatin interactions from Hi-C data via aggregated Cauchy test.

Genome-wide chromatin conformation capture technologies such as Hi-C are commonly employed to study chromatin spatial organization. In particular, to identify statistically significant long-range chromatin interactions from Hi-C data, most existing methods such as Fit-Hi-C/FitHiC2 and HiCCUPS assume that all chromatin interactions are statistically independent. Such an independence assumption is reasonable at low resolution (e.g., 40 kb bin) but is invalid at high resolution (e.g., 5 or 10 kb bins) because spatial dependency of neighboring chromatin interactions is non-negligible at high resolution. Our previous hidden Markov random field-based methods accommodate spatial dependency but are computationally intensive. It is urgent to develop approaches that can model spatial dependence in a computationally efficient and scalable manner. Here, we develop HiC-ACT, an aggregated Cauchy test (ACT)-based approach, to improve the detection of chromatin interactions by post-processing results from methods assuming independence. To benchmark the performance of HiC-ACT, we re-analyzed deeply sequenced Hi-C data from a human lymphoblastoid cell line, GM12878, and mouse embryonic stem cells (mESCs). Our results demonstrate advantages of HiC-ACT in improving sensitivity with controlled type I error. By leveraging information from neighboring chromatin interactions, HiC-ACT enhances the power to detect interactions with lower signal-to-noise ratio and similar (if not stronger) epigenetic signatures that suggest regulatory roles. We further demonstrate that HiC-ACT peaks show higher overlap with known enhancers than Fit-Hi-C/FitHiC2 peaks in both GM12878 and mESCs. HiC-ACT, effectively a summary statistics-based approach, is computationally efficient (∼6 min and ∼2 GB memory to process 25,000 pairwise interactions).

SnapHiC: a computational pipeline to identify chromatin loops from single-cell Hi-C data.

Single-cell Hi-C (scHi-C) analysis has been increasingly used to map chromatin architecture in diverse tissue contexts, but computational tools to define chromatin loops at high resolution from scHi-C data are still lacking. Here, we describe Single-Nucleus Analysis Pipeline for Hi-C (SnapHiC), a method that can identify chromatin loops at high resolution and accuracy from scHi-C data. Using scHi-C data from 742 mouse embryonic stem cells, we benchmark SnapHiC against a number of computational tools developed for mapping chromatin loops and interactions from bulk Hi-C. We further demonstrate its use by analyzing single-nucleus methyl-3C-seq data from 2,869 human prefrontal cortical cells, which uncovers cell type-specific chromatin loops and predicts putative target genes for noncoding sequence variants associated with neuropsychiatric disorders. Our results indicate that SnapHiC could facilitate the analysis of cell type-specific chromatin architecture and gene regulatory programs in complex tissues.

A systematic evaluation of Hi-C data enhancement methods for enhancing PLAC-seq and HiChIP data.

The three-dimensional organization of chromatin plays a critical role in gene regulation. Recently developed technologies, such as HiChIP and proximity ligation-assisted ChIP-Seq (PLAC-seq) (hereafter referred to as HP for brevity), can measure chromosome spatial organization by interrogating chromatin interactions mediated by a protein of interest. While offering cost-efficiency over genome-wide unbiased high-throughput chromosome conformation capture (Hi-C) data, HP data remain sparse at kilobase (Kb) resolution with the current sequencing depth in the order of 108 reads per sample. Deep learning models, including HiCPlus, HiCNN, HiCNN2, DeepHiC and Variationally Encoded Hi-C Loss Enhancer (VEHiCLE), have been developed to enhance the sequencing depth of Hi-C data, but their performance on HP data has not been benchmarked. Here, we performed a comprehensive evaluation of HP data sequencing depth enhancement using models developed for Hi-C data. Specifically, we analyzed various HP data, including Smc1a HiChIP data of the human lymphoblastoid cell line GM12878, H3K4me3 PLAC-seq data of four human neural cell types as well as of mouse embryonic stem cells (mESC), and mESC CCCTC-binding factor (CTCF) PLAC-seq data. Our evaluations lead to the following three findings: (i) most models developed for Hi-C data achieve reasonable performance when applied to HP data (e.g. with Pearson correlation ranging 0.76-0.95 for pairs of loci within 300 Kb), and the enhanced datasets lead to improved statistical power for detecting long-range chromatin interactions, (ii) models trained on HP data outperform those trained on Hi-C data and (iii) most models are transferable across cell types. Our results provide a general guideline for HP data enhancement using existing methods designed for Hi-C data.

Transcriptional network orchestrating regional patterning of cortical progenitors.

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.

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

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.

Global analysis of histone modifications and long-range chromatin interactions revealed the differential cistrome changes and novel transcriptional players in human dilated cardiomyopathy.

BACKGROUND: Acetylation and methylation of histones alter the chromatin structure and accessibility that affect transcriptional regulators binding to enhancers and promoters. The binding of transcriptional regulators enables the interaction between enhancers and promoters, thus affecting gene expression. However, our knowledge of these epigenetic alternations in patients with heart failure remains limited. METHODS AND RESULTS: From the comprehensive analysis of major histone modifications, 3-dimensional chromatin interactions, and transcriptome in left ventricular (LV) tissues from dilated cardiomyopathy (DCM) patients and non-heart failure (NF) donors, differential active enhancer and promoter regions were identified between NF and DCM. Moreover, the genome-wide average promoter signal is significantly lower in DCM than in NF. Super-enhancer (SE) analysis revealed that fewer SEs were found in DCM LVs than in NF ones, and three unique SE-associated genes between NF and DCM were identified. Moreover, SEs are enriched within the genomic region associated with long-range chromatin interactions. The differential enhancer-promoter interactions were observed in the known heart failure gene loci and are correlated with the gene expression levels. Motif analysis identified known cardiac factors and possible novel players for DCM. CONCLUSIONS: We have established the cistrome of four histone modifications and chromatin interactome for enhancers and promoters in NF and DCM tissues. Differential histone modifications and enhancer-promoter interactions were found in DCM, which were associated with gene expression levels of a subset of disease-associated genes in human heart failure.

MAPS: Model-based analysis of long-range chromatin interactions from PLAC-seq and HiChIP experiments.

Hi-C and chromatin immunoprecipitation (ChIP) have been combined to identify long-range chromatin interactions genome-wide at reduced cost and enhanced resolution, but extracting information from the resulting datasets has been challenging. Here we describe a computational method, MAPS, Model-based Analysis of PLAC-seq and HiChIP, to process the data from such experiments and identify long-range chromatin interactions. MAPS adopts a zero-truncated Poisson regression framework to explicitly remove systematic biases in the PLAC-seq and HiChIP datasets, and then uses the normalized chromatin contact frequencies to identify significant chromatin interactions anchored at genomic regions bound by the protein of interest. MAPS shows superior performance over existing software tools in the analysis of chromatin interactions from multiple PLAC-seq and HiChIP datasets centered on different transcriptional factors and histone marks. MAPS is freely available at https://github.com/ijuric/MAPS.

Alignment-free clustering of large data sets of unannotated protein conserved regions using minhashing.

BACKGROUND: Clustering of protein sequences is of key importance in predicting the structure and function of newly sequenced proteins and is also of use for their annotation. With the advent of multiple high-throughput sequencing technologies, new protein sequences are becoming available at an extraordinary rate. The rapid growth rate has impeded deployment of existing protein clustering/annotation tools which depend largely on pairwise sequence alignment. RESULTS: In this paper, we propose an alignment-free clustering approach, coreClust, for annotating protein sequences using detected conserved regions. The proposed algorithm uses Min-Wise Independent Hashing for identifying similar conserved regions. Min-Wise Independent Hashing works by generating a (w,c)-sketch for each document and comparing these sketches. Our algorithm fits well within the MapReduce framework, permitting scalability. We show that coreClust generates results comparable to existing known methods. In particular, we show that the clusters generated by our algorithm capture the subfamilies of the Pfam domain families for which the sequences in a cluster have a similar domain architecture. We show that for a data set of 90,000 sequences (about 250,000 domain regions), the clusters generated by our algorithm give a 75% average weighted F1 score, our accuracy metric, when compared to the clusters generated by a semi-exhaustive pairwise alignment algorithm. CONCLUSIONS: The new clustering algorithm can be used to generate meaningful clusters of conserved regions. It is a scalable method that when paired with our prior work, NADDA for detecting conserved regions, provides a complete end-to-end pipeline for annotating protein sequences.

HPTAD: A computational method to identify topologically associating domains from HiChIP and PLAC-seq datasets.

High-throughput chromatin conformation capture technologies, such as Hi-C and Micro-C, have enabled genome-wide view of chromatin spatial organization. Most recently, Hi-C-derived enrichment-based technologies, including HiChIP and PLAC-seq, offer attractive alternatives due to their high signal-to-noise ratio and low cost. While a series of computational tools have been developed for Hi-C data, methods tailored for HiChIP and PLAC-seq data are still under development. Here we present HPTAD, a computational method to identify topologically associating domains (TADs) from HiChIP and PLAC-seq data. We performed comprehensive benchmark analysis to demonstrate its superior performance over existing TAD callers designed for Hi-C data. HPTAD is freely available at https://github.com/yunliUNC/HPTAD.

Combinatorial transcription factor binding encodes cis-regulatory wiring of forebrain GABAergic neurogenesis.

Transcription factors (TFs) bind combinatorially to genomic cis-regulatory elements (cREs), orchestrating transcription programs. While studies of chromatin state and chromosomal interactions have revealed dynamic neurodevelopmental cRE landscapes, parallel understanding of the underlying TF binding lags. To elucidate the combinatorial TF-cRE interactions driving mouse basal ganglia development, we integrated ChIP-seq for twelve TFs, H3K4me3-associated enhancer-promoter interactions, chromatin and transcriptional state, and transgenic enhancer assays. We identified TF-cREs modules with distinct chromatin features and enhancer activity that have complementary roles driving GABAergic neurogenesis and suppressing other developmental fates. While the majority of distal cREs were bound by one or two TFs, a small proportion were extensively bound, and these enhancers also exhibited exceptional evolutionary conservation, motif density, and complex chromosomal interactions. Our results provide new insights into how modules of combinatorial TF-cRE interactions activate and repress developmental expression programs and demonstrate the value of TF binding data in modeling gene regulatory wiring.

SnapHiC2: A computationally efficient loop caller for single cell Hi-C data.

Single cell Hi-C (scHi-C) technologies enable the study of chromatin spatial organization directly from complex tissues at single cell resolution. However, the identification of chromatin loops from single cells is challenging, largely due to the extremely sparse data. Our recently developed SnapHiC pipeline provides the first tool to map chromatin loops from scHi-C data, but it is computationally intensive. Here we introduce SnapHiC2, which adapts a sliding window approximation when imputing missing contacts in each single cell and reduces both memory usage and computational time by 70%. SnapHiC2 can identify 5 Kb resolution chromatin loops with high sensitivity and accuracy and help to suggest target genes for GWAS variants in a cell-type-specific manner. SnapHiC2 is freely available at: https://github.com/HuMingLab/SnapHiC/releases/tag/v0.2.2.

Proximity Ligation-Assisted ChIP-Seq (PLAC-Seq).

Proximity ligation-assisted ChIP-Seq (PLAC-Seq), also known as HiChIP, is a method to detect and quantify chromatin contacts anchored at genomic regions bound by specific proteins or histone modifications. By combining in situ Hi-C and chromatin immunoprecipitation (ChIP) using antibodies against transcription factors (TFs) or histone marks of interest, the method achieves targeted interrogation of chromatin organization at a subset of genomic regions. PLAC-Seq is able to identify long-range chromatin interactions at kilobase-scale resolution with significantly reduced sequencing cost.

MUNIn: A statistical framework for identifying long-range chromatin interactions from multiple samples.

Chromatin spatial organization (interactome) plays a critical role in genome function. Deep understanding of chromatin interactome can shed insights into transcriptional regulation mechanisms and human disease pathology. One essential task in the analysis of chromatin interactomic data is to identify long-range chromatin interactions. Existing approaches, such as HiCCUPS, FitHiC/FitHiC2, and FastHiC, are all designed for analyzing individual cell types or samples. None of them accounts for unbalanced sequencing depths and heterogeneity among multiple cell types or samples in a unified statistical framework. To fill in the gap, we have developed a novel statistical framework MUNIn (multiple-sample unifying long-range chromatin-interaction detector) for identifying long-range chromatin interactions from multiple samples. MUNIn adopts a hierarchical hidden Markov random field (H-HMRF) model, in which the status (peak or background) of each interacting chromatin loci pair depends not only on the status of loci pairs in its neighborhood region but also on the status of the same loci pair in other samples. To benchmark the performance of MUNIn, we performed comprehensive simulation studies and real data analysis and showed that MUNIn can achieve much lower false-positive rates for detecting sample-specific interactions (33.1%-36.2%), and much enhanced statistical power for detecting shared peaks (up to 74.3%), compared to uni-sample analysis. Our data demonstrated that MUNIn is a useful tool for the integrative analysis of interactomic data from multiple samples.

FIREcaller: Detecting frequently interacting regions from Hi-C data.

Hi-C experiments have been widely adopted to study chromatin spatial organization, which plays an essential role in genome function. We have recently identified frequently interacting regions (FIREs) and found that they are closely associated with cell-type-specific gene regulation. However, computational tools for detecting FIREs from Hi-C data are still lacking. In this work, we present FIREcaller, a stand-alone, user-friendly R package for detecting FIREs from Hi-C data. FIREcaller takes raw Hi-C contact matrices as input, performs within-sample and cross-sample normalization, and outputs continuous FIRE scores, dichotomous FIREs, and super-FIREs. Applying FIREcaller to Hi-C data from various human tissues, we demonstrate that FIREs and super-FIREs identified, in a tissue-specific manner, are closely related to gene regulation, are enriched for enhancer-promoter (E-P) interactions, tend to overlap with regions exhibiting epigenomic signatures of cis-regulatory roles, and aid the interpretation or GWAS variants. The FIREcaller package is implemented in R and freely available at https://yunliweb.its.unc.edu/FIREcaller.

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

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-thioguanine­induced 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.

An ultra high-throughput method for single-cell joint analysis of open chromatin and transcriptome.

Simultaneous profiling of transcriptome and chromatin accessibility within single cells is a powerful approach to dissect gene regulatory programs in complex tissues. However, current tools are limited by modest throughput. We now describe an ultra high-throughput method, Paired-seq, for parallel analysis of transcriptome and accessible chromatin in millions of single cells. We demonstrate the utility of Paired-seq for analyzing the dynamic and cell-type-specific gene regulatory programs in complex tissues by applying it to mouse adult cerebral cortex and fetal forebrain. The joint profiles of a large number of single cells allowed us to deconvolute the transcriptome and open chromatin landscapes in the major cell types within these brain tissues, infer putative target genes of candidate enhancers, and reconstruct the trajectory of cellular lineages within the developing forebrain.