Acute depletion of BRG1 reveals its primary function as an activator of transcription.

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

Evaluating Machine Learning Stability in Predicting Depression and Anxiety Amidst Subjective Response Errors.

Major Depressive Disorder (MDD) and Generalized Anxiety Disorder (GAD) pose significant burdens on individuals and society, necessitating accurate prediction methods. Machine learning (ML) algorithms utilizing electronic health records and survey data offer promising tools for forecasting these conditions. However, potential bias and inaccuracies inherent in subjective survey responses can undermine the precision of such predictions. This research investigates the reliability of five prominent ML algorithms-a Convolutional Neural Network (CNN), Random Forest, XGBoost, Logistic Regression, and Naive Bayes-in predicting MDD and GAD. A dataset rich in biomedical, demographic, and self-reported survey information is used to assess the algorithms' performance under different levels of subjective response inaccuracies. These inaccuracies simulate scenarios with potential memory recall bias and subjective interpretations. While all algorithms demonstrate commendable accuracy with high-quality survey data, their performance diverges significantly when encountering erroneous or biased responses. Notably, the CNN exhibits superior resilience in this context, maintaining performance and even achieving enhanced accuracy, Cohen's kappa score, and positive precision for both MDD and GAD. This highlights the CNN's superior ability to handle data unreliability, making it a potentially advantageous choice for predicting mental health conditions based on self-reported data. These findings underscore the critical importance of algorithmic resilience in mental health prediction, particularly when relying on subjective data. They emphasize the need for careful algorithm selection in such contexts, with the CNN emerging as a promising candidate due to its robustness and improved performance under data uncertainties.

The Drosophila histone methyl-transferase SET1 coordinates multiple signaling pathways in regulating male germline stem cell maintenance and differentiation.

Many cell types come from tissue-specific adult stem cells that maintain the balance between proliferation and differentiation. Here, we study how the H3K4me3 methyltransferase, Set1, regulates early-stage male germ cell proliferation and differentiation in Drosophila. Early-stage germline-specific knockdown of set1 results in a temporally progressed defects, arising as germ cell loss and developing to overpopulated early-stage germ cells. These germline defects also impact the niche architecture and cyst stem cell lineage in a non-cell-autonomous manner. Additionally, wild-type Set1, but not the catalytically inactive Set1, could rescue the set1 knockdown phenotypes, highlighting the functional importance of the methyl-transferase activity of the Set1 enzyme. Further, RNA-seq experiments reveal key signaling pathway components, such as the JAK-STAT pathway gene stat92E and the BMP pathway gene mad, that are upregulated upon set1 knockdown. Genetic interaction assays support the functional relationships between set1 and JAK-STAT or BMP pathways, as mutations of both the stat92E and mad genes suppress the set1 knockdown phenotypes. These findings enhance our understanding of the balance between proliferation and differentiation in an adult stem cell lineage. The germ cell loss followed by over-proliferation phenotypes when inhibiting a histone methyl-transferase raise concerns about using their inhibitors in cancer therapy.

A single N-terminal amino acid determines the distinct roles of histones H3 and H3.3 in the Drosophila male germline stem cell lineage.

Adult stem cells undergo asymmetric cell divisions to produce 2 daughter cells with distinct cell fates: one capable of self-renewal and the other committed for differentiation. Misregulation of this delicate balance can lead to cancer and tissue degeneration. During asymmetric division of Drosophila male germline stem cells (GSCs), preexisting (old) and newly synthesized histone H3 are differentially segregated, whereas old and new histone variant H3.3 are more equally inherited. However, what underlies these distinct inheritance patterns remains unknown. Here, we report that the N-terminal tails of H3 and H3.3 are critical for their inheritance patterns, as well as GSC maintenance and proper differentiation. H3 and H3.3 differ at the 31st position in their N-termini with Alanine for H3 and Serine for H3.3. By swapping these 2 amino acids, we generated 2 mutant histones (i.e., H3A31S and H3.3S31A). Upon expressing them in the early-stage germline, we identified opposing phenotypes: overpopulation of early-stage germ cells in the H3A31S-expressing testes and significant germ cell loss in testes expressing the H3.3S31A. Asymmetric H3 inheritance is disrupted in the H3A31S-expressing GSCs, due to misincorporation of old histones between sister chromatids during DNA replication. Furthermore, H3.3S31A mutation accelerates old histone turnover in the GSCs. Finally, using a modified Chromatin Immunocleavage assay on early-stage germ cells, we found that H3A31S has enhanced occupancy at promoters and transcription starting sites compared with H3, while H3.3S31A is more enriched at transcriptionally silent intergenic regions compared to H3.3. Overall, these results suggest that the 31st amino acids for both H3 and H3.3 are critical for their proper genomic occupancy and function. Together, our findings indicate a critical role for the different amino acid composition of the N-terminal tails between H3 and H3.3 in an endogenous stem cell lineage and provide insights into the importance of proper histone inheritance in specifying cell fates and regulating cellular differentiation.

Opposing functions of circadian protein DBP and atypical E2F family E2F8 in anti-tumor Th9 cell differentiation.

Interleukin-9 (IL-9)-producing CD4+ T helper cells (Th9) have been implicated in allergy/asthma and anti-tumor immunity, yet molecular insights on their differentiation from activated T cells, driven by IL-4 and transforming growth factor-beta (TGF-ß), is still lacking. Here we show opposing functions of two transcription factors, D-binding protein (DBP) and E2F8, in controlling Th9 differentiation. Specifically, TGF-ß and IL-4 signaling induces phosphorylation of the serine 213 site in the linker region of the Smad3 (pSmad3L-Ser213) via phosphorylated p38, which is necessary and sufficient for Il9 gene transcription. We identify DBP and E2F8 as an activator and repressor, respectively, for Il9 transcription by pSmad3L-Ser213. Notably, Th9 cells with siRNA-mediated knockdown for Dbp or E2f8 promote and suppress tumor growth, respectively, in mouse tumor models. Importantly, DBP and E2F8 also exhibit opposing functions in regulating human TH9 differentiation in vitro. Thus, our data uncover a molecular mechanism of Smad3 linker region-mediated, opposing functions of DBP and E2F8 in Th9 differentiation.

scPCOR-seq enables co-profiling of chromatin occupancy and RNAs in single cells.

Cell-to-cell variation in gene expression is a widespread phenomenon, which may play important roles in cellular differentiation, function, and disease development1-9. Chromatin is implicated in contributing to the cellular heterogeneity in gene expression10-16. Fully understanding the mechanisms of cellular heterogeneity requires simultaneous measurement of RNA and occupancy of histone modifications and transcription factors on chromatin due to their critical roles in transcriptional regulation17,18. We generally term the occupancy of histone modifications and transcription factors as Chromatin occupancy. Here, we report a technique, termed scPCOR-seq (single-cell Profiling of Chromatin Occupancy and RNAs Sequencing), for simultaneously profiling genome-wide chromatin protein binding or histone modification marks and RNA expression in the same cell. We demonstrated that scPCOR-seq can profile either H3K4me3 or RNAPII and RNAs in a mixture of human H1, GM12878 and 293 T cells at a single-cell resolution and either H3K4me3, RNAPII, or RNA profile can correctly separate the cells. Application of scPCOR-seq to the in vitro differentiation of the erythrocyte precursor CD36 cells from human CD34 stem or progenitor cells revealed that H3K4me3 and RNA exhibit distinct properties in clustering cells during differentiation. Overall, our work provides a promising approach to understand the relationships among different omics layers.

A dual-activity topoisomerase complex regulates mRNA translation and turnover.

Topoisomerase 3ß (TOP3B) and TDRD3 form a dual-activity topoisomerase complex that interacts with FMRP and can change the topology of both DNA and RNA. Here, we investigated the post-transcriptional influence of TOP3B and associated proteins on mRNA translation and turnover. First, we discovered that in human HCT116 colon cancer cells, knock-out (KO) of TOP3B had similar effects on mRNA turnover and translation as did TDRD3-KO, while FMRP-KO resulted in rather distinct effects, indicating that TOP3B had stronger coordination with TDRD3 than FMRP in mRNA regulation. Second, we identified TOP3B-bound mRNAs in HCT116 cells; we found that while TOP3B did not directly influence the stability or translation of most TOP3B target mRNAs, it stabilized a subset of target mRNAs but had a more complex effect on translation-enhancing for some mRNAs whereas reducing for others. Interestingly, a point mutation that specifically disrupted TOP3B catalytic activity only partially recapitulated the effects of TOP3B-KO on mRNA stability and translation, suggesting that the impact of TOP3B on target mRNAs is partly linked to its ability to change topology of mRNAs. Collectively, our data suggest that TOP3B-TDRD3 can regulate mRNA translation and turnover by mechanisms that are dependent and independent of topoisomerase activity.

Concurrent mapping of multiple epigenetic marks and co-occupancy using ACT2-seq.

BACKGROUND: Genome-wide profiling of epigenetic marks is a core technology in molecular genetics. Co-occupancy of different epigenetic marks or protein factors at the same genomic locations must often be inferred from multiple independently collected data sets. However, this strategy does not provide direct evidence of co-enrichment in the same cells due to the existence of cellular heterogeneity. To address this issue, we have developed a technique termed ACT2-seq that is capable of concurrently profiling multiple epigenetic marks in a single biological sample. In addition to reducing the numbers of samples required for experiments, ACT2-seq is capable of mapping co-occupancy of epigenetic factors on chromatin. This strategy provides direct evidence of co-enrichment without requiring complex single-molecule, single-cell, or magnetic bead-based approaches. RESULTS: We concurrently profiled pairs of two epigenetic marks using ACT2-seq as well as three marks in individual samples. Data obtained using ACT2-seq were found to be reproducible and robust. ACT2-seq was capable of cleanly partitioning concurrently mapped data sets that exhibited distinct enrichment patterns. Using ACT2-seq, we identified distinct relationships between co-occupancy of specific histone modifications and gene expression patterns. CONCLUSIONS: We conclude that ACT2-seq presents an attractive option for epigenomic profiling due to its ease of use, potential for reducing sample and sequencing costs, and ability to simultaneously profile co-occupancy of multiple histone marks and/or chromatin-associated proteins.

Profiling single-cell histone modifications using indexing chromatin immunocleavage sequencing.

Recently, multiple single-cell assays were developed for detecting histone marks at the single-cell level. These techniques are either limited by the low cell throughput or sparse reads which limit their applications. To address these problems, we introduce indexing single-cell immunocleavage sequencing (iscChIC-seq), a multiplex indexing method based on TdT terminal transferase and T4 DNA ligase-mediated barcoding strategy and single-cell ChIC-seq, which is capable of readily analyzing histone modifications across tens of thousands of single cells in one experiment. Application of iscChIC-seq to profiling H3K4me3 and H3K27me3 in human white blood cells (WBCs) enabled successful detection of more than 10,000 single cells for each histone modification with 11 K and 45 K nonredundant reads per cell, respectively. Cluster analysis of these data allowed identification of monocytes, T cells, B cells, and NK cells from WBCs. The cell types annotated from H3K4me3 single-cell data are specifically correlated with the cell types annotated from H3K27me3 single-cell data. Our data indicate that iscChIC-seq is a reliable technique for profiling histone modifications in a large number of single cells, which may find broad applications in studying cellular heterogeneity and differentiation status in complex developmental and disease systems.

Multiplex indexing approach for the detection of DNase I hypersensitive sites in single cells.

Single cell chromatin accessibility assays reveal epigenomic variability at cis-regulatory elements among individual cells. We previously developed a single-cell DNase-seq assay (scDNase-seq) to profile accessible chromatin in a limited number of single cells. Here, we report a novel indexing strategy to resolve single-cell DNase hypersensitivity profiles based on bulk cell analysis. This new technique, termed indexing single-cell DNase sequencing (iscDNase-seq), employs the activities of terminal DNA transferase (TdT) and T4 DNA ligase to add unique cell barcodes to DNase-digested chromatin ends. By a three-layer indexing strategy, it allows profiling genome-wide DHSs for >15 000 single-cells in a single experiment. Application of iscDNase-seq to human white blood cells accurately revealed specific cell types and inferred regulatory transcription factors (TF) specific to each cell type. We found that iscDNase-seq detected DHSs with specific properties related to gene expression and conservation missed by scATAC-seq for the same cell type. Also, we found that the cell-to-cell variation in accessibility computed using iscDNase-seq data is significantly correlated with the cell-to-cell variation in gene expression. Importantly, this correlation is significantly higher than that between scATAC-seq and scRNA-seq, suggesting that iscDNase-seq data can better predict the cellular heterogeneity in gene expression compared to scATAC-seq. Thus, iscDNase-seq is an attractive alternative method for single-cell epigenomics studies.

Author Correction: Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Topoisomerase 3ß knockout mice show transcriptional and behavioural impairments associated with neurogenesis and synaptic plasticity.

Topoisomerase 3ß (Top3ß) is the only dual-activity topoisomerase in animals that can change topology for both DNA and RNA, and facilitate transcription on DNA and translation on mRNAs. Top3ß mutations have been linked to schizophrenia, autism, epilepsy, and cognitive impairment. Here we show that Top3ß knockout mice exhibit behavioural phenotypes related to psychiatric disorders and cognitive impairment. The mice also display impairments in hippocampal neurogenesis and synaptic plasticity. Notably, the brains of the mutant mice exhibit impaired global neuronal activity-dependent transcription in response to fear conditioning stress, and the affected genes include many with known neuronal functions. Our data suggest that Top3ß is essential for normal brain function, and that defective neuronal activity-dependent transcription may be a mechanism by which Top3ß deletion causes cognitive impairment and psychiatric disorders.

TGF-ß induces ST2 and programs ILC2 development.

The molecular pathways underlying the development of innate lymphoid cells (ILCs) are mostly unknown. Here we show that TGF-ß signaling programs the development of ILC2s from their progenitors. Specifically, the deficiency of TGF-ß receptor II in bone marrow progenitors results in inefficient development of ILC2s, but not ILC1s or ILC3s. Mechanistically, TGF-ß signaling is required for the generation and maintenance of ILC2 progenitors (ILC2p). In addition, TGF-ß upregulates the expression of the IL-33 receptor gene Il1rl1 (encoding IL-1 receptor-like 1, also known as ST2) in ILC2p and common helper-like innate lymphoid progenitors (CHILP), at least partially through the MEK-dependent pathway. These findings identify a function of TGF-ß in the development of ILC2s from their progenitors.

Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq).

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification.

Our method for analyzing histone modifications, scChIC-seq (single-cell chromatin immunocleavage sequencing), involves targeting of the micrococcal nuclease (MNase) to a histone mark of choice by tethering to a specific antibody. Cleaved target sites are then selectively PCR amplified. We show that scChIC-seq reliably detects H3K4me3 and H3K27me3 target sites in single human white blood cells. The resulting data are used for clustering of blood cell types.

Topoisomerase 3ß interacts with RNAi machinery to promote heterochromatin formation and transcriptional silencing in Drosophila.

Topoisomerases solve topological problems during DNA metabolism, but whether they participate in RNA metabolism remains unclear. Top3ß represents a family of topoisomerases carrying activities for both DNA and RNA. Here we show that in Drosophila, Top3ß interacts biochemically and genetically with the RNAi-induced silencing complex (RISC) containing AGO2, p68 RNA helicase, and FMRP. Top3ß and RISC mutants are similarly defective in heterochromatin formation and transcriptional silencing by position-effect variegation assay. Moreover, both Top3ß and AGO2 mutants exhibit reduced levels of heterochromatin protein HP1 in heterochromatin. Furthermore, expression of several genes and transposable elements in heterochromatin is increased in the Top3ß mutant. Notably, Top3ß mutants defective in either RNA binding or catalytic activity are deficient in promoting HP1 recruitment and silencing of transposable elements. Our data suggest that Top3ß may act as an RNA topoisomerase in siRNA-guided heterochromatin formation and transcriptional silencing.

Dominant-negative SMARCA4 mutants alter the accessibility landscape of tissue-unrestricted enhancers.

Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induces pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in disorders with SMARCA4 mutations in a wide variety of tissues.

Genome-wide identification of histone H2A and histone variant H2A.Z-interacting proteins by bPPI-seq.

H2A is a nucleosome core subunit involved in organizing DNA into a chromatin structure that is often inaccessible to regulatory enzymes. Replacement of H2A by its variant H2A.Z renders chromatin accessible at enhancers and promoters. However, it remains unclear how H2A.Z functions so differently from canonical H2A. Here we report the genome-wide identification of proteins that directly interact with H2A and H2A.Z in vivo using a novel strategy, bPPI-seq. We show that bPPI-seq is a sensitive and robust technique to identify protein-protein interactions in vivo. Our data indicate that H2A.Z-interacting proteins and H2A-interacting proteins participate in distinct biological processes. In contrast to H2A-interacting proteins, the H2A.Z-interacting proteins are involved in transcriptional regulation. We found that the transcription factor Osr1 interacts with H2A.Z both in vitro and in vivo. It also mediates H2A.Z incorporation to a large number of target sites and regulates gene expression. Our data indicate that bPPI-seq can be widely applied to identify genome-wide interacting proteins under physiological conditions.

SMARCB1 is required for widespread BAF complex-mediated activation of enhancers and bivalent promoters.

Perturbations to mammalian SWI/SNF (mSWI/SNF or BAF) complexes contribute to more than 20% of human cancers, with driving roles first identified in malignant rhabdoid tumor, an aggressive pediatric cancer characterized by biallelic inactivation of the core BAF complex subunit SMARCB1 (BAF47). However, the mechanism by which this alteration contributes to tumorigenesis remains poorly understood. We find that BAF47 loss destabilizes BAF complexes on chromatin, absent significant changes in complex assembly or integrity. Rescue of BAF47 in BAF47-deficient sarcoma cell lines results in increased genome-wide BAF complex occupancy, facilitating widespread enhancer activation and opposition of Polycomb-mediated repression at bivalent promoters. We demonstrate differential regulation by two distinct mSWI/SNF assemblies, BAF and PBAF complexes, enhancers and promoters, respectively, suggesting that each complex has distinct functions that are perturbed upon BAF47 loss. Our results demonstrate collaborative mechanisms of mSWI/SNF-mediated gene activation, identifying functions that are co-opted or abated to drive human cancers and developmental disorders.

Smarca4 ATPase mutations disrupt direct eviction of PRC1 from chromatin.

Trithorax-group proteins and their mammalian homologs, including those in BAF (mSWI/SNF) complexes, are known to oppose the activity of Polycomb repressive complexes (PRCs). This opposition underlies the tumor-suppressive role of BAF subunits and is expected to contribute to neurodevelopmental disorders. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease-associated mutations in BAF subunits induce genome-wide increases in PRC deposition and activity. We show that point mutations in SMARCA4 (also known as BRG1) mapping to the ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction is ATP dependent, consistent with a transient eviction mechanism. Using a new chemical-induced proximity assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, thereby establishing a new mechanism for the widespread BAF-PRC opposition underlying development and disease.