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Cardiovascular disease (CVD) remains a leading cause of global morbidity and mortality. Timely diagnosis is important in reducing both short and long-term health complications. Saliva has emerged as a potential source for biomarker discovery, offering a non-invasive tool for early detection of individuals at elevated risk for CVD, yet large-scale extensive proteomic analysis using saliva for a comprehensive biomarker discovery remains limited. In an effort to develop a diagnostic tool using saliva samples, our study aims to assess the salivary and plasma proteomes in subjects with high risk of developing CVD using a large-scale proteomic approach. Leveraging on the SOMAscan platform, we analyzed 1,317 proteins in saliva and plasma collected from subjects at a high risk of CVD (HR-CVD) and compared the profiles to subjects with low risk of CVD (LR-CVD). Our analysis revealed significant differences in the plasma and salivary proteins between the two groups. Pathway enrichment analysis of the differentially detected proteins revealed that the immune system activation and extracellular matrix remodeling are the most enriched pathways in the CVD-HR group. Comparing proteomic signatures between plasma and saliva, we found approximately 42 and 17 differentially expressed proteins associated with CVD-HR uniquely expressed in plasma and saliva respectively. Additionally, we identified eight common CVD-risk biomarkers shared between both plasma and saliva, demonstrating promising diagnostic tools for identifying individuals at high risk of developing CVD. In conclusion, saliva proteomics holds a significant promise to identify subjects with a high risk to develop CVD. Further studies are needed to validate our findings., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Introduction: For years, the placenta was believed to be sterile, but recent studies reveal it hosts a unique microbiome. Despite these findings, significant questions remain about the origins of the placental microbiome and its effects on pregnancy and fetal health. Some studies suggest it may originate from the vaginal tract, while others indicate that oral bacteria can enter the maternal bloodstream and seed the placenta. However, research analyzing the vaginal, oral, and placental microbiomes within the same cohort is lacking. Additionally, it's unclear whether the placental microbiome differs between healthy pregnancies and those with complications like preterm birth (PTB), which remains a leading cause of neonatal morbidity and mortality worldwide., Methods: In this study, we performed 16S rRNA gene sequencing to investigate the composition of the oral and placental microbiome in samples collected from 18 women who experienced PTB and 36 matched controls who delivered at term (TB), all of whom were part of the Molecular Signature in Pregnancy (MSP) study. We leveraged on the multisite microbiome sampling from the MSP participants and on our previously published vaginal microbiome data to investigate the potential origins of the placental microbiome and assess whether its composition varies between healthy and complicated pregnancies., Results and Discussion: Our analysis revealed distinct profiles in the oral microbiome of PTB subjects compared to those who delivered at term. Specifically, we observed an increased abundance of Treponema maltophilum, Bacteroides sp, Mollicutes, Prevotella buccae, Leptotrichia, Prevotella_sp_Alloprevotella, in the PTB group. Importantly, Treponema maltophilum species showed higher abundance in the PTB group during the second trimester, suggesting its potential use as biomarkers. When we assessed the placenta microbiome composition, we found that Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria were the most dominant phyla. Interestingly, microorganisms such as Ureaplasma urealyticum were more abundant in PTB placenta samples. Our findings suggest that the placenta microbiome could originate from the oral or vaginal cavities, with a notable increase in the crosstalk between the vaginal and placental sites in cases of PTB. Specifically, our data revealed that in PTB cases, the placental microbiome exhibited a closer resemblance to the vaginal microbiome, whereas in term pregnancies, the placental microbiome was similar to the oral microbiome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2025 Saadaoui, Djekidel, Murugesan, Kumar, Elhag, Singh, Kabeer, Marr, Kino, Brummaier, McGready, Nosten, Chaussabel, Terranegra and Al Khodor.)

Background: The rising prevalence of Type 2 diabetes mellitus (T2D) in the Qatari population presents a significant public health challenge, highlighting the need for innovative approaches to early detection and management. While most efforts are centered on using blood samples for biomarker discovery, the use of saliva remains underexplored., Methods: Using noninvasive saliva samples from 2974 Qatari subjects, we analyzed the microbial communities from diabetic, pre-diabetic, and non-diabetic participants based on their HbA1C levels. The salivary microbiota was assessed in all subjects by sequencing the V1-V3 regions of 16S rRNA gene. For the proteomics profiling, we randomly selected 50 gender and age-matched non-diabetic and diabetic subjects and compared their proteome with SOMAscan. Microbiota and proteome profiles were then integrated to reveal candidate biomarkers for T2D., Results: Our results indicate that the salivary microbiota of pre-diabetic and diabetic individuals differs significantly from that of non-diabetic subjects. Specifically, a significant increase in the abundance of Campylobacter, Dorea, and Bacteroidales was observed in the diabetic subjects compared to their non-diabetic controls. Metabolic pathway prediction analysis for these bacteria revealed a significant overrepresentation of genes associated with fatty acid and lipid biosynthesis, as well as aromatic amino acid metabolism in the diabetic group. Additionally, we observed distinct differences in salivary proteomic profiles between diabetic and non-diabetic subjects. Notably, levels of Haptoglobin, Plexin-C1, and MCL-1 were elevated, while Osteopontin (SPP1), Histone1H3A (HIST3H2A), and Histone H1.2 were reduced in diabetic individuals. Furthermore, integrated correlation analysis of salivary proteome and microbiota data demonstrated a strong positive correlation between HIST1H3A and HIST3H2A with Porphyromonas sp., all of which were decreased in the diabetic group., Conclusion: This is the first study to assess the salivary microbiota in T2D patients from a large cohort of the Qatari population. We found significant differences in the salivary microbiota of pre-diabetic and diabetic individuals compared to non-diabetic controls. Our study is also the first to assess the salivary proteome using SOMAScan in diabetic and non-diabetic subjects. Integration of the microbiota and proteome profiles revealed a unique signature for T2D that can be used as potential T2D biomarkers., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by the Institutional Review Board (IRB) of Sidra Medicine under (protocol #1510001907) and by Qatar Biobank (QBB) (protocol #E/2018/QBB-RES-ACC-0063/0022. All study participants signed an informed consent prior to sample collection. All experiments were performed in accordance with the approved guidelines. Consent for publication: All authors reviewed the final version of the manuscript and approved it for publication. Competing interests: The authors declare no conflict of interests., (© 2024. The Author(s).)

53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Nuclear factor Foxp3 determines regulatory T (Treg) cell fate and function via mechanisms that remain unclear. Here, we investigate the nature of Foxp3-mediated gene regulation in suppressing autoimmunity and antitumor immune response. Contrasting with previous models, we find that Foxp3-chromatin binding is regulated by Treg activation states, tumor microenvironment, and antigen and cytokine stimulations. Proteomics studies uncover dynamic proteins within Foxp3 proximity upon TCR or IL-2 receptor signaling in vitro, reflecting intricate interactions among Foxp3, signal transducers, and chromatin. Pharmacological inhibition and genetic knockdown experiments indicate that NFAT and AP-1 protein Batf are required for enhanced Foxp3-chromatin binding in activated Treg cells and tumor-infiltrating Treg cells to modulate target gene expression. Furthermore, mutations at the Foxp3 DNA-binding domain destabilize the Foxp3-chromatin association. These representative settings delineate context-dependent Foxp3-chromatin interaction, suggesting that Foxp3 associates with chromatin by hijacking DNA-binding proteins resulting from Treg activation or differentiation, which is stabilized by direct Foxp3-DNA binding, to dynamically regulate Treg cell function according to immunological contexts., (© 2024 He et al.)

Sequence-based genetic testing identifies causative variants in ~ 50% of individuals with developmental and epileptic encephalopathies (DEEs). Aberrant changes in DNA methylation are implicated in various neurodevelopmental disorders but remain unstudied in DEEs. We interrogate the diagnostic utility of genome-wide DNA methylation array analysis on peripheral blood samples from 582 individuals with genetically unsolved DEEs. We identify rare differentially methylated regions (DMRs) and explanatory episignatures to uncover causative and candidate genetic etiologies in 12 individuals. Using long-read sequencing, we identify DNA variants underlying rare DMRs, including one balanced translocation, three CG-rich repeat expansions, and four copy number variants. We also identify pathogenic variants associated with episignatures. Finally, we refine the CHD2 episignature using an 850 K methylation array and bisulfite sequencing to investigate potential insights into CHD2 pathophysiology. Our study demonstrates the diagnostic yield of genome-wide DNA methylation analysis to identify causal and candidate variants as 2% (12/582) for unsolved DEE cases., (© 2024. The Author(s).)

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8 + T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8 + T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The prefrontal cortex (PFC) is a complex brain region that regulates diverse functions ranging from cognition, emotion and executive action to even pain processing. To decode the cellular and circuit organization of such diverse functions, we employed spatially resolved single-cell transcriptome profiling of the adult mouse PFC. Results revealed that PFC has distinct cell-type composition and gene-expression patterns relative to neighboring cortical areas-with neuronal excitability-regulating genes differently expressed. These cellular and molecular features are further segregated within PFC subregions, alluding to the subregion-specificity of several PFC functions. PFC projects to major subcortical targets through combinations of neuronal subtypes, which emerge in a target-intrinsic fashion. Finally, based on these features, we identified distinct cell types and circuits in PFC underlying chronic pain, an escalating healthcare challenge with limited molecular understanding. Collectively, this comprehensive map will facilitate decoding of discrete molecular, cellular and circuit mechanisms underlying specific PFC functions in health and disease., (© 2023. The Author(s).)

Stem cell survival versus death is a developmentally programmed process essential for morphogenesis, sizing, and quality control of genome integrity and cell fates. Cell death is pervasive during development, but its programming is little known. Here, we report that Smad nuclear interacting protein 1 (SNIP1) promotes neural progenitor cell survival and neurogenesis and is, therefore, integral to brain development. The SNIP1-depleted brain exhibits dysplasia with robust induction of caspase 9-dependent apoptosis. Mechanistically, SNIP1 regulates target genes that promote cell survival and neurogenesis, and its activities are influenced by TGFβ and NFκB signaling pathways. Further, SNIP1 facilitates the genomic occupancy of Polycomb complex PRC2 and instructs H3K27me3 turnover at target genes. Depletion of PRC2 is sufficient to reduce apoptosis and brain dysplasia and to partially restore genetic programs in the SNIP1-depleted brain in vivo. These findings suggest a loci-specific regulation of PRC2 and H3K27 marks to toggle cell survival and death in the developing brain., (© 2023. Springer Nature Limited.)

53BP1 is a well-established DNA damage repair factor recently shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.

Bromo- and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activities in many cancers. However, the mechanisms controlling BETi response and resistance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a combination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combination treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.

Background: CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF., Results: We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF., Conclusions: By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time., (© 2023. The Author(s).)

Schizophrenia (SCZ) is a chronic, serious mental disorder. Although more than 200 SCZ-associated genes have been identified, the underlying molecular and cellular mechanisms remain largely unknown. Here, we generated a Setd1a (SET domain containing 1A) haploinsufficiency mouse model to understand how this SCZ-associated epigenetic factor affects gene expression in brain regions highly relevant to SCZ. Single-cell RNA sequencing revealed that Setd1a heterozygosity causes highly variable transcriptional adaptations across different cell types in prefrontal cortex (PFC) and striatum. The Foxp2 + neurons exhibit the most prominent gene expression changes among the different neuron subtypes in PFC, which correlate with changes in histone H3 lysine 4 trimethylation. Many of the genes dysregulated in Setd1a +/- mice are involved in neuron morphogenesis and synaptic function. Consistently, Setd1a +/- mice exhibit certain behavioral features of patients with SCZ. Collectively, our study establishes Setd1a +/- mice as a model for understanding SCZ and uncovers a complex brain region- and cell type-specific dysregulation that potentially underlies SCZ pathogenesis.

The nucleus accumbens (NAc) plays an important role in regulating multiple behaviors, and its dysfunction has been linked to many neural disorders. However, the molecular, cellular and anatomic heterogeneity underlying its functional diversity remains incompletely understood. In this study, we generated a cell census of the mouse NAc using single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization, revealing a high level of cell heterogeneity in this brain region. Here we show that the transcriptional and spatial diversity of neuron subtypes underlie the NAc's anatomic and functional heterogeneity. These findings explain how the seemingly simple neuronal composition of the NAc achieves its highly heterogenous structure and diverse functions. Collectively, our study generates a spatially resolved cell taxonomy for understanding the structure and function of the NAc, which demonstrates the importance of combining molecular and spatial information in revealing the fundamental features of the nervous system., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species 1,2 . However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription., Results: Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription., Conclusions: This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation., (© 2021. The Author(s).)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans could cause coronavirus disease 2019 (COVID-19). Since its first discovery in Dec 2019, SARS-CoV-2 has become a global pandemic and caused 3.3 million direct/indirect deaths (2021 May). Amongst the scientific community's response to COVID-19, data sharing has emerged as an essential aspect of the combat against SARS-CoV-2. Despite the ever-growing studies about SARS-CoV-2 and COVID-19, to date, only a few databases were curated to enable access to gene expression data. Furthermore, these databases curated only a small set of data and do not provide easy access for investigators without computational skills to perform analyses. To fill this gap and advance open-access to the growing gene expression data on this deadly virus, we collected about 1,500 human bulk RNA-seq datasets from publicly available resources, developed a database and visualization tool, named CovidExpress (https://stjudecab.github.io/covidexpress). This open access database will allow research investigators to examine the gene expression in various tissues, cell lines, and their response to SARS-CoV-2 under different experimental conditions, accelerating the understanding of the etiology of this disease to inform the drug and vaccine development. Our integrative analysis of this big dataset highlights a set of commonly regulated genes in SARS-CoV-2 infected lung and Rhinovirus infected nasal tissues, including OASL that were under-studied in COVID-19 related reports. Our results also suggested a potential FURIN positive feedback loop that might explain the evolutional advantage of SARS-CoV-2.

Polycomb repressive complexes 1 and 2 (PRC1/2) maintain transcriptional silencing of developmental genes largely by catalyzing the formation of mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3), respectively. How Polycomb domains are reprogrammed during mammalian preimplantation development remains largely unclear. Here we show that, although H2AK119ub1 and H3K27me3 are highly colocalized in gametes, they undergo differential reprogramming dynamics following fertilization. H3K27me3 maintains thousands of maternally biased domains until the blastocyst stage, whereas maternally biased H2AK119ub1 distribution in zygotes is largely equalized at the two-cell stage. Notably, while maternal PRC2 depletion has a limited effect on global H2AK119ub1 in early embryos, it disrupts allelic H2AK119ub1 at H3K27me3 imprinting loci including Xist. By contrast, acute H2AK119ub1 depletion in zygotes does not affect H3K27me3 imprinting maintenance, at least by the four-cell stage. Importantly, loss of H2AK119ub1, but not H3K27me3, causes premature activation of developmental genes during zygotic genome activation (ZGA) and subsequent embryonic arrest. Thus, our study reveals distinct dynamics and functions of H3K27me3 and H2AK119ub1 in mouse preimplantation embryos.

In mouse embryonic stem cell (ESC), a small cell population displays totipotent features by expressing a set of genes that are transiently active in 2-cell-stage embryos. These 2-cell-like (2C-like) cells spontaneously transit back into the pluripotent state. We previously dissected the transcriptional dynamics of the transition from pluripotency to the totipotent 2C-like state and identified factors that modulate the process. However, how 2C-like cells transit back into the pluripotent state remains largely unknown. In this study, we analyzed the transcriptional dynamics from the 2C-like state to pluripotent ESCs and identified an intermediate state. The intermediate state characterized by two-wave step up-regulation of pluripotent genes is different from the one observed during the 2C-like entry transition. Nonsense-mediated Dux mRNA decay plays an important role in the 2C-like state exit. Thus, our study not only provides a transcriptional roadmap for 2C-like-to-pluripotent state transition but also reveals a key molecular event driving the transition., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Coordinated activity-induced transcriptional changes across multiple neuron subtypes of the prefrontal cortex (PFC) play a pivotal role in encoding and regulating major cognitive behaviors. Yet, the specific transcriptional programs in each neuron subtype remain unknown. Using single-cell RNA sequencing (scRNA-seq), here we comprehensively classify all unique cell subtypes in the PFC. We analyze transcriptional dynamics of each cell subtype under a naturally adaptive and an induced condition. Adaptive changes during adolescence (between P21 and P60), a highly dynamic phase of postnatal neuroplasticity, profoundly impacted transcription in each neuron subtype, including cell type-specific regulation of genes implicated in major neuropsychiatric disorders. On the other hand, an induced plasticity evoked by chronic cocaine addiction resulted in progressive transcriptional changes in multiple neuron subtypes and became most pronounced upon prolonged drug withdrawal. Our findings lay a foundation for understanding cell type-specific postnatal transcriptional dynamics under normal PFC function and in neuropsychiatric disease states.

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.

Midbrain dopamine (mDA) neurons play a central role in reward signaling and are widely implicated in psychiatric and neurodegenerative disorders. To understand how mDA neurons perform these functions, it is important to understand how mDA-specific genes are regulated. However, cellular heterogeneity in the mammalian brain presents a major challenge to obtaining this understanding. To this end, we developed a virus-based approach to label and capture mDA nuclei for transcriptome (RNA-Seq), and low-input chromatin accessibility (liDNase-Seq) profiling, followed by predictive modeling to identify putative transcriptional regulators of mDA neurons. Using this method, we identified Gmeb1, a transcription factor predicted to regulate expression of Th and Dat, genes critical for dopamine synthesis and reuptake, respectively. Gmeb1 knockdown in mDA neurons resulted in downregulation of Th and Dat, as well as in severe motor deficits. This study thus identifies Gmeb1 as a master regulator of mDA gene expression and function, and provides a general method for identifying cell type-specific transcriptional regulators.

Mammalian oocytes have the ability to reset the transcriptional program of differentiated somatic cells into that of totipotent embryos through somatic cell nuclear transfer (SCNT). However, the mechanisms underlying SCNT-mediated reprogramming are largely unknown. To understand the mechanisms governing chromatin reprogramming during SCNT, we profiled DNase I hypersensitive sites (DHSs) in donor cumulus cells and one-cell stage SCNT embryos. To our surprise, the chromatin accessibility landscape of the donor cells is drastically changed to recapitulate that of the in vitro fertilization (IVF)-derived zygotes within 12 hr. Interestingly, this DHS reprogramming takes place even in the presence of a DNA replication inhibitor, suggesting that SCNT-mediated DHS reprogramming is independent of DNA replication. Thus, this study not only reveals the rapid and drastic nature of the changes in chromatin accessibility through SCNT but also establishes a DNA replication-independent model for studying cellular reprogramming., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Polymer-based simulations and experimental studies indicate the existence of a spatial dependency between the adjacent DNA fibers involved in the formation of chromatin loops. However, the existing strategies for detecting differential chromatin interactions assume that the interacting segments are spatially independent from the other segments nearby. To resolve this issue, we developed a new computational method, FIND, which considers the local spatial dependency between interacting loci. FIND uses a spatial Poisson process to detect differential chromatin interactions that show a significant difference in their interaction frequency and the interaction frequency of their neighbors. Simulation and biological data analysis show that FIND outperforms the widely used count-based methods and has a better signal-to-noise ratio., (© 2018 Djekidel et al.; Published by Cold Spring Harbor Laboratory Press.)

Chromatin conformation plays a key role in regulating gene expression and controlling cell differentiation. However, the whole-genome chromatin conformation changes that occur during leukemia cell differentiation are poorly understood. Here, we characterized the changes in chromatin conformation, histone states, chromatin accessibility, and gene expression using an all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation model. The results showed that the boundaries of topological associated domains (TADs) were stable during differentiation; however, the chromatin conformations within several specific TADs were obviously changed. By combining H3K4me3, H3K27ac, and Hi-C signals, we annotated the differential gene-regulatory chromatin interactions upon ATRA induction. The gains and losses of the gene-regulatory chromatin interactions are significantly correlated with gene expression and chromatin accessibility. Finally, we found that the loss of GATA2 expression and DNA binding are crucial for the differentiation process, and changes in the chromatin structure around the GATA2 regulate its expression upon ATRA induction. This study provided both statistical insights and experimental details regarding the relationship between chromatin conformation changes and transcription regulation during leukemia cell differentiation, and the results suggested that the chromatin conformation is a new type of potential drug target for cancer therapy.

In human cells, DNA is hierarchically organized and assembled with histones and DNA-binding proteins in three dimensions. Chromatin interactions play important roles in genome architecture and gene regulation, including robustness in the developmental stages and flexibility during the cell cycle. Here we propose in situ Hi-C method named Bridge Linker-Hi-C (BL-Hi-C) for capturing structural and regulatory chromatin interactions by restriction enzyme targeting and two-step proximity ligation. This method improves the sensitivity and specificity of active chromatin loop detection and can reveal the regulatory enhancer-promoter architecture better than conventional methods at a lower sequencing depth and with a simpler protocol. We demonstrate its utility with two well-studied developmental loci: the beta-globin and HOXC cluster regions.

Various efforts have been made to elucidate the cooperating proteins involved in maintaining chromatin interactions; however, many are still unknown. Here, we present 3CPET, a tool based on a non-parametric Bayesian approach, to infer the set of the most probable protein complexes involved in maintaining chromatin interactions and the regions that they may control, making it a valuable downstream analysis tool in chromatin conformation studies. 3CPET does so by combining data from ChIA-PET, transcription factor binding sites, and protein interactions. 3CPET results show biologically significant and accurate predictions when validated against experimental and simulation data.