Mechanisms Of Gene Regulation Research Articles

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Enhancer Recognition: A Transformer Encoder-Based Method with WGAN-GP for Data Augmentation.

Enhancers are located upstream or downstream of key deoxyribonucleic acid (DNA) sequences in genes and can adjust the transcription activity of neighboring genes. Identifying enhancers and determining their functions are important for understanding gene regulatory networks and expression regulatory mechanisms. However, traditional enhancer recognition relies on manual feature engineering, which is time-consuming and labor-intensive, making it difficult to perform large-scale recognition analysis. In addition, if the original dataset is too small, there is a risk of overfitting. In recent years, emerging methods, such as deep learning, have provided new insights for enhancing identification. However, these methods also present certain challenges. Deep learning models typically require a large amount of high-quality data, and data acquisition demands considerable time and resources. To address these challenges, in this paper, we propose a data-augmentation method based on generative adversarial networks to solve the problem of small datasets. Moreover, we used regularization methods such as weight decay to improve the generalizability of the model and alleviate overfitting. The Transformer encoder was used as the main component to capture the complex relationships and dependencies in enhancer sequences. The encoding layer was designed based on the principle of k-mers to preserve more information from the original DNA sequence. Compared with existing methods, the proposed approach made significant progress in enhancing the accuracy and strength of enhancer identification and prediction, demonstrating the effectiveness of the proposed method. This paper provides valuable insights for enhancer analysis and is of great significance for understanding gene regulatory mechanisms and studying disease correlations.

Data integration and inference of gene regulation using single-cell temporal multimodal data with scTIE.

Single-cell technologies offer unprecedented opportunities to dissect gene regulatory mechanisms in context-specific ways. Although there are computational methods for extracting gene regulatory relationships from scRNA-seq and scATAC-seq data, the data integration problem, essential for accurate cell type identification, has been mostly treated as a standalone challenge. Here we present scTIE, a unified method that integrates temporal multimodal data and infers regulatory relationships predictive of cellular state changes. scTIE uses an autoencoder to embed cells from all time points into a common space by using iterative optimal transport, followed by extracting interpretable information to predict cell trajectories. Using a variety of synthetic and real temporal multimodal data sets, we show scTIE achieves effective data integration while preserving more biological signals than existing methods, particularly in the presence of batch effects and noise. Furthermore, on the exemplar multiome data set we generated from differentiating mouse embryonic stem cells over time, we show scTIE captures regulatory elements highly predictive of cell transition probabilities, providing new potentials to understand the regulatory landscape driving developmental processes.

Multi-ancestry genetic analysis of gene regulation in coronary arteries prioritizes disease risk loci

Genome-wide association studies (GWASs) have identified hundreds of risk loci for coronary artery disease (CAD). However, non-European populations are underrepresented in GWASs, and the causal gene-regulatory mechanisms of these risk loci during atherosclerosis remain unclear. We incorporated local ancestry and haplotypes to identify quantitative trait loci for expression (eQTLs) and splicing (sQTLs) in coronary arteries from 138 ancestrally diverse Americans. Of 2,132 eQTL-associated genes (eGenes), 47% were previously unreported in coronary artery; 19% exhibited cell-type-specific expression. Colocalization revealed subgroups of eGenes unique to CAD and blood pressure GWAS. Fine-mapping highlighted additional eGenes, including TBX20 and IL5. We also identified sQTLs for 1,690 genes, among which TOR1AIP1 and ULK3 sQTLs demonstrated the importance of evaluating splicing to accurately identify disease-relevant isoform expression. Our work provides a patient-derived coronary artery eQTL resource and exemplifies the need for diverse study populations and multifaceted approaches to characterize gene regulation in disease processes.

Gene regulation supporting sociality shared across lineages and variation in complexity.

Across evolutionary lineages, insects vary in social complexity, from those that exhibit extended parental care to those with elaborate divisions of labor. Here, we synthesize the sociogenomic resources from hundreds of species to describe common gene regulatory mechanisms in insects that regulate social organization across phylogeny and levels of social complexity. Different social phenotypes expressed by insects can be linked to the organization of co-expressing gene networks and features of the epigenetic landscape. Insect sociality also stems from processes like the emergence of parental care and the decoupling of ancestral genetic programs. One underexplored avenue is how variation in a group's social environment affects the gene expression of individuals. Additionally, an experimental reduction of gene expression would demonstrate how the activity of specific genes contributes to insect social phenotypes. While tissue specificity provides greater localization of the gene expression underlying social complexity, emerging transcriptomic analysis of insect brains at the cellular level provides even greater resolution to understand the molecular basis of social insect evolution.

A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR.

Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.

Time series-based hybrid ensemble learning model with multivariate multidimensional feature coding for DNA methylation prediction

BackgroundDNA methylation is a form of epigenetic modification that impacts gene expression without modifying the DNA sequence, thereby exerting control over gene function and cellular development. The prediction of DNA methylation is vital for understanding and exploring gene regulatory mechanisms. Currently, machine learning algorithms are primarily used for model construction. However, several challenges remain to be addressed, including limited prediction accuracy, constrained generalization capability, and insufficient learning capacity.ResultsIn response to the aforementioned challenges, this paper leverages the similarities between DNA sequences and time series to introduce a time series-based hybrid ensemble learning model, called Multi2-Con-CAPSO-LSTM. The model utilizes multivariate and multidimensional encoding approach, combining three types of time series encodings with three kinds of genetic feature encodings, resulting in a total of nine types of feature encoding matrices. Convolutional Neural Networks are utilized to extract features from DNA sequences, including temporal, positional, physicochemical, and genetic information, thereby creating a comprehensive feature matrix. The Long Short-Term Memory model is then optimized using the Chaotic Accelerated Particle Swarm Optimization algorithm for predicting DNA methylation.ConclusionsThrough cross-validation experiments conducted on 17 species involving three types of DNA methylation (6 mA, 5hmC, and 4mC), the results demonstrate the robust predictive capabilities of the Multi2-Con-CAPSO-LSTM model in DNA methylation prediction across various types and species. Compared with other benchmark models, the Multi2-Con-CAPSO-LSTM model demonstrates significant advantages in sensitivity, specificity, accuracy, and correlation. The model proposed in this paper provides valuable insights and inspiration across various disciplines, including sequence alignment, genetic evolution, time series analysis, and structure–activity relationships.

Mechanism of Wendan decoction in preventing obesity by regulating multiple signal pathway networks based on gene promoter methylation

ObjectiveTo investigate the potential mechanism of Wendan decoction in obesity by screening target genes with promoter region methylation changes and constructing a multiple signaling pathways network based on promoter methylation. MethodsThe methylation degree of Itgad, Col8a1, Adra2b, Jund, Rab2a, Wnt8b, Fzd9, B4galt7, Pik3cd, Creb1, Stard8, and Mmp1 in the abdominal adipose tissue of obese rats was determined using the Agena MassARRAY system. Western blot was performed to assess protein expression levels. Target genes were identified based on the methylation degree in the promoter region and protein expression. Enrichment analysis of signaling pathways was conducted to identify relevant target genes and obtain a multiple signaling pathway network associated with obesity. Core and terminal effector molecules in the pathway networks were selected as research targets for reverse transcription-polymerase chain reaction (RT-PCR) analysis. ResultsFour genes (Adra2b, Creb1, Itgad, and Pik3cd) showed a degree of promoter methylation consistent with their respective protein expression levels. Among them, Adra2b, Creb1, and Pik3cd expression increased, while that of Itgad decreased. Enrichment analysis revealed that Creb1 and Pik3cd were involved in 6 signaling pathways related to obesity: tumor necrosis factor (TNF) signaling pathway, growth hormone synthesis/secretion and action, adenosine 5′-monophosphate-activated protein kinase (AMPK) signaling pathway, relaxin signaling pathway, cyclic nucleotide (cAMP) signaling pathway, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Subsequently, a multiple signaling pathways network was constructed based on promoter methylation. Key molecules including protein kinase B (AKT), mechanistic target of rapamycin complex 1 (mTORC1), and unc-51 like autophagy activating kinase 1 (ULK1), as well as terminal effector molecules interleukin-1β (IL-1β), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 2 (CXCL2) were selected as research targets. Wendan decoction decreased the expressions of AKT, mTORC1, IL-1β, IL-6, and CXCL2 while up-regulating ULK1 expression. ConclusionThe mechanism of Wendan decoction in preventing obesity involves the regulation of multiple signaling pathways through the control of Creb1 and Pik3cd gene promoter methylation. However, the associated multi-path gene regulation mechanism in preventing obesity is complex. Thus, further exploration is needed to elucidate the role of methylation changes in this mechanism.

Gene expression and alternative splicing contribute to adaptive divergence of ecotypes.

Regulation of gene expression is a critical link between genotype and phenotype explaining substantial heritable variation within species. However, we are only beginning to understand the ways that specific gene regulatory mechanisms contribute to adaptive divergence of populations. In plants, the post-transcriptional regulatory mechanism of alternative splicing (AS) plays an important role in both development and abiotic stress response, making it a compelling potential target of natural selection. AS allows organisms to generate multiple different transcripts/proteins from a single gene and thus may provide a source of evolutionary novelty. Here, we examine whether variation in alternative splicing and gene expression levels might contribute to adaptation and incipient speciation of dune-adapted prairie sunflowers in Great Sand Dunes National Park, Colorado, USA. We conducted a common garden experiment to assess transcriptomic variation among ecotypes and analyzed differential expression, differential splicing, and gene coexpression. We show that individual genes are strongly differentiated for both transcript level and alternative isoform proportions, even when grown in a common environment, and that gene coexpression networks are disrupted between ecotypes. Furthermore, we examined how genome-wide patterns of sequence divergence correspond to divergence in transcript levels and isoform proportions and find evidence for both cis and trans-regulation. Together, our results emphasize that alternative splicing has been an underappreciated mechanism providing source material for natural selection at short evolutionary time scales.

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.

Transcriptional changes are tightly coupled to chromatin reorganization during cellular aging.

Human life expectancy is constantly increasing and aging has become a major risk factor for many diseases, although the underlying gene regulatory mechanisms are still unclear. Using transcriptomic and chromosomal conformation capture (Hi-C) data from human skin fibroblasts from individuals across different age groups, we identified a tight coupling between the changes in co-regulation and co-localization of genes. We obtained transcription factors, cofactors, and chromatin regulators that could drive the cellular aging process by developing a time-course prize-collecting Steiner tree algorithm. In particular, by combining RNA-Seq data from different age groups and protein-protein interaction data we determined the key transcription regulators and gene regulatory changes at different life stage transitions. We then mapped these transcription regulators to the 3D reorganization of chromatin in young and old skin fibroblasts. Collectively, we identified key transcription regulators whose target genes are spatially rearranged and correlate with changes in their expression, thereby providing potential targets for reverting cellular aging.

Lactate-induced histone lactylation by p300 promotes osteoblast differentiation.

Lactate, which is synthesized as an end product by lactate dehydrogenase A (LDHA) from pyruvate during anaerobic glycolysis, has attracted attention for its energy metabolism and oxidant effects. A novel histone modification-mediated gene regulation mechanism termed lactylation by lactate was recently discovered. The present study examined the involvement of histone lactylation in undifferentiated cells that underwent differentiation into osteoblasts. C2C12 cells cultured in medium with a high glucose content (4500 mg/L) showed increases in marker genes (Runx2, Sp7, Tnap) indicating BMP-2-induced osteoblast differentiation and ALP staining activity, as well as histone lactylation as compared to those cultured in medium with a low glucose content (900 mg/L). Furthermore, C2C12 cells stimulated with the LDH inhibitor oxamate had reduced levels of BMP-2-induced osteoblast differentiation and histone lactylation, while addition of lactate to C2C12 cells cultured in low glucose medium resulted in partial restoration of osteoblast differentiation and histone lactylation. These results indicate that lactate synthesized by LDHA during glucose metabolism is important for osteoblast differentiation of C2C12 cells induced by BMP-2. Additionally, silencing of p300, a possible modifier of histone lactylation, also inhibited osteoblast differentiation and reduced histone lactylation. Together, these findings suggest a role of histone lactylation in promotion of undifferentiated cells to undergo differentiation into osteoblasts.

Combinatorial single-cell profiling of major chromatin types with MAbID

Gene expression programs result from the collective activity of numerous regulatory factors. Studying their cooperative mode of action is imperative to understand gene regulation, but simultaneously measuring these factors within one sample has been challenging. Here we introduce Multiplexing Antibodies by barcode Identification (MAbID), a method for combinatorial genomic profiling of histone modifications and chromatin-binding proteins. MAbID employs antibody–DNA conjugates to integrate barcodes at the genomic location of the epitope, enabling combined incubation of multiple antibodies to reveal the distributions of many epigenetic markers simultaneously. We used MAbID to profile major chromatin types and multiplexed measurements without loss of individual data quality. Moreover, we obtained joint measurements of six epitopes in single cells of mouse bone marrow and during mouse in vitro differentiation, capturing associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially for low-input samples and in single cells.

Dynamic regulation of BDNF gene expression by estradiol and lncRNA HOTAIR

Brain derived neurotrophic factor (BDNF) is a major neurotransmitter that controls growth and maintenance of neurons and its misregulation is linked to neurodegeneration and human diseases. Estradiol (E2) is well-known to regulate the process of differentiation and plasticity of hippocampal neurons. Here we examined the mechanisms of BDNF gene regulation under basal conditions and under stimuli such as E2. Our results demonstrated that BDNF expression is induced by E2 in vitro in HT22 cells (hippocampal neuronal cells) and in vivo (in ovariectomized mouse brain under E2-treatment). Using chromatin immunoprecipitation assay, we demonstrated that estrogen receptors (ERα, ERβ) were enriched at the BDNF promoter in presence of E2. Additionally, ER-coregulators (e.g., CBP/p300, MLL3), histone acetylation, H3K4-trimethylation, and RNA polymerase II levels were also elevated at the BDNF promoter in an E2-dependent manner. Additionally, under the basal conditions (in the absence of E2), the long noncoding RNA HOTAIR and its interacting partners PRC2 and LSD1 complexes binds to the promoter of BDNF and represses its expression. HOTAIR knockdown -relieves the repression resulting in elevation of BDNF expression. Further, levels of HOTAIR-interacting partners, EZH2 and LSD1 were reduced at the BDNF promoter upon HOTAIR-knockdown revealing that HOTAIR plays a regulatory role in BDNF gene expression by modulating promoter histone modifications. Additionally, we showed that E2 induced-BDNF expression is mediated by the displacement of silencing factors, EZH2 and LSD1 at BDNF promoter and subsequent recruitment of active transcription machinery. These results reveal the mechanisms of BDNF gene regulation under the basal condition and in presence of a positive regulator such as E2 in neuronal cells.

Transcriptomic analysis reveals candidate genes associated with salinity stress tolerance during the early vegetative stage in fababean genotype, Hassawi-2

Abiotic stresses are a significant constraint to plant production globally. Identifying stress-related genes can aid in the development of stress-tolerant elite genotypes and facilitate trait and crop manipulation. The primary aim of this study was to conduct whole transcriptome analyses of the salt-tolerant faba bean genotype, Hassawi-2, under different durations of salt stress (6 h, 12 h, 24 h, 48 h, and 72 h) at the early vegetative stage, to better understand the molecular basis of salt tolerance. After de novo assembly, a total of 140,308 unigenes were obtained. The up-regulated differentially expressed genes (DEGs) were 2380, 2863, 3057, 3484, and 4820 at 6 h, 12 h, 24 h, 48 h, and 72 h of salt stress, respectively. Meanwhile, 1974, 3436, 2371, 3502, and 5958 genes were downregulated at 6 h, 12 h, 24 h, 48 h, and 72 h of salt stress, respectively. These DEGs encoded various regulatory and functional proteins, including kinases, plant hormone proteins, transcriptional factors (TFs) basic helix-loop-helix (bHLH), Myeloblastosis (MYB), and (WRKY), heat shock proteins (HSPs), late embryogenesis abundant (LEA) proteins, dehydrin, antioxidant enzymes, and aquaporin proteins. This suggests that the faba bean genome possesses an abundance of salinity resistance genes, which trigger different adaptive mechanisms under salt stress. Some selected DEGs validated the RNA sequencing results, thus confirming similar gene expression levels. This study represents the first transcriptome analysis of faba bean leaves subjected to salinity stress offering valuable insights into the mechanisms governing salt tolerance in faba bean during the vegetative stage. This comprehensive investigation enhances our understanding of precise gene regulatory mechanisms and holds promise for the development of novel salt-tolerant faba bean salt-tolerant cultivars.

Modulation of Expression of Drug Metabolizing Enzymes and Augmentation of Anti-cancer Drug Effects: Through Epigenetics and Three-dimensional Cancer Cell Culture Systems

Since commencing my role as a professor in a newly established Department of Pharmacodynamics and Molecular Genetics at the School of Pharmacy, Iwate Medical University, on April 1, 2007, my research has focused on modifying gene expression of cytochrome P-450 (CYP) in established human colon cancer cells. Additionally, I have been investigating methods to enhance the anti-tumor effects of irinotecan (CPT-11) and 5-fluorouracil (5-FU) using epigenetic modifying inhibitors of DNA methyltransferase and histone deacetylase. Treating colon cancer cells with a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC), led to elevated expression levels of CYP1B1 and CYP3A4 through demethylation of the promoter regions of related genes. Furthermore, the administration of DAC and the histone deacetylase inhibitor depsipeptide [(DEP), an anti-cancer drug romidepsin] significantly increased the cellular sensitivities of human colon cancer cells to CPT-11 and 5-FU, respectively. Remarkably, DAC treatment also increased colon cancer cell sensitivity to SN-38, an active metabolite of CPT-11, through the suppression of the anti-apoptotic protein Bcl-2. DEP increased colon cancer cell sensitivity to 5-FU in association with increased expressions of tumor-suppressor p21 and major histocompatibility complex class II genes. Another facet of my research is centered around understanding the gene regulatory mechanisms of the CYP1 family through aryl hydrocarbon receptors (AhR)s under glucose-deprivation stress and in three-dimensional (3D) culture systems of human solid tumor cells. In the 3D culture of human liver cancer cells, I found Pregnane X Receptor being implicated in the regulation of CYP1A2, which aligns with the in vivo mode of CYP1A2 expression.

Whole Genome Deconvolution Unveils Alzheimer’s Resilient Epigenetic Signature.

AbstractBackgroundThe pathological hallmarks of Alzheimer’s disease (AD) are amyloid beta plaques and neurofibrillary tangles that usually progressively affect characteristic brain regions as individuals advance from latent to prodromal to severe AD dementia (ADD). An uncommon exception to coupled progression of disease burden and clinical impairment are resilient individuals who harbor a high pathological load sufficient to cause ADD yet remain cognitively normal. We hypothesize that determining the biological mechanisms of such underlying resilience to AD (RAD) will provide unique insights into therapeutic opportunities.MethodHere, we combined epigenetic sequencing data and an innovative computational algorithm to identify gene regulatory mechanisms and putative mediators distinguishing RAD from normal control and ADD at an unprecedented scale. We developed and validated a novel machine learning method, Cellformer, for deconvoluting bulk ATAC‐seq into cell type‐specific expression across the whole genome, leading to extensive regional and cellular chromatin profiling of RAD.ResultCellformer successfully deconvoluted in silico bulk ATAC‐seq data across cross‐validation iterations, achieving strong performances with a mean Spearman coefficient of 0.82 between deconvoluted and ground truth expressions, outperforming other approaches. Validation was performed using external single nucleus datasets and showed a significant correlation above 0.75 (P‐value&lt;0.05) between deconvoluted cell type‐specific ATAC‐seq and single‐ATAC‐seq expressions for each cell type. Applied on 191 samples from normal control, RAD and ADD’s 3 brain regions, CellFormer predicted cell type‐specific expression of more than 41954 open chromatin regions (OCR), preserving regional and phenotypic variations. Downstream analysis of the derived RAD‐specific epigenetic features pinpointed the hippocampus, a region critical for learning and memory, as the major player in the resilience, modulating synaptic and neuronal process in a cell‐type specific manner.ConclusionCellformer opens new perspectives to advance bulk analysis and understand biological mechanisms using cost effective bulk sequencing. Through this study, we showed that cell type‐resolved data nominated potential epigenetic and transcriptional mediators underlying RAD that may illuminate therapeutic opportunities to limit the cognitive impact of this highly prevalent yet stubbornly incurable disease. Cellformer will be made publicly available to advance analysis of high‐throughput bulk ATAC‐seq in future investigations.

Cross-regulation in a three-component cell envelope stress signaling system of Brucella.

As intracellular pathogens, Brucella must contend with a variety of host-derived stressors when infecting a host cell. The inner membrane, cell wall, and outer membrane, i.e. the cell envelope, of Brucella provide a critical barrier to host assault. A conserved regulatory mechanism known as two-component signaling (TCS) commonly controls transcription of genes that determine the structure and biochemical composition of the cell envelope during stress. We report the identification of previously uncharacterized TCS genes that determine Brucella ovis fitness in the presence of cell envelope disruptors and within infected mammalian host cells. Our study reveals a new molecular mechanism of TCS-dependent gene regulation, and thereby advances fundamental understanding of transcriptional regulatory processes in bacteria.

Nuclear Condensates Are a Therapeutic Vulnerability in NPM1-Mutant Acute Myeloid Leukemia

The development of targeted therapies against drivers of malignancy requires a robust mechanistic understanding of the pathogenic axis underlying disease. Several subtypes of acute myeloid leukemias (AML), such as those driven by mutations in IDH1/2, FLT3, and KMT2A, have benefitted from the development of novel therapeutics that disrupt their transcriptional program. In contrast, there are no approved therapies that target the mutant version of Nucleophosmin (NPM1), which is the most common driver in adult AML. Mutations in NPM1 result in export of the mutant protein (NPM1c) to the cytoplasm, prompting the suggestion that aberrant localization leads to neomorphic and pathogenic functions of NPM1c in the cytoplasm. In parallel, recent data from several groups has suggested that NPM1c directly regulates gene expression within the nucleus. Despite this progress, a well-defined mechanism for NPM1c-mediated gene regulation is lacking, hindering the development of NPM1c-targeted therapies. To investigate the function of NPM1c, we generated over a dozen AML cell lines expressing unique NPM1c truncations and characterized their impact on the leukemic transcriptome and cell morphology. To our surprise, we found that although the majority of these truncated proteins reside in the cytoplasm, most were unable to recapitulate the transcriptional program driven by full-length NPM1c, and cells subsequently underwent differentiation and growth arrest. We further explored the chromatin binding ability of these truncated proteins to key genomic regions such as the HOXA cluster and MEIS1 through CUT&amp;RUN analysis. Next, we identified a region of the NPM1c protein critical for active nuclear import (NLS). Strikingly, disruption of this region enforces cytoplasmic sequestration of the truncated NPM1c protein. However, cells harboring this truncation fail to maintain leukemic gene expression and differentiate. Together these results suggest that aberrant localization and/or shuttling of NPM1c between cellular compartments is insufficient to maintain leukemic gene expression and cell-stemness. We next considered whether NPM1c had a de-novo function that may explain its role in gene regulation. We have recently shown that NPM1c forms phase-separated condensates in the nucleus that regulate HOX gene expression and are necessary for stem-ness. These NPM1c condensates recruit multiple gene regulatory factors including XPO1, NUP98, and KMT2A (Figure 1). Through selective degradation of NPM1c, or pharmacological inhibition of XPO1, we found that disruption of nuclear condensates abrogates recruitment of these protein co-factors. We further explore the effect of drug compounds known to inhibit NPM1-mutant AML on condensate behavior and chromatin binding. This condensate behavior is a defining feature of heterotypic phase separation, a process in which two or more biomolecules form multivalent interactions to create a condensate. Importantly, we found that significant overexpression of NPM1c causes an imbalance in the ratio between NPM1c and its binding partners, disrupting normal condensate formation, and causing subsequent differentiation and growth arrest. Collectively, these results suggest that therapeutic targeting of NPM1c, or a host of interacting partners, may disrupt condensate formation and the leukemic transcriptional program, providing an exciting new therapeutic vulnerability in NPM1-mutant AML.

Divergent Roles of MLL3 and MLL4 in Hematopoietic Stem Cell Fate Decision

Complex gene regulatory mechanisms, orchestrated by transcription factors (TFs) and epigenetic regulators, govern cell fate decisions in normal and malignant hematopoiesis. While prior works have extensively characterized the TF-networks in blood development, we currently have limited understanding of epigenetic regulation, in particular molecular rules underlying cofactor usage, in hematopoietic cell fate decisions. Kmt2c and Kmt2d are homologous genes that encode transcriptional coactivators, MLL3 and MLL4, respectively. They have been shown to coordinate embryonic stem cell fate transitions and are recurrently mutated in leukemias, offering an ideal paradigm to study epigenetic regulation of hematopoiesis. While MLL3 and MLL4 are often believed to play redundant roles in stem cell fate decisions, few has characterized their molecular distinctions. MLL3 and MLL4 each nucleate a chromatin-bound complex called the Complex of Proteins Associated with Set1 (COMPASS) and both deposit H3K4me1 at enhancers. Despite similar biochemical functions, Kmt2c and Kmt2d loss-of-function mutations have been reported with contrasting phenotypes in hematopoiesis. Kmt2d loss impairs HSC self-renewal and enhances myeloid differentiation at the expense of lymphopoiesis, whereas Kmt2c deletions enhance HSC self-renewal and impair differentiation. We hypothesize that MLL3 and MLL4 are recruited by different transcription factors and bind chromatin at both overlapping and distinct enhancers to convey specific hematopoietic fates. To test whether MLL3 and MLL4 are redundant in licensing myeloid differentiation, we transplanted wildtype, Kmt2c/d single knockout, and Kmt2c/d double knockout (DKO) donor cells into lethally irradiated recipients. Surprisingly, DKO recipients showed significant expansion of phenotypic HSCs, MPPs, and pre-granulocyte-monocyte progenitors (pGMs), typically associated with a myeloid differentiation block. DKO mice progressively developed bone marrow failure 9-months after transplantation, indicating that MLL3/4-COMPASS is necessary to sustain hematopoiesis. To dissect how Kmt2c/d deletions shape lineage specification and whether DKO imposes a differentiation block, we performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) on donor Lin -Kit +Sca1 + (LSK) cells from the transplantation described above. While Kmt2c deletion does not markedly reprogram HSC/MPP identity, Kmt2d deletion results in precocious myeloid differentiation. Most notably, we observed that compound Kmt2c/d deletions fundamentally reprogramed HSCs/MPPs toward a B-lymphoid-like state, instead of imposing a myeloid differentiation block. The data establish a model wherein MLL3 and MLL4 have redundant roles in licensing HSC identity and myeloid potential. However, within the HSC to myeloid differentiation trajectory, MLL3 promotes differentiation while MLL4 promotes self-renewal. To understand how MLL3- and MLL4-COMPASS differentially regulate enhancer accessibility, we performed single-cell transposase-accessible chromatin sequencing (scATAC-seq) on the same donor LSK cells as in the CITE-seq. Compound Kmt2c/d deletions impose a unique epigenomic state on HSCs/MPPs (Figure 1A). Motif enrichment analyses revealed that DKO cells (i) were enriched for PAX5 and EBF1 motifs, consistent with B-lymphoid priming. HSCs (ii) were enriched for AP-1 and NFE2L2 motifs, suggesting that MLL4 may preferentially bind AP-1 and antioxidant response elements associated with HSC identity. Clusters of myeloid progenitors (iii and iv) were populated primarily by Kmt2d-deficient cells and enriched for CEBP and SPI1 motifs, suggesting that MLL3 is preferentially recruited to enhancers bound by myeloid TFs. This study provides several novel insights into epigenetic regulation of hematopoiesis. We have shown that HSC identity and myeloid fate specification are licensed by redundant MLL3/4-COMPASS-dependent TF-cofactor interactions, but once HSCs are established, MLL3- and MLL4-COMPASS engage distinct, antagonistic programs. Furthermore, early B-lymphoid priming is mediated via MLL3/4-COMPASS-independent mechanisms (Figure 1B). Ongoing work will link precise transcriptional codes to divergent cofactor utilization patterns and provide critical insights into how epigenetic regulators are deployed to effect distinct cell fates.