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1.
J Phys Chem B ; 128(14): 3329-3339, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38557033

RESUMO

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins: histones in somatic nuclei and protamines in sperm chromatin. The extreme, nearly crystalline packaging of DNA by protamines in spermatozoa is thought to be essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species. The protective role of protamines is required in sperm, as they are sensitive to ROS damage due to the progressive loss of DNA repair mechanisms during maturation. The degree to which DNA packaging directly relates to DNA protection in the condensed state, however, is poorly understood. Here, we utilized different polycation condensing agents to achieve varying DNA packaging densities and quantify DNA damage by free radical oxidation within the condensates. Although we see that tighter DNA packaging generally leads to better protection, the length of the polycation also plays a significant role. Molecular dynamics simulations suggest that longer polyarginine chains offer increased protection by occupying more space on the DNA surface and forming more stable interactions. Taken together, our results suggest a complex interplay among polycation properties, DNA packaging density, and DNA protection against free radical damage within condensed states.


Assuntos
DNA , Polieletrólitos , Sêmen , Masculino , Humanos , DNA/química , Cromatina , Protaminas/química , Espermatozoides , Empacotamento do DNA , Dano ao DNA
2.
J Clin Invest ; 134(7)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38557491

RESUMO

Mutations in genes encoding chromatin modifiers are enriched among mutations causing intellectual disability. The continuing development of the brain postnatally, coupled with the inherent reversibility of chromatin modifications, may afford an opportunity for therapeutic intervention following a genetic diagnosis. Development of treatments requires an understanding of protein function and models of the disease. Here, we provide a mouse model of Say-Barber-Biesecker-Young-Simpson syndrome (SBBYSS) (OMIM 603736) and demonstrate proof-of-principle efficacy of postnatal treatment. SBBYSS results from heterozygous mutations in the KAT6B (MYST4/MORF/QFK) gene and is characterized by intellectual disability and autism-like behaviors. Using human cells carrying SBBYSS-specific KAT6B mutations and Kat6b heterozygous mice (Kat6b+/-), we showed that KAT6B deficiency caused a reduction in histone H3 lysine 9 acetylation. Kat6b+/- mice displayed learning, memory, and social deficits, mirroring SBBYSS individuals. Treatment with a histone deacetylase inhibitor, valproic acid, or an acetyl donor, acetyl-carnitine (ALCAR), elevated histone acetylation levels in the human cells with SBBYSS mutations and in brain and blood cells of Kat6b+/- mice and partially reversed gene expression changes in Kat6b+/- cortical neurons. Both compounds improved sociability in Kat6b+/- mice, and ALCAR treatment restored learning and memory. These data suggest that a subset of SBBYSS individuals may benefit from postnatal therapeutic interventions.


Assuntos
Anormalidades Múltiplas , Acetilcarnitina , Hipotireoidismo Congênito , Anormalidades Craniofaciais , Histona Acetiltransferases , Deficiência Intelectual , Instabilidade Articular , Animais , Humanos , Camundongos , Anormalidades Múltiplas/tratamento farmacológico , Anormalidades Múltiplas/genética , Acetilação , Acetilcarnitina/farmacologia , Acetilcarnitina/uso terapêutico , Blefarofimose , Cromatina , Anormalidades Craniofaciais/tratamento farmacológico , Anormalidades Craniofaciais/genética , Éxons , Facies , Cardiopatias Congênitas , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/genética
3.
Elife ; 122024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38578680

RESUMO

Heterogeneity in endothelial cell (EC) sub-phenotypes is becoming increasingly appreciated in atherosclerosis progression. Still, studies quantifying EC heterogeneity across whole transcriptomes and epigenomes in both in vitro and in vivo models are lacking. Multiomic profiling concurrently measuring transcriptomes and accessible chromatin in the same single cells was performed on six distinct primary cultures of human aortic ECs (HAECs) exposed to activating environments characteristic of the atherosclerotic microenvironment in vitro. Meta-analysis of single-cell transcriptomes across 17 human ex vivo arterial specimens was performed and two computational approaches quantitatively evaluated the similarity in molecular profiles between heterogeneous in vitro and ex vivo cell profiles. HAEC cultures were reproducibly populated by four major clusters with distinct pathway enrichment profiles and modest heterogeneous responses: EC1-angiogenic, EC2-proliferative, EC3-activated/mesenchymal-like, and EC4-mesenchymal. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG and IL1B. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for coronary artery disease (CAD)-associated single-nucleotide polymorphisms from Genome Wide Association Studies (GWAS), suggesting that these cell phenotypes harbor CAD-modulating mechanisms. Primary EC cultures contain markedly heterogeneous cell subtypes defined by their molecular profiles. Surprisingly, the perturbations used here only modestly shifted cells between subpopulations, suggesting relatively stable molecular phenotypes in culture. Identifying consistently heterogeneous EC subpopulations between in vitro and ex vivo models should pave the way for improving in vitro systems while enabling the mechanisms governing heterogeneous cell state decisions.


Assuntos
Aterosclerose , Doença da Artéria Coronariana , Humanos , Células Endoteliais/metabolismo , Estudo de Associação Genômica Ampla , Aterosclerose/metabolismo , Doença da Artéria Coronariana/genética , Cromatina/metabolismo
4.
Nat Commun ; 15(1): 3074, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38594255

RESUMO

Although DNA methylation data yields highly accurate age predictors, little is known about the dynamics of this quintessential epigenomic biomarker during lifespan. To narrow the gap, we investigate the methylation trajectories of male mouse colon at five different time points of aging. Our study indicates the existence of sudden hypermethylation events at specific stages of life. Precisely, we identify two epigenomic switches during early-to-midlife (3-9 months) and mid-to-late-life (15-24 months) transitions, separating the rodents' life into three stages. These nonlinear methylation dynamics predominantly affect genes associated with the nervous system and enrich in bivalently marked chromatin regions. Based on groups of nonlinearly modified loci, we construct a clock-like classifier STageR (STage of aging estimatoR) that accurately predicts murine epigenetic stage. We demonstrate the universality of our clock in an independent mouse cohort and with publicly available datasets.


Assuntos
Metilação de DNA , Epigênese Genética , Humanos , Masculino , Animais , Camundongos , Metilação de DNA/genética , Envelhecimento/genética , Longevidade , Cromatina
5.
Proc Natl Acad Sci U S A ; 121(15): e2322563121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38557192

RESUMO

Mammalian switch/sucrose nonfermentable (mSWI/SNF) ATPase degraders have been shown to be effective in enhancer-driven cancers by functioning to impede oncogenic transcription factor chromatin accessibility. Here, we developed AU-24118, an orally bioavailable proteolysis-targeting chimera (PROTAC) degrader of mSWI/SNF ATPases (SMARCA2 and SMARCA4) and PBRM1. AU-24118 demonstrated tumor regression in a model of castration-resistant prostate cancer (CRPC) which was further enhanced with combination enzalutamide treatment, a standard of care androgen receptor (AR) antagonist used in CRPC patients. Importantly, AU-24118 exhibited favorable pharmacokinetic profiles in preclinical analyses in mice and rats, and further toxicity testing in mice showed a favorable safety profile. As acquired resistance is common with targeted cancer therapeutics, experiments were designed to explore potential mechanisms of resistance that may arise with long-term mSWI/SNF ATPase PROTAC treatment. Prostate cancer cell lines exposed to long-term treatment with high doses of a mSWI/SNF ATPase degrader developed SMARCA4 bromodomain mutations and ABCB1 (ATP binding cassette subfamily B member 1) overexpression as acquired mechanisms of resistance. Intriguingly, while SMARCA4 mutations provided specific resistance to mSWI/SNF degraders, ABCB1 overexpression provided broader resistance to other potent PROTAC degraders targeting bromodomain-containing protein 4 and AR. The ABCB1 inhibitor, zosuquidar, reversed resistance to all three PROTAC degraders tested. Combined, these findings position mSWI/SNF degraders for clinical translation for patients with enhancer-driven cancers and define strategies to overcome resistance mechanisms that may arise.


Assuntos
Adenosina Trifosfatases , Neoplasias de Próstata Resistentes à Castração , Masculino , Humanos , Ratos , Camundongos , Animais , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Linhagem Celular , Cromatina , Mamíferos/genética , Antagonistas de Receptores de Andrógenos , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética
6.
Proc Natl Acad Sci U S A ; 121(15): e2321338121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38568969

RESUMO

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.


Assuntos
Proteínas de Drosophila , Drosophila , Animais , Cromatina/metabolismo , Ritmo Circadiano/genética , Proteínas CLOCK/genética , DNA/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , RNA/metabolismo
7.
Sci Adv ; 10(15): eadm8167, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38598632

RESUMO

Even when split into several chromosomes, DNA molecules that make up our genome are too long to fit into the cell nuclei unless massively folded. Such folding must accommodate the need for timely access to selected parts of the genome by transcription factors, RNA polymerases, and DNA replication machinery. Here, we review our current understanding of the genome folding inside the interphase nuclei. We consider the resulting genome architecture at three scales with a particular focus on the intermediate (meso) scale and summarize the insights gained from recent experimental observations and diverse computational models.


Assuntos
Núcleo Celular , Cromatina , Cromatina/genética , Núcleo Celular/genética , Cromossomos/genética , DNA/genética , Genoma
8.
Sci Data ; 11(1): 380, 2024 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-38615081

RESUMO

Rice blast caused by Pyricularia oryzae (syn., Magnaporthe oryzae) was one of the most destructive diseases of rice throughout the world. Genome assembly was fundamental to genetic variation identification and critically impacted the understanding of its ability to overcome host resistance. Here, we report a gapless genome assembly of rice blast fungus P. oryzae strain P131 using PacBio, Illumina and high throughput chromatin conformation capture (Hi-C) sequencing data. This assembly contained seven complete chromosomes (43,237,743 bp) and a circular mitochondrial genome (34,866 bp). Approximately 14.31% of this assembly carried repeat sequences, significantly greater than its previous assembled version. This assembly had a 99.9% complement in BUSCO evaluation. A total of 14,982 genes protein-coding genes were predicted. In summary, we assembled the first telomere-to-telomere gapless genome of P. oryzae, which would be a valuable genome resource for future research on the genome evolution and host adaptation.


Assuntos
Ascomicetos , Genoma Fúngico , Ascomicetos/genética , Cromatina , Telômero/genética
9.
Epigenetics ; 19(1): 2337085, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38595049

RESUMO

The PhiC31 integration system allows for targeted and efficient transgene integration and expression by recognizing pseudo attP sites in mammalian cells and integrating the exogenous genes into the open chromatin regions of active chromatin. In order to investigate the regulatory patterns of efficient gene expression in the open chromatin region of PhiC31 integration, this study utilized Ubiquitous Chromatin Opening Element (UCOE) and activating RNA (saRNA) to modulate the chromatin structure in the promoter region of the PhiC31 integration vector. The study analysed the effects of DNA methylation and nucleosome occupancy changes in the integrated promoter on gene expression levels. The results showed that for the OCT4 promoter with moderate CG density, DNA methylation had a smaller impact on expression compared to changes in nucleosome positioning near the transcription start site, which was crucial for enhancing downstream gene expression. On the other hand, for the SOX2 promoter with high CG density, increased methylation in the CpG island upstream of the transcription start site played a key role in affecting high expression, but the positioning and clustering of nucleosomes also had an important influence. In conclusion, analysing the DNA methylation patterns, nucleosome positioning, and quantity distribution of different promoters can determine whether the PhiC31 integration site possesses the potential to further enhance expression or overcome transgene silencing effects by utilizing chromatin regulatory elements.


Assuntos
Cromatina , Nucleossomos , Animais , Cromatina/genética , Nucleossomos/genética , Metilação de DNA , Ilhas de CpG , Regiões Promotoras Genéticas , Mamíferos/genética
10.
Indian J Tuberc ; 71(2): 204-212, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38589125

RESUMO

Mycobacterium tuberculosis (Mtb) employs several sophisticated strategies to evade host immunity and facilitate its intracellular survival. One of them is the epigenetic manipulation of host chromatin by three strategies i.e., DNA methylation, histone modifications and miRNA involvement. A host-directed therapeutic can be an attractive approach that targets these host epigenetics or gene regulations and circumvent manipulation of host cell machinery by Mtb. Given the complexity of the nature of intracellular infection by Mtb, there are challenges in identifying the important host proteins, non-coding RNA or the secretory proteins of Mtb itself that directly or indirectly bring upon the epigenetic modifications in the host chromatin. Equally challenging is developing the methods of targeting these epigenetic factors through chemical or non-chemical approaches as host-directed therapeutics. The current review article briefly summarizes several of the epigenetic factors that serve to bring upon potential changes in the host transcriptional machinery and targets the immune system for immunosuppression and disease progression in Mtb infection.


Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Epigênese Genética , Tuberculose/tratamento farmacológico , Regulação da Expressão Gênica , Cromatina/metabolismo
11.
Clin Epigenetics ; 16(1): 50, 2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38561804

RESUMO

BACKGROUND: Nucleosome repositioning in cancer is believed to cause many changes in genome organisation and gene expression. Understanding these changes is important to elucidate fundamental aspects of cancer. It is also important for medical diagnostics based on cell-free DNA (cfDNA), which originates from genomic DNA regions protected from digestion by nucleosomes. RESULTS: We have generated high-resolution nucleosome maps in paired tumour and normal tissues from the same breast cancer patients using MNase-assisted histone H3 ChIP-seq and compared them with the corresponding cfDNA from blood plasma. This analysis has detected single-nucleosome repositioning at key regulatory regions in a patient-specific manner and common cancer-specific patterns across patients. The nucleosomes gained in tumour versus normal tissue were particularly informative of cancer pathways, with ~ 20-fold enrichment at CpG islands, a large fraction of which marked promoters of genes encoding DNA-binding proteins. The tumour tissues were characterised by a 5-10 bp decrease in the average distance between nucleosomes (nucleosome repeat length, NRL), which is qualitatively similar to the differences between pluripotent and differentiated cells. This effect was correlated with gene activity, differential DNA methylation and changes in local occupancy of linker histone variants H1.4 and H1X. CONCLUSIONS: Our study offers a novel resource of high-resolution nucleosome maps in breast cancer patients and reports for the first time the effect of systematic decrease of NRL in paired tumour versus normal breast tissues from the same patient. Our findings provide a new mechanistic understanding of nucleosome repositioning in tumour tissues that can be valuable for patient diagnostics, stratification and monitoring.


Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , Humanos , Feminino , Nucleossomos/genética , Neoplasias da Mama/genética , Metilação de DNA , Histonas/genética , Histonas/metabolismo , DNA/metabolismo , Ácidos Nucleicos Livres/metabolismo , Cromatina
12.
Genome Biol ; 25(1): 90, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38589969

RESUMO

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Núcleo Celular
13.
Protein Sci ; 33(5): e4970, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38591484

RESUMO

Histone H1 is involved in chromatin compaction and dynamics. In human cells, the H1 complement is formed by different amounts of somatic H1 subtypes, H1.0-H1.5 and H1X. The amount of each variant depends on the cell type, the cell cycle phase, and the time of development and can be altered in disease. However, the mechanisms regulating H1 protein levels have not been described. We have analyzed the contribution of the proteasome to the degradation of H1 subtypes in human cells using two different inhibitors: MG132 and bortezomib. H1 subtypes accumulate upon treatment with both drugs, indicating that the proteasome is involved in the regulation of H1 protein levels. Proteasome inhibition caused a global increase in cytoplasmatic H1, with slight changes in the composition of H1 bound to chromatin and chromatin accessibility and no alterations in the nucleosome repeat length. The analysis of the proteasome degradation pathway showed that H1 degradation is ubiquitin-independent. The whole protein and its C-terminal domain can be degraded directly by the 20S proteasome in vitro. Partial depletion of PA28γ revealed that this regulatory subunit contributes to H1 degradation within the cell. Our study shows that histone H1 protein levels are under tight regulation to prevent its accumulation in the nucleus. We revealed a new regulatory mechanism for histone H1 degradation, where the C-terminal disordered domain is responsible for its targeting and degradation by the 20S proteasome, a process enhanced by the regulatory subunit PA28γ.


Assuntos
Histonas , Complexo de Endopeptidases do Proteassoma , Humanos , Histonas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Cromatina
14.
Proc Natl Acad Sci U S A ; 121(16): e2403316121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38593082

RESUMO

Compact chromatin is closely linked with gene silencing in part by sterically masking access to promoters, inhibiting transcription factor binding and preventing polymerase from efficiently transcribing a gene. However, a broader hypothesis suggests that chromatin compaction can be both a cause and a consequence of the locus histone modification state, with a tight bidirectional interaction underpinning bistable transcriptional states. To rigorously test this hypothesis, we developed a mathematical model for the dynamics of the HMR locus in Saccharomyces cerevisiae, that incorporates activating histone modifications, silencing proteins, and a dynamic, acetylation-dependent, three-dimensional locus size. Chromatin compaction enhances silencer protein binding, which in turn feeds back to remove activating histone modifications, leading to further compaction. The bistable output of the model was in good agreement with prior quantitative data, including switching rates from expressed to silent states (and vice versa), and protein binding/histone modification levels within the locus. We then tested the model by predicting changes in switching rates as the genetic length of the locus was increased, which were then experimentally verified. Such bidirectional feedback between chromatin compaction and the histone modification state may be a widespread and important regulatory mechanism given the hallmarks of many heterochromatic regions: physical chromatin compaction and dimerizing (or multivalent) silencing proteins.


Assuntos
Cromatina , Proteínas de Saccharomyces cerevisiae , Cromatina/genética , Cromatina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Código das Histonas , Retroalimentação , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
Methods Mol Biol ; 2795: 169-182, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38594538

RESUMO

DNA methylation and posttranslational modifications of histones instruct gene expression in eukaryotes. Besides canonical histones, histone variants also play a critical role in transcriptional regulation. One of the best studied histone variants in plants is H2A.Z whose removal from gene bodies correlates with increased transcriptional activity. The eviction of H2A.Z is regulated by environmental cues such as increased ambient temperatures, and current models suggest that H2A.Z functions as a transcriptional buffer preventing environmentally responsive genes from undesired activation. To monitor temperature-dependent H2A.Z dynamics, chromatin immunoprecipitation (ChIP) of H2A.Z-occupied DNA can be performed. The following protocol describes a quick and easy ChIP approach to study in vivo H2A.Z occupancy.


Assuntos
Regulação da Expressão Gênica , Histonas , Histonas/genética , Histonas/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Temperatura , Cromatina/genética , Nucleossomos
16.
Brief Bioinform ; 25(3)2024 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-38581422

RESUMO

Reliable cell type annotations are crucial for investigating cellular heterogeneity in single-cell omics data. Although various computational approaches have been proposed for single-cell RNA sequencing (scRNA-seq) annotation, high-quality cell labels are still lacking in single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) data, because of extreme sparsity and inconsistent chromatin accessibility between datasets. Here, we present a novel automated cell annotation method that transfers cell type information from a well-labeled scRNA-seq reference to an unlabeled scATAC-seq target, via a parallel graph neural network, in a semi-supervised manner. Unlike existing methods that utilize only gene expression or gene activity features, HyGAnno leverages genome-wide accessibility peak features to facilitate the training process. In addition, HyGAnno reconstructs a reference-target cell graph to detect cells with low prediction reliability, according to their specific graph connectivity patterns. HyGAnno was assessed across various datasets, showcasing its strengths in precise cell annotation, generating interpretable cell embeddings, robustness to noisy reference data and adaptability to tumor tissues.


Assuntos
Cromatina , Redes Neurais de Computação , Reprodutibilidade dos Testes
17.
BMC Biol ; 22(1): 78, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600550

RESUMO

BACKGROUND: Regulation of transcription is central to the emergence of new cell types during development, and it often involves activation of genes via proximal and distal regulatory regions. The activity of regulatory elements is determined by transcription factors (TFs) and epigenetic marks, but despite extensive mapping of such patterns, the extraction of regulatory principles remains challenging. RESULTS: Here we study differentially and similarly expressed genes along with their associated epigenomic profiles, chromatin accessibility and DNA methylation, during lineage specification at gastrulation in mice. Comparison of the three lineages allows us to identify genomic and epigenomic features that distinguish the two classes of genes. We show that differentially expressed genes are primarily regulated by distal elements, while similarly expressed genes are controlled by proximal housekeeping regulatory programs. Differentially expressed genes are relatively isolated within topologically associated domains, while similarly expressed genes tend to be located in gene clusters. Transcription of differentially expressed genes is associated with differentially open chromatin at distal elements including enhancers, while that of similarly expressed genes is associated with ubiquitously accessible chromatin at promoters. CONCLUSION: Based on these associations of (linearly) distal genes' transcription start sites (TSSs) and putative enhancers for developmental genes, our findings allow us to link putative enhancers to their target promoters and to infer lineage-specific repertoires of putative driver transcription factors, within which we define subgroups of pioneers and co-operators.


Assuntos
Epigenômica , Genes Essenciais , Animais , Camundongos , Cromatina/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Perfilação da Expressão Gênica
18.
Stem Cell Res Ther ; 15(1): 104, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38600587

RESUMO

BACKGROUND: Microglia, the brain's resident immune cells, play vital roles in brain development, and disorders like Alzheimer's disease (AD). Human iPSC-derived microglia (iMG) provide a promising model to study these processes. However, existing iMG generation protocols face challenges, such as prolonged differentiation time, lack of detailed characterization, and limited gene function investigation via CRISPR-Cas9. METHODS: Our integrated toolkit for in-vitro microglia functional genomics optimizes iPSC differentiation into iMG through a streamlined two-step, 20-day process, producing iMG with a normal karyotype. We confirmed the iMG's authenticity and quality through single-cell RNA sequencing, chromatin accessibility profiles (ATAC-Seq), proteomics and functional tests. The toolkit also incorporates a drug-dependent CRISPR-ON/OFF system for temporally controlled gene expression. Further, we facilitate the use of multi-omic data by providing online searchable platform that compares new iMG profiles to human primary microglia: https://sherlab.shinyapps.io/IPSC-derived-Microglia/ . RESULTS: Our method generates iMG that closely align with human primary microglia in terms of transcriptomic, proteomic, and chromatin accessibility profiles. Functionally, these iMG exhibit Ca2 + transients, cytokine driven migration, immune responses to inflammatory signals, and active phagocytosis of CNS related substrates including synaptosomes, amyloid beta and myelin. Significantly, the toolkit facilitates repeated iMG harvesting, essential for large-scale experiments like CRISPR-Cas9 screens. The standalone ATAC-Seq profiles of our iMG closely resemble primary microglia, positioning them as ideal tools to study AD-associated single nucleotide variants (SNV) especially in the genome regulatory regions. CONCLUSIONS: Our advanced two-step protocol rapidly and efficiently produces authentic iMG. With features like the CRISPR-ON/OFF system and a comprehensive multi-omic data platform, our toolkit equips researchers for robust microglial functional genomic studies. By facilitating detailed SNV investigation and offering a sustainable cell harvest mechanism, the toolkit heralds significant progress in neurodegenerative disease drug research and therapeutic advancement.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Humanos , Microglia/metabolismo , Proteômica , Peptídeos beta-Amiloides , Genômica , Doença de Alzheimer/genética , Cromatina/genética , Cromatina/metabolismo
19.
Bull Environ Contam Toxicol ; 112(4): 59, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38602569

RESUMO

Environmental pollutants produce adverse effects on organisms and ecosystems. Biomonitoring and biomarkers offer a reasonable approach to make these assessments. Induced genetic changes can be using as a biomarker in organisms that react to a given compound in the ecosystem. Monitoring environmental genotoxicity necessitates the choice of model animals known as "sentinels or biological monitors" and the suitability of validated tests for DNA damage evaluation. We aimed to estimate the DNA damage produced by thermal stress in the leukocytes of the Mexican free-tailed bat (Tadarida brasiliensis). The DNA damage in bat leukocytes exposed to different temperatures (35 °C, 45 °C, and 55 °C) was evaluated by the adapted chromatin dispersion test (CDT) and the results were confirmed by the alkaline comet test. The CDT permitted a clear representation of leukocytes with fragmented DNA and of nonfragmented DNA. In addition, we detected nuclear anomalies in relation to cell death cellular swelling, nuclear fragmentation, and chromatin lysis. The alkaline comet assay revealed that the halos of diffuse chromatin include fragmented DNA. The assay of the method employing the CDT is well established, precise, and cost-effective for the routine quantitative analysis of DNA damage on the effect of the leukocytes of bats exposed to thermal stress. This could also apply as a sensitive screening tool for the evaluation of genotoxicity in environmental protection programs.


Assuntos
Quirópteros , Animais , Ecossistema , Dano ao DNA , Leucócitos , Biomarcadores , Cromatina , DNA
20.
Nat Commun ; 15(1): 2960, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580649

RESUMO

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.


Assuntos
Metilação de DNA , Neoplasias , Humanos , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Neoplasias/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
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