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1.
Genome Biol ; 25(1): 175, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961490

RESUMO

BACKGROUND: Transposable elements play a critical role in maintaining genome architecture during neurodevelopment. Short Interspersed Nuclear Elements (SINEs), a major subtype of transposable elements, are known to harbor binding sites for the CCCTC-binding factor (CTCF) and pivotal in orchestrating chromatin organization. However, the regulatory mechanisms controlling the activity of SINEs in the developing brain remains elusive. RESULTS: In our study, we conduct a comprehensive genome-wide epigenetic analysis in mouse neural precursor cells using ATAC-seq, ChIP-seq, whole genome bisulfite sequencing, in situ Hi-C, and RNA-seq. Our findings reveal that the SET domain bifurcated histone lysine methyltransferase 1 (SETDB1)-mediated H3K9me3, in conjunction with DNA methylation, restricts chromatin accessibility on a selective subset of SINEs in neural precursor cells. Mechanistically, loss of Setdb1 increases CTCF access to these SINE elements and contributes to chromatin loop reorganization. Moreover, de novo loop formation contributes to differential gene expression, including the dysregulation of genes enriched in mitotic pathways. This leads to the disruptions of cell proliferation in the embryonic brain after genetic ablation of Setdb1 both in vitro and in vivo. CONCLUSIONS: In summary, our study sheds light on the epigenetic regulation of SINEs in mouse neural precursor cells, suggesting their role in maintaining chromatin organization and cell proliferation during neurodevelopment.


Assuntos
Cromatina , Histona-Lisina N-Metiltransferase , Células-Tronco Neurais , Elementos Nucleotídeos Curtos e Dispersos , Animais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Camundongos , Cromatina/metabolismo , Metilação de DNA , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Epigênese Genética , Histonas/metabolismo , Encéfalo/metabolismo , Encéfalo/citologia
2.
Proc Natl Acad Sci U S A ; 121(28): e2400737121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38968127

RESUMO

In recent years, the exploration of genome three-dimensional (3D) conformation has yielded profound insights into the regulation of gene expression and cellular functions in both animals and plants. While animals exhibit a characteristic genome topology defined by topologically associating domains (TADs), plants display similar features with a more diverse conformation across species. Employing advanced high-throughput sequencing and microscopy techniques, we investigated the landscape of 26 histone modifications and RNA polymerase II distribution in tomato (Solanum lycopersicum). Our study unveiled a rich and nuanced epigenetic landscape, shedding light on distinct chromatin states associated with heterochromatin formation and gene silencing. Moreover, we elucidated the intricate interplay between these chromatin states and the overall topology of the genome. Employing a genetic approach, we delved into the role of the histone modification H3K9ac in genome topology. Notably, our investigation revealed that the ectopic deposition of this chromatin mark triggered a reorganization of the 3D chromatin structure, defining different TAD-like borders. Our work emphasizes the critical role of H3K9ac in shaping the topology of the tomato genome, providing valuable insights into the epigenetic landscape of this agriculturally significant crop species.


Assuntos
Epigenoma , Histonas , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Histonas/metabolismo , Histonas/genética , Epigênese Genética , Genoma de Planta , Cromatina/metabolismo , Cromatina/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Heterocromatina/metabolismo , Heterocromatina/genética , Código das Histonas/genética
3.
Proc Natl Acad Sci U S A ; 121(28): e2319772121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38968124

RESUMO

Transcription has a mechanical component, as the translocation of the transcription machinery or RNA polymerase (RNAP) on DNA or chromatin is dynamically coupled to the chromatin torsion. This posits chromatin mechanics as a possible regulator of eukaryotic transcription, however, the modes and mechanisms of this regulation are elusive. Here, we first take a statistical mechanics approach to model the torsional response of topology-constrained chromatin. Our model recapitulates the experimentally observed weaker torsional stiffness of chromatin compared to bare DNA and proposes structural transitions of nucleosomes into chirally distinct states as the driver of the contrasting torsional mechanics. Coupling chromatin mechanics with RNAP translocation in stochastic simulations, we reveal a complex interplay of DNA supercoiling and nucleosome dynamics in governing RNAP velocity. Nucleosomes play a dual role in controlling the transcription dynamics. The steric barrier aspect of nucleosomes in the gene body counteracts transcription via hindering RNAP motion, whereas the chiral transitions facilitate RNAP motion via driving a low restoring torque upon twisting the DNA. While nucleosomes with low dissociation rates are typically transcriptionally repressive, highly dynamic nucleosomes offer less of a steric barrier and enhance the transcription elongation dynamics of weakly transcribed genes via buffering DNA twist. We use the model to predict transcription-dependent levels of DNA supercoiling in segments of the budding yeast genome that are in accord with available experimental data. The model unveils a paradigm of DNA supercoiling-mediated interaction between genes and makes testable predictions that will guide experimental design.


Assuntos
RNA Polimerases Dirigidas por DNA , Nucleossomos , Transcrição Gênica , Nucleossomos/metabolismo , Nucleossomos/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , DNA/metabolismo , DNA/química , DNA/genética , Cromatina/metabolismo , Cromatina/genética , DNA Super-Helicoidal/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
4.
Proc Natl Acad Sci U S A ; 121(28): e2407077121, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38954553

RESUMO

An array of motor proteins consumes chemical energy in setting up the architectures of chromosomes. Here, we explore how the structure of ideal polymer chains is influenced by two classes of motors. The first class which we call "swimming motors" acts to propel the chromatin fiber through three-dimensional space. They represent a caricature of motors such as RNA polymerases. Previously, they have often been described by adding a persistent flow onto Brownian diffusion of the chain. The second class of motors, which we call "grappling motors" caricatures the loop extrusion processes in which segments of chromatin fibers some distance apart are brought together. We analyze these models using a self-consistent variational phonon approximation to a many-body Master equation incorporating motor activities. We show that whether the swimming motors lead to contraction or expansion depends on the susceptibility of the motors, that is, how their activity depends on the forces they must exert. Grappling motors in contrast to swimming motors lead to long-ranged correlations that resemble those first suggested for fractal globules and that are consistent with the effective interactions inferred by energy landscape analyses of Hi-C data on the interphase chromosome.


Assuntos
Cromossomos , Cromatina/química , Cromatina/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas Motores Moleculares/química
5.
Commun Biol ; 7(1): 783, 2024 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-38951619

RESUMO

Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.


Assuntos
Cromatina , Proteínas de Drosophila , Interfase , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Animais , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/embriologia , Núcleo Celular/metabolismo , Sítios de Ligação
6.
Nat Commun ; 15(1): 5629, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38965223

RESUMO

Mutations that decrease or increase the activity of the tyrosine phosphatase, SHP2 (encoded by PTPN11), promotes developmental disorders and several malignancies by varying phosphatase activity. We uncovered that SHP2 is a distinct class of an epigenetic enzyme; upon phosphorylation by the kinase ACK1/TNK2, pSHP2 was escorted by androgen receptor (AR) to chromatin, erasing hitherto unidentified pY54-H3 (phosphorylation of histones H3 at Tyr54) epigenetic marks to trigger a transcriptional program of AR. Noonan Syndrome with Multiple Lentigines (NSML) patients, SHP2 knock-in mice, and ACK1 knockout mice presented dramatic increase in pY54-H3, leading to loss of AR transcriptome. In contrast, prostate tumors with high pSHP2 and pACK1 activity exhibited progressive downregulation of pY54-H3 levels and higher AR expression that correlated with disease severity. Overall, pSHP2/pY54-H3 signaling acts as a sentinel of AR homeostasis, explaining not only growth retardation, genital abnormalities and infertility among NSML patients, but also significant AR upregulation in prostate cancer patients.


Assuntos
Epigênese Genética , Histonas , Homeostase , Camundongos Knockout , Neoplasias da Próstata , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Receptores Androgênicos , Animais , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Histonas/metabolismo , Masculino , Humanos , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fosforilação , Síndrome de Noonan/genética , Síndrome de Noonan/metabolismo , Transdução de Sinais , Cromatina/metabolismo
7.
PLoS One ; 19(7): e0305809, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38954704

RESUMO

Chromatin exhibits non-random distribution within the nucleus being arranged into discrete domains that are spatially organized throughout the nuclear space. Both the spatial distribution and structural rearrangement of chromatin domains in the nucleus depend on epigenetic modifications of DNA and/or histones and structural elements such as the nuclear envelope. These components collectively contribute to the organization and rearrangement of chromatin domains, thereby influencing genome architecture and functional regulation. This study develops an innovative, user-friendly, ImageJ-based plugin, called IsoConcentraChromJ, aimed quantitatively delineating the spatial distribution of chromatin regions in concentric patterns. The IsoConcentraChromJ can be applied to quantitative chromatin analysis in both two- and three-dimensional spaces. After DNA and histone staining with fluorescent probes, high-resolution images of nuclei have been obtained using advanced fluorescence microscopy approaches, including confocal and stimulated emission depletion (STED) microscopy. IsoConcentraChromJ workflow comprises the following sequential steps: nucleus segmentation, thresholding, masking, normalization, and trisection with specified ratios for either 2D or 3D acquisitions. The effectiveness of the IsoConcentraChromJ has been validated and demonstrated using experimental datasets consisting in nuclei images of pre-adipocytes and mature adipocytes, encompassing both 2D and 3D imaging. The outcomes allow to characterize the nuclear architecture by calculating the ratios between specific concentric nuclear areas/volumes of acetylated chromatin with respect to total acetylated chromatin and/or total DNA. The novel IsoConcentrapChromJ plugin could represent a valuable resource for researchers investigating the rearrangement of chromatin architecture driven by epigenetic mechanisms using nuclear images obtained by different fluorescence microscopy methods.


Assuntos
Núcleo Celular , Cromatina , Microscopia de Fluorescência , Cromatina/metabolismo , Cromatina/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Animais , Camundongos , Microscopia de Fluorescência/métodos , Humanos , Histonas/metabolismo , Histonas/genética , Software , Imageamento Tridimensional/métodos , Processamento de Imagem Assistida por Computador/métodos
8.
Sci Data ; 11(1): 725, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38956385

RESUMO

Teratoma, due to its remarkable ability to differentiate into multiple cell lineages, is a valuable model for studying human embryonic development. The similarity of the gene expression and chromatin accessibility patterns in these cells to those observed in vivo further underscores its potential as a research tool. Notably, teratomas derived from human naïve (pre-implantation epiblast-like) pluripotent stem cells (PSCs) have larger embryonic cell diversity and contain extraembryonic lineages, making them more suitable to study developmental processes. However, the cell type-specific epigenetic profiles of naïve PSC teratomas have not been yet characterized. Using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), we analyzed 66,384 cell profiles from five teratomas derived from human naïve PSCs and their post-implantation epiblast-like (primed) counterparts. We observed 17 distinct cell types from both embryonic and extraembryonic lineages, resembling the corresponding cell types in human fetal tissues. Additionally, we identified key transcription factors specific to different cell types. Our dataset provides a resource for investigating gene regulatory programs in a relevant model of human embryonic development.


Assuntos
Cromatina , Células-Tronco Pluripotentes , Análise de Célula Única , Teratoma , Humanos , Teratoma/genética , Teratoma/patologia , Células-Tronco Pluripotentes/metabolismo , Linhagem da Célula , Fatores de Transcrição/genética
9.
Nat Commun ; 15(1): 5693, 2024 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-38972954

RESUMO

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias.


Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Redes Reguladoras de Genes , Humanos , Metilação de DNA/genética , Ilhas de CpG/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Regulação Leucêmica da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Cromatina/genética , Masculino , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Feminino , Hematopoese/genética , Criança , Transcriptoma , Proteínas Proto-Oncogênicas , Transativadores
10.
Mol Cell ; 84(13): 2511-2524.e8, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38996460

RESUMO

BCL6, an oncogenic transcription factor (TF), forms polymers in the presence of a small-molecule molecular glue that stabilizes a complementary interface between homodimers of BCL6's broad-complex, tramtrack, and bric-à-brac (BTB) domain. The BTB domains of other proteins, including a large class of TFs, have similar architectures and symmetries, raising the possibility that additional BTB proteins self-assemble into higher-order structures. Here, we surveyed 189 human BTB proteins with a cellular fluorescent reporter assay and identified 18 ZBTB TFs that show evidence of polymerization. Through biochemical and cryoelectron microscopy (cryo-EM) studies, we demonstrate that these ZBTB TFs polymerize into filaments. We found that BTB-domain-mediated polymerization of ZBTB TFs enhances chromatin occupancy within regions containing homotypic clusters of TF binding sites, leading to repression of target genes. Our results reveal a role of higher-order structures in regulating ZBTB TFs and suggest an underappreciated role for TF polymerization in modulating gene expression.


Assuntos
Cromatina , Microscopia Crioeletrônica , Humanos , Cromatina/metabolismo , Cromatina/genética , Multimerização Proteica , Sítios de Ligação , Ligação Proteica , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Polimerização , Células HEK293 , Regulação da Expressão Gênica
11.
Cell ; 187(14): 3541-3562.e51, 2024 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-38996487

RESUMO

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.


Assuntos
Genoma , Mamutes , Pele , Animais , Mamutes/genética , Genoma/genética , Feminino , Elefantes/genética , Cromatina/genética , Fósseis , DNA Antigo/análise , Camundongos , Humanos , Cromossomo X/genética
12.
Commun Biol ; 7(1): 834, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38982263

RESUMO

Chromatin spatial organization plays a crucial role in gene regulation. Recently developed and prospering multiplexed DNA FISH technologies enable direct visualization of chromatin conformation in the nucleus. However, incomplete data caused by limited detection efficiency can substantially complicate and impair downstream analysis. Here, we present SnapFISH-IMPUTE that imputes missing values in multiplexed DNA FISH data. Analysis on multiple published datasets shows that the proposed method preserves the distribution of pairwise distances between imaging loci, and the imputed chromatin conformations are indistinguishable from the observed conformations. Additionally, imputation greatly improves downstream analyses such as identifying enhancer-promoter loops and clustering cells into distinct cell types. SnapFISH-IMPUTE is freely available at https://github.com/hyuyu104/SnapFISH-IMPUTE .


Assuntos
Cromatina , DNA , Hibridização in Situ Fluorescente , Hibridização in Situ Fluorescente/métodos , Cromatina/genética , DNA/genética , Humanos , Animais , Software
13.
Phys Rev Lett ; 132(24): 248403, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38949344

RESUMO

The 3D folding of a mammalian gene can be studied by a polymer model, where the chromatin fiber is represented by a semiflexible polymer which interacts with multivalent proteins, representing complexes of DNA-binding transcription factors and RNA polymerases. This physical model leads to the natural emergence of clusters of proteins and binding sites, accompanied by the folding of chromatin into a set of topologies, each associated with a different network of loops. Here, we combine numerics and analytics to first classify these networks and then find their relative importance or statistical weight, when the properties of the underlying polymer are those relevant to chromatin. Unlike polymer networks previously studied, our chromatin networks have finite average distances between successive binding sites, and this leads to giant differences between the weights of topologies with the same number of edges and nodes but different wiring. These weights strongly favor rosettelike structures with a local cloud of loops with respect to more complicated nonlocal topologies. Our results suggest that genes should overwhelmingly fold into a small fraction of all possible 3D topologies, which can be robustly characterized by the framework we propose here.


Assuntos
Cromatina , Entropia , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Modelos Moleculares
14.
Methods Mol Biol ; 2830: 81-91, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38977570

RESUMO

Chromatin immunoprecipitation (ChIP) is used to analyze the targeting of a protein to a specific region of chromatin in vivo. Here, we present an instructive ChIP protocol for Arabidopsis imbibed seeds. The protocol covers all steps, from the sampling of imbibed seeds to the reverse crosslinking of immunoprecipitated protein-DNA complexes, and includes experimental tips and notes. The targeting of the protein to DNA is determined by quantitative PCR (qPCR) using reverse crosslinked DNA. The protocol can be further scaled up for ChIP-sequencing (ChIP-seq) analysis. As an example of the protocol, we include a ChIP-quantitative PCR (ChIP-qPCR) analysis demonstrating the targeting of PIF1 to the ABI5 promoter.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Imunoprecipitação da Cromatina , Sementes , Arabidopsis/genética , Arabidopsis/metabolismo , Imunoprecipitação da Cromatina/métodos , Sementes/genética , Sementes/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regiões Promotoras Genéticas , DNA de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos
15.
Development ; 151(13)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38958075

RESUMO

Development is regulated by coordinated changes in gene expression. Control of these changes in expression is largely governed by the binding of transcription factors to specific regulatory elements. However, the packaging of DNA into chromatin prevents the binding of many transcription factors. Pioneer factors overcome this barrier owing to unique properties that enable them to bind closed chromatin, promote accessibility and, in so doing, mediate binding of additional factors that activate gene expression. Because of these properties, pioneer factors act at the top of gene-regulatory networks and drive developmental transitions. Despite the ability to bind target motifs in closed chromatin, pioneer factors have cell type-specific chromatin occupancy and activity. Thus, developmental context clearly shapes pioneer-factor function. Here, we discuss this reciprocal interplay between pioneer factors and development: how pioneer factors control changes in cell fate and how cellular environment influences pioneer-factor binding and activity.


Assuntos
Cromatina , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromatina/metabolismo , Humanos , Redes Reguladoras de Genes , Ligação Proteica
16.
Nat Commun ; 15(1): 5604, 2024 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-38961054

RESUMO

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics.


Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Coesinas , Dano ao DNA , Replicação do DNA , Leucemia Mieloide Aguda , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Linhagem Celular Tumoral , Acetilação , Animais , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Camundongos , Cromatina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Apoptose , Proliferação de Células , Células HEK293
17.
Science ; 385(6704): eadd8394, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38963856

RESUMO

Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.


Assuntos
Linfócitos T CD4-Positivos , Elementos Facilitadores Genéticos , Predisposição Genética para Doença , Sítio de Iniciação de Transcrição , Transcrição Gênica , Humanos , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Cromatina/metabolismo , Cromatina/genética , Regiões Promotoras Genéticas , Linfócitos T Auxiliares-Indutores/imunologia , Análise da Expressão Gênica de Célula Única , Atlas como Assunto
18.
Syst Biol Reprod Med ; 70(1): 195-203, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38972054

RESUMO

The presence of cyclic adenosine monophosphate (cAMP) has been considered to be a fundamental factor in ensuring meiotic arrest prior to ovulation. cAMP is regarded as a key molecule in the regulation of oocyte maturation. However, it has been reported that increased levels of intracellular cAMP can result in abnormal cytokinesis, with some MI oocytes leading to symmetrically cleaved 2-cell MII oocytes. Consequently, we aimed to investigate the effects of elevated intracellular cAMP levels on abnormal cytokinesis and oocyte maturation during the meiosis of mouse oocytes. This study found that a high concentration of isobutylmethylxanthine (IBMX) also caused chromatin/chromosomes aggregation (AC) after the first meiosis. The rates of AC increased the greater the concentration of IBMX. In addition, AC formation was found to be reversible, showing that the re-formation of the spindle chromosome complex was possible after the IBMX was removed. In human oocytes, the chromosomes aggregate after the germinal vesicle breakdown and following the first and second polar body extrusions (the AC phase), while mouse oocytes do not have this AC phase. The results of our current study may indicate that the AC phase in human oocytes could be related to elevated levels of intracytoplasmic cAMP.


Assuntos
1-Metil-3-Isobutilxantina , Cromatina , Oócitos , Animais , Oócitos/metabolismo , Feminino , Cromatina/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Camundongos , Humanos , Meiose/efeitos dos fármacos , AMP Cíclico/metabolismo , Inibidores de Fosfodiesterase/farmacologia
19.
Methods Mol Biol ; 2805: 127-135, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39008178

RESUMO

The modulation of cis-regulatory elements (e.g., enhancers and promoters) is a major mechanism by which gene expression can be controlled in a temporal and spatially restricted manner. However, methods for both identifying these elements and inferring their activity are limited and often require a substantial investment of time, money, and resources. Here, using mammalian skin as a model, we demonstrate a streamlined protocol by which these hurdles can be overcome using a novel chromatin profiling technique (CUT&RUN) to map histone modifications genome-wide. This protocol can be used to map the location and activity of putative cis-regulatory elements, providing mechanistic insight into how differential gene expression is controlled in mammalian tissues.


Assuntos
Regiões Promotoras Genéticas , Pele , Animais , Pele/metabolismo , Elementos Facilitadores Genéticos , Cromatina/genética , Cromatina/metabolismo , Humanos , Mamíferos/genética , Camundongos , Regulação da Expressão Gênica , Sequências Reguladoras de Ácido Nucleico/genética , Histonas/metabolismo , Histonas/genética , Genoma/genética , Perfilação da Expressão Gênica/métodos , Imunoprecipitação da Cromatina/métodos
20.
Life Sci Alliance ; 7(9)2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38960623

RESUMO

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.


Assuntos
Anáfase , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Oócitos , Fuso Acromático , Tubulina (Proteína) , Animais , Caenorhabditis elegans/metabolismo , Tubulina (Proteína)/metabolismo , Fuso Acromático/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Oócitos/metabolismo , Prometáfase , Meiose/fisiologia , Proteína ran de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos
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