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1.
Sci China Life Sci ; 67(6): 1089-1105, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38842635

RESUMO

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Histonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Humanos , Metilação , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Proteína de Replicação A/metabolismo , Proteína de Replicação A/genética , Recombinação Homóloga , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Reparo do DNA , Cromatina/metabolismo , Cromatina/genética
2.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38830772

RESUMO

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.


Assuntos
Montagem e Desmontagem da Cromatina , DNA , Regulação da Expressão Gênica , Nucleossomos , Transcrição Gênica , Nucleossomos/metabolismo , Nucleossomos/genética , Humanos , Montagem e Desmontagem da Cromatina/genética , DNA/genética , DNA/metabolismo , Cromatina/metabolismo , Cromatina/genética , Genoma Humano , Sequência de Bases
3.
Biochemistry (Mosc) ; 89(4): 626-636, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38831500

RESUMO

Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study functioning of the Su(Hw)-dependent complexes, we used the previously described su(Hw)E8 mutation with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)E8 protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)E8 protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.


Assuntos
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Repressoras , Fatores de Transcrição , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/química , Animais , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Ligação Proteica , Proteínas Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dedos de Zinco , Proteínas Associadas aos Microtúbulos
4.
PLoS One ; 19(6): e0298965, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38829854

RESUMO

Familial Dysautonomia (FD) is a rare disease caused by ELP1 exon 20 skipping. Here we clarify the role of RNA Polymerase II (RNAPII) and chromatin on this splicing event. A slow RNAPII mutant and chromatin-modifying chemicals that reduce the rate of RNAPII elongation induce exon skipping whereas chemicals that create a more relaxed chromatin exon inclusion. In the brain of a mouse transgenic for the human FD-ELP1 we observed on this gene an age-dependent decrease in the RNAPII density profile that was most pronounced on the alternative exon, a robust increase in the repressive marks H3K27me3 and H3K9me3 and a decrease of H3K27Ac, together with a progressive reduction in ELP1 exon 20 inclusion level. In HEK 293T cells, selective drug-induced demethylation of H3K27 increased RNAPII elongation on ELP1 and SMN2, promoted the inclusion of the corresponding alternative exons, and, by RNA-sequencing analysis, induced changes in several alternative splicing events. These data suggest a co-transcriptional model of splicing regulation in which age-dependent changes in H3K27me3/Ac modify the rate of RNAPII elongation and affect processing of ELP1 alternative exon 20.


Assuntos
Processamento Alternativo , Cromatina , Disautonomia Familiar , Éxons , RNA Polimerase II , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Disautonomia Familiar/genética , Disautonomia Familiar/metabolismo , Humanos , Éxons/genética , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Células HEK293 , Histonas/metabolismo , Camundongos Transgênicos , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Cinética , Splicing de RNA , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo
5.
Nat Commun ; 15(1): 4914, 2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38851846

RESUMO

FOXA family proteins act as pioneer factors by remodeling compact chromatin structures. FOXA1 is crucial for the chromatin binding of the androgen receptor (AR) in both normal prostate epithelial cells and the luminal subtype of prostate cancer (PCa). Recent studies have highlighted the emergence of FOXA2 as an adaptive response to AR signaling inhibition treatments. However, the role of the FOXA1 to FOXA2 transition in regulating cancer lineage plasticity remains unclear. Our study demonstrates that FOXA2 binds to distinct classes of developmental enhancers in multiple AR-independent PCa subtypes, with its binding depending on LSD1. Moreover, we reveal that FOXA2 collaborates with JUN at chromatin and promotes transcriptional reprogramming of AP-1 in lineage-plastic cancer cells, thereby facilitating cell state transitions to multiple lineages. Overall, our findings underscore the pivotal role of FOXA2 as a pan-plasticity driver that rewires AP-1 to induce the differential transcriptional reprogramming necessary for cancer cell lineage plasticity.


Assuntos
Linhagem da Célula , Regulação Neoplásica da Expressão Gênica , Fator 3-beta Nuclear de Hepatócito , Neoplasias da Próstata , Fator de Transcrição AP-1 , Masculino , Humanos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição AP-1/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Histona Desmetilases/metabolismo , Histona Desmetilases/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Animais , Cromatina/metabolismo , Cromatina/genética , Plasticidade Celular/genética , Reprogramação Celular/genética , Camundongos , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Elementos Facilitadores Genéticos/genética , Transcrição Gênica
6.
Biochemistry (Mosc) ; 89(4): 637-652, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38831501

RESUMO

Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development; it is also necessary for the choice of targeted therapy. The Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements, such as inversions and translocations. We propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues. The developed protocol allows to generate high-quality Hi-C data and detect all types of chromosomal rearrangements. We have analyzed various databases to compile a comprehensive list of translocations that hold clinical importance for the targeted therapy selection. The practical value of molecular genetic testing is its ability to influence the treatment strategies and to provide prognostic insights. Detecting specific chromosomal rearrangements can guide the choice of the targeted therapies, which is a critical aspect of personalized medicine in oncology.


Assuntos
Formaldeído , Neoplasias , Inclusão em Parafina , Humanos , Neoplasias/genética , Neoplasias/patologia , Formaldeído/química , Translocação Genética , Fixação de Tecidos , Cromatina/genética , Cromatina/metabolismo , Cromatina/química
7.
Biochemistry (Mosc) ; 89(4): 653-662, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38831502

RESUMO

Chromosome conformation capture techniques have revolutionized our understanding of chromatin architecture and dynamics at the genome-wide scale. In recent years, these methods have been applied to a diverse array of species, revealing fundamental principles of chromosomal organization. However, structural organization of the extrachromosomal entities, like viral genomes or plasmids, and their interactions with the host genome, remain relatively underexplored. In this work, we introduce an enhanced 4C-protocol tailored for probing plasmid DNA interactions. We design specific plasmid vector and optimize protocol to allow high detection rate of contacts between the plasmid and host DNA.


Assuntos
Plasmídeos , Plasmídeos/metabolismo , Plasmídeos/genética , DNA/química , DNA/genética , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Genoma
8.
Sci Adv ; 10(23): eadn2955, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38848364

RESUMO

The hierarchical chromatin organization begins with formation of nucleosomes, which fold into chromatin domains punctuated by boundaries and ultimately chromosomes. In a hierarchal organization, lower levels shape higher levels. However, the dependence of higher-order 3D chromatin organization on the nucleosome-level organization has not been studied in cells. We investigated the relationship between nucleosome-level organization and higher-order chromatin organization by perturbing nucleosomes across the genome by deleting Imitation SWItch (ISWI) and Chromodomain Helicase DNA-binding (CHD1) chromatin remodeling factors in budding yeast. We find that changes in nucleosome-level properties are accompanied by changes in 3D chromatin organization. Short-range chromatin contacts up to a few kilo-base pairs decrease, chromatin domains weaken, and boundary strength decreases. Boundary strength scales with accessibility and moderately with width of nucleosome-depleted region. Change in nucleosome positioning seems to alter the stiffness of chromatin, which can affect formation of chromatin contacts. Our results suggest a biomechanical "bottom-up" mechanism by which nucleosome distribution across genome shapes 3D chromatin organization.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Genoma Fúngico , Nucleossomos , Saccharomyces cerevisiae , Nucleossomos/genética , Nucleossomos/metabolismo , Cromatina/genética , Cromatina/metabolismo , Cromatina/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Adenosina Trifosfatases
9.
J Exp Med ; 221(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38842525

RESUMO

The proliferation marker Ki67 has been attributed critical functions in maintaining mitotic chromosome morphology and heterochromatin organization during the cell cycle, indicating a potential role in developmental processes requiring rigid cell-cycle control. Here, we discovered that despite normal fecundity and organogenesis, germline deficiency in Ki67 resulted in substantial defects specifically in peripheral B and T lymphocytes. This was not due to impaired cell proliferation but rather to early lymphopoiesis at specific stages where antigen-receptor gene rearrangements occurred. We identified that Ki67 was required for normal global chromatin accessibility involving regulatory regions of genes critical for checkpoint stages in B cell lymphopoiesis. In line with this, mRNA expression of Rag1 was diminished and gene rearrangement was less efficient in the absence of Ki67. Transgenes encoding productively rearranged immunoglobulin heavy and light chains complemented Ki67 deficiency, completely rescuing early B cell development. Collectively, these results identify a unique contribution from Ki67 to somatic antigen-receptor gene rearrangement during lymphopoiesis.


Assuntos
Linfócitos B , Cromatina , Antígeno Ki-67 , Antígeno Ki-67/metabolismo , Animais , Cromatina/metabolismo , Cromatina/genética , Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfopoese/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Camundongos , Rearranjo Gênico , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Linfócitos T/metabolismo , Linfócitos T/imunologia , Camundongos Endogâmicos C57BL , Proliferação de Células/genética
10.
Nat Commun ; 15(1): 5032, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38866770

RESUMO

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA por Junção de Extremidades , Transdução de Sinais , Proteína 28 com Motivo Tripartido , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Ubiquitinação , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Fosforilação , Proteína 28 com Motivo Tripartido/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Células HEK293 , Telômero/metabolismo , Dano ao DNA , Cromatina/metabolismo , Animais
11.
Sci Data ; 11(1): 616, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38866804

RESUMO

The development and aging of the brain constitute a lifelong dynamic process, marked by structural and functional changes that entail highly coordinated cellular differentiation and epigenetic regulatory mechanisms. Chromatin accessibility serves as the foundational basis for genetic activity. However, the holistic and dynamic chromatin landscape that spans various brain regions throughout development and ageing remains predominantly unexplored. In this study, we employed single-nucleus ATAC-seq to generate comprehensive chromatin accessibility maps, incorporating data from 69,178 cells obtained from four distinct brain regions - namely, the olfactory bulb (OB), cerebellum (CB), prefrontal cortex (PFC), and hippocampus (HP) - across key developmental time points at 7 P, 3 M, 12 M, and 18 M. We delineated the distribution of cell types across different age stages and brain regions, providing insight into chromatin accessible regions and key transcription factors specific to different cell types. Our data contribute to understanding the epigenetic basis of the formation of different brain regions, providing a dynamic landscape and comprehensive resource for revealing gene regulatory programs during brain development and aging.


Assuntos
Envelhecimento , Encéfalo , Cromatina , Animais , Cromatina/metabolismo , Camundongos , Envelhecimento/genética , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Epigênese Genética , Hipocampo/metabolismo , Hipocampo/crescimento & desenvolvimento , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/crescimento & desenvolvimento
12.
Commun Biol ; 7(1): 721, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862613

RESUMO

The genome folds into complex configurations and structures thought to profoundly impact its function. The intricacies of this dynamic structure-function relationship are not well understood particularly in the context of viral infection. To unravel this interplay, here we provide a comprehensive investigation of simultaneous host chromatin structural (via Hi-C and ATAC-seq) and functional changes (via RNA-seq) in response to vaccinia virus infection. Over time, infection significantly impacts global and local chromatin structure by increasing long-range intra-chromosomal interactions and B compartmentalization and by decreasing chromatin accessibility and inter-chromosomal interactions. Local accessibility changes are independent of broad-scale chromatin compartment exchange (~12% of the genome), underscoring potential independent mechanisms for global and local chromatin reorganization. While infection structurally condenses the host genome, there is nearly equal bidirectional differential gene expression. Despite global weakening of intra-TAD interactions, functional changes including downregulated immunity genes are associated with alterations in local accessibility and loop domain restructuring. Therefore, chromatin accessibility and local structure profiling provide impactful predictions for host responses and may improve development of efficacious anti-viral counter measures including the optimization of vaccine design.


Assuntos
Cromatina , Vaccinia virus , Cromatina/metabolismo , Cromatina/genética , Animais , Vaccinia virus/genética , Vaccinia virus/fisiologia , Chlorocebus aethiops , Células Vero , Vacínia/virologia , Vacínia/imunologia , Interações Hospedeiro-Patógeno/genética , Multiômica
13.
Proc Natl Acad Sci U S A ; 121(24): e2404383121, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38843184

RESUMO

Transcription is extremely important for cellular processes but can be hindered by RNA polymerase II (RNAPII) pausing and stalling. Cockayne syndrome protein B (CSB) promotes the progression of paused RNAPII or initiates transcription-coupled nucleotide excision repair (TC-NER) to remove stalled RNAPII. However, the specific mechanism by which CSB initiates TC-NER upon damage remains unclear. In this study, we identified the indispensable role of the ARK2N-CK2 complex in the CSB-mediated initiation of TC-NER. The ARK2N-CK2 complex is recruited to damage sites through CSB and then phosphorylates CSB. Phosphorylation of CSB enhances its binding to stalled RNAPII, prolonging the association of CSB with chromatin and promoting CSA-mediated ubiquitination of stalled RNAPII. Consistent with this finding, Ark2n-/- mice exhibit a phenotype resembling Cockayne syndrome. These findings shed light on the pivotal role of the ARK2N-CK2 complex in governing the fate of RNAPII through CSB, bridging a critical gap necessary for initiating TC-NER.


Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Reparo do DNA , Proteínas de Ligação a Poli-ADP-Ribose , RNA Polimerase II , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Humanos , Animais , Camundongos , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Cockayne/genética , Síndrome de Cockayne/metabolismo , Transcrição Gênica , Fosforilação , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Camundongos Knockout , Dano ao DNA , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Cromatina/metabolismo , Ubiquitinação , Reparo por Excisão
14.
JCI Insight ; 9(11)2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38855864

RESUMO

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.


Assuntos
Displasia Campomélica , Diferenciação Celular , Condrócitos , Condrogênese , Fatores de Transcrição SOX9 , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Animais , Condrócitos/metabolismo , Camundongos , Displasia Campomélica/genética , Displasia Campomélica/patologia , Displasia Campomélica/metabolismo , Condrogênese/genética , Diferenciação Celular/genética , Elementos Facilitadores Genéticos/genética , Cromatina/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Knockout , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/genética , Humanos , Desenvolvimento Ósseo/genética
15.
Methods Mol Biol ; 2832: 33-46, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38869785

RESUMO

Nucleosome occupancy plays an important role in chromatin compaction, affecting biological processes by hampering the binding of cis-acting elements such as transcription factors, RNA polymerase machinery, and coregulatory. Accessible regions allow for cis-acting elements to bind DNA and regulate transcription. Here, we detail our protocol to profile nucleosome occupancy and chromatin structure dynamics under drought stress at the genome-wide scale using micrococcal nuclease (MNase) digestion. Combining variable MNase concentration treatments and high-throughput sequencing, we investigate the changes in the overall chromatin state using bread wheat samples from an exemplary drought experiment.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Nuclease do Micrococo , Nucleossomos , Estresse Fisiológico , Triticum , Nucleossomos/metabolismo , Nucleossomos/genética , Nuclease do Micrococo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Estresse Fisiológico/genética , Triticum/genética , Triticum/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Cromatina/metabolismo , Cromatina/genética , Plantas/genética , Plantas/metabolismo
16.
Nature ; 630(8016): 466-474, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38839952

RESUMO

Histone acetylation regulates gene expression, cell function and cell fate1. Here we study the pattern of histone acetylation in the epithelial tissue of the Drosophila wing disc. H3K18ac, H4K8ac and total lysine acetylation are increased in the outer rim of the disc. This acetylation pattern is controlled by nuclear position, whereby nuclei continuously move from apical to basal locations within the epithelium and exhibit high levels of H3K18ac when they are in proximity to the tissue surface. These surface nuclei have increased levels of acetyl-CoA synthase, which generates the acetyl-CoA for histone acetylation. The carbon source for histone acetylation in the rim is fatty acid ß-oxidation, which is also increased in the rim. Inhibition of fatty acid ß-oxidation causes H3K18ac levels to decrease in the genomic proximity of genes involved in disc development. In summary, there is a physical mark of the outer rim of the wing and other imaginal epithelia in Drosophila that affects gene expression.


Assuntos
Acetilcoenzima A , Núcleo Celular , Cromatina , Drosophila melanogaster , Ácidos Graxos , Histonas , Asas de Animais , Animais , Acetilcoenzima A/metabolismo , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Acetilação , Núcleo Celular/metabolismo , Asas de Animais/metabolismo , Ácidos Graxos/metabolismo , Discos Imaginais/metabolismo , Discos Imaginais/citologia , Oxirredução , Lisina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Acetato-CoA Ligase/metabolismo , Acetato-CoA Ligase/genética
17.
Front Immunol ; 15: 1403018, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38881893

RESUMO

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease primarily affecting premature neonates, marked by poorly understood pro-inflammatory signaling cascades. Recent advancements have shed light on a subset of endogenous molecular patterns, termed chromatin-associated molecular patterns (CAMPs), which belong to the broader category of damage-associated molecular patterns (DAMPs). CAMPs play a crucial role in recognizing pattern recognition receptors and orchestrating inflammatory responses. This review focuses into the realm of CAMPs, highlighting key players such as extracellular cold-inducible RNA-binding protein (eCIRP), high mobility group box 1 (HMGB1), cell-free DNA, neutrophil extracellular traps (NETs), histones, and extracellular RNA. These intrinsic molecules, often perceived as foreign, have the potential to trigger immune signaling pathways, thus contributing to NEC pathogenesis. In this review, we unravel the current understanding of the involvement of CAMPs in both preclinical and clinical NEC scenarios. We also focus on elucidating the downstream signaling pathways activated by these molecular patterns, providing insights into the mechanisms that drive inflammation in NEC. Moreover, we scrutinize the landscape of targeted therapeutic approaches, aiming to mitigate the impact of tissue damage in NEC. This in-depth exploration offers a comprehensive overview of the role of CAMPs in NEC, bridging the gap between preclinical and clinical insights.


Assuntos
Alarminas , Cromatina , Enterocolite Necrosante , Humanos , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/imunologia , Alarminas/metabolismo , Alarminas/imunologia , Cromatina/metabolismo , Animais , Transdução de Sinais , Recém-Nascido , Proteína HMGB1/metabolismo
18.
Nat Commun ; 15(1): 5048, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38871723

RESUMO

Despite the advent of genomic sequencing, molecular diagnosis remains unsolved in approximately half of patients with Mendelian disorders, largely due to unclarified functions of noncoding regions and the difficulty in identifying complex structural variations. In this study, we map a unique form of central iris hypoplasia in a large family to 6q15-q23.3 and 18p11.31-q12.1 using a genome-wide linkage scan. Long-read sequencing reveals a balanced translocation t(6;18)(q22.31;p11.22) with intergenic breakpoints. By performing Hi-C on induced pluripotent stem cells from a patient, we identify two chromatin topologically associating domains spanning across the breakpoints. These alterations lead the ectopic chromatin interactions between APCDD1 on chromosome 18 and enhancers on chromosome 6, resulting in upregulation of APCDD1. Notably, APCDD1 is specifically localized in the iris of human eyes. Our findings demonstrate that noncoding structural variations can lead to Mendelian diseases by disrupting the 3D genome structure and resulting in altered gene expression.


Assuntos
Cromatina , Iris , Linhagem , Translocação Genética , Humanos , Cromatina/metabolismo , Cromatina/genética , Iris/metabolismo , Masculino , Feminino , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 18/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Adulto , Doenças da Íris/genética , Doenças da Íris/metabolismo , Doenças da Íris/patologia , Ligação Genética
19.
BMC Bioinformatics ; 25(1): 212, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38872103

RESUMO

BACKGROUND: A vital step in analyzing single-cell data is ascertaining which cell types are present in a dataset, and at what abundance. In many diseases, the proportions of varying cell types can have important implications for health and prognosis. Most approaches for cell type annotation have centered around cell typing for single-cell RNA-sequencing (scRNA-seq) and have had promising success. However, reliable methods are lacking for many other single-cell modalities such as single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), which quantifies the extent to which genes of interest in each cell are epigenetically "open" for expression. RESULTS: To leverage the informative potential of scATAC-seq data, we developed CAMML with the integration of chromatin accessibility (CAraCAl), a bioinformatic method that performs cell typing on scATAC-seq data. CAraCAl performs cell typing by scoring each cell for its enrichment of cell type-specific gene sets. These gene sets are composed of the most upregulated or downregulated genes present in each cell type according to projected gene activity. CONCLUSIONS: We found that CAraCAl does not improve performance beyond CAMML when scRNA-seq is present, but if only scATAC-seq is available, CAraCAl performs cell typing relatively successfully. As such, we also discuss best practices for cell typing and the strengths and weaknesses of various cell annotation options.


Assuntos
Cromatina , Biologia Computacional , Cromatina/metabolismo , Cromatina/genética , Cromatina/química , Biologia Computacional/métodos , Humanos , Análise de Célula Única/métodos , Software , Análise de Sequência de RNA/métodos , Transposases/metabolismo , Transposases/genética
20.
Nat Cell Biol ; 26(6): 991-1002, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38866970

RESUMO

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.


Assuntos
Envelhecimento , Linfócitos B , Montagem e Desmontagem da Cromatina , Cadeias Pesadas de Imunoglobulinas , Linfopoese , Animais , Envelhecimento/genética , Envelhecimento/metabolismo , Linfócitos B/metabolismo , Linfopoese/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Transativadores/metabolismo , Transativadores/genética , Cromatina/metabolismo , Cromatina/genética , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia , Camundongos Endogâmicos C57BL , Camundongos , Diferenciação Celular , Camundongos Knockout
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