RESUMO
Long known as the site of ribosome biogenesis, the nucleolus is increasingly recognized for its role in shaping three-dimensional (3D) genome organization. Still, the mechanisms governing the targeting of selected regions of the genome to nucleolus-associated domains (NADs) remain enigmatic. Here, we reveal the essential role of ZNF274, a SCAN-bearing member of the Krüppel-associated box (KRAB)-containing zinc finger protein (KZFP) family, in sequestering lineage-specific gene clusters within NADs. Ablation of ZNF274 triggers transcriptional activation across entire genomic neighborhoods-encompassing, among others, protocadherin and KZFP-encoding genes-with loss of repressive chromatin marks, altered the 3D genome architecture and de novo CTCF binding. Mechanistically, ZNF274 anchors target DNA sequences at the nucleolus and facilitates their compartmentalization via a previously uncharted function of the SCAN domain. Our findings illuminate the mechanisms underlying NAD organization and suggest that perinucleolar entrapment into repressive hubs constrains the activation of tandemly arrayed genes to enable selective expression and modulate cell differentiation programs during development.
Assuntos
Nucléolo Celular , Família Multigênica , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , Animais , Humanos , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Cromatina/metabolismo , Cromatina/genética , Linhagem da Célula/genética , Dedos de Zinco/genética , Diferenciação Celular/genética , Ligação ProteicaRESUMO
Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer-promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer-promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer-promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer-promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1's architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage.
Assuntos
Proteínas Cromossômicas não Histona , Coesinas , Regiões Promotoras Genéticas , Transcrição Gênica , Fator de Transcrição YY1 , Animais , Humanos , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Elementos Facilitadores Genéticos , Células Eritroides/metabolismo , Células Eritroides/citologia , Fase G1/genética , Regulação da Expressão Gênica , Mitose/genética , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
Eukaryotic genomes are organized into chromatin domains through long-range chromatin interactions which are mediated by the binding of architectural proteins, such as CTCF and cohesin, and histone modifications. Based on the published Hi-C and ChIP-seq datasets in human monocyte-derived macrophages, we identified 206 and 127 differential chromatin interactions (DCIs) that were not located within transcription readthrough regions in influenza A virus- and interferon ß-treated cells, respectively, and found that the binding positions of CTCF and RAD21 within more than half of the DCI sites did not change. However, five histone modifications, H3K4me3, H3K27ac, H3K36me3, H3K9me3, and H3K27me3, showed significantly more dramatic changes than CTCF and RAD21 within the DCI sites. For H3K4me3, H3K27ac, H3K36me3, and H3K27me3, significantly more dramatic changes were observed outside than within the DCI sites. We further applied a motif scanning approach to discover proteins that might correlate with changes in histone modifications and chromatin interactions and found that PRDM9, ZNF384, and STAT2 frequently bound to DNA sequences corresponding to 1 kb genomic intervals with gains or losses of a histone modification within the DCI sites. This study explores the dynamic regulation of chromatin interactions and extends the current knowledge of the relationship between histone modifications and chromatin interactions.
Assuntos
Cromatina , Código das Histonas , Histonas , Humanos , Cromatina/metabolismo , Cromatina/genética , Histonas/metabolismo , Histonas/genética , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Macrófagos/metabolismoRESUMO
Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated.
Assuntos
Fator de Ligação a CCCTC , Cromatina , Elementos Isolantes , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Elementos Isolantes/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Genoma/genética , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Coesinas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genéticaRESUMO
While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterized. This is partly due to the genomic context of CTCF sites greatly influencing their roles and activity. Here we have developed an experimental system to determine the ability of minimal, consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly, each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the degree of evolutionary conservation; their resemblance to the consensus core sequences; or the number of CTCF core motifs harboured in the element. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNaseI footprints located close to the core motif that may affect function. We have developed an assay of CTCF sequences which will enable researchers to sub-classify individual CTCF elements in a uniform and unbiased way.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Sítios de Ligação/genética , Humanos , Animais , Camundongos , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Elementos Isolantes/genética , Ligação Proteica , Motivos de Nucleotídeos , Linhagem Celular , Regulação da Expressão Gênica , Diferenciação Celular/genéticaRESUMO
Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ligação a DNA , Animais , Cromatina/metabolismo , Cromatina/genética , Camundongos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Neurônios Motores/metabolismo , Diferenciação Celular , Dedos de Zinco , Humanos , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genéticaRESUMO
A number of factors contribute to cell type-specific CTCF chromatin binding, but how they act in concert to determine binding stability and functionality has not been fully elucidated. In this review, we tie together different layers of regulation to provide a holistic view of what is known. What emerges from these studies is a multifaceted system in which DNA sequence, DNA and chromatin accessibility, and cell type-specific transcription factors together contribute to CTCF binding profile and function. We discuss these findings in the light of disease settings in which changes in the chromatin landscape and transcriptional programming can disrupt CTCF's binding profile and involvement in looping.
Assuntos
Fator de Ligação a CCCTC , Cromatina , Proteínas Repressoras , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Cromatina/genética , Cromatina/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ligação Proteica , Animais , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Regulação da Expressão Gênica , Sítios de LigaçãoRESUMO
The specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow some physical crosstalk across regulatory domains, which is at odds with the specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites cooperatively foster the robust insulation of regulatory domains. By genetically dissecting a couple of representative loci in mouse embryonic stem cells, we show that CTCF sites prevent undesirable enhancer-gene contacts (i.e. physical insulation), while developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the insulation of regulatory domains, which in turn might help interpreting the pathological consequences of certain structural variants.
Assuntos
Fator de Ligação a CCCTC , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas , Regiões Promotoras Genéticas , Animais , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Camundongos , Regiões Promotoras Genéticas/genética , Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Murinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Elementos Isolantes/genéticaRESUMO
TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes.
Assuntos
Fator de Ligação a CCCTC , Proteínas de Ciclo Celular , Cromatina , Proteínas Cromossômicas não Histona , Coesinas , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromatina/metabolismo , Cromatina/genética , Animais , Sítios de Ligação , Genoma/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas/genética , DNA/metabolismo , DNA/genéticaRESUMO
CCCTC-binding factor (CTCF) is an insulator protein that binds to a highly conserved DNA motif and facilitates regulation of three-dimensional (3D) nuclear architecture and transcription. CTCF binding sites (CTCF-BSs) reside in non-coding DNA and are frequently mutated in cancer. Our previous study identified a small subclass of CTCF-BSs that are resistant to CTCF knock down, termed persistent CTCF binding sites (P-CTCF-BSs). P-CTCF-BSs show high binding conservation and potentially regulate cell-type constitutive 3D chromatin architecture. Here, using ICGC sequencing data we made the striking observation that P-CTCF-BSs display a highly elevated mutation rate in breast and prostate cancer when compared to all CTCF-BSs. To address whether P-CTCF-BS mutations are also enriched in other cell-types, we developed CTCF-INSITE-a tool utilising machine learning to predict persistence based on genetic and epigenetic features of experimentally-determined P-CTCF-BSs. Notably, predicted P-CTCF-BSs also show a significantly elevated mutational burden in all 12 cancer-types tested. Enrichment was even stronger for P-CTCF-BS mutations with predicted functional impact to CTCF binding and chromatin looping. Using in vitro binding assays we validated that P-CTCF-BS cancer mutations, predicted to be disruptive, indeed reduced CTCF binding. Together this study reveals a new subclass of cancer specific CTCF-BS DNA mutations and provides insights into their importance in genome organization in a pan-cancer setting.
Assuntos
Fator de Ligação a CCCTC , Aprendizado de Máquina , Mutação , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Sítios de Ligação/genética , Cromatina/metabolismo , Cromatina/genética , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Masculino , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismoRESUMO
Human cell line models, including the neuronal precursor line LUHMES, are important for investigating developmental transcriptional dynamics within imprinted regions, particularly the 15q11-q13 Angelman (AS) and Prader-Willi (PWS) syndrome locus. AS results from loss of maternal UBE3A in neurons, where the paternal allele is silenced by a convergent antisense transcript UBE3A-ATS, a lncRNA that terminates at PWAR1 in non-neurons. qRT-PCR analysis confirmed the exclusive and progressive increase in UBE3A-ATS in differentiating LUHMES neurons, validating their use for studying UBE3A silencing. Genome-wide transcriptome analyses revealed changes to 11 834 genes during neuronal differentiation, including the upregulation of most genes within the 15q11-q13 locus. To identify dynamic changes in chromatin loops linked to transcriptional activity, we performed a HiChIP validated by 4C, which identified two neuron-specific CTCF loops between MAGEL2-SNRPN and PWAR1-UBE3A. To determine if allele-specific differentially methylated regions (DMR) may be associated with CTCF loop anchors, whole genome long-read nanopore sequencing was performed. We identified a paternally hypomethylated DMR near the SNRPN upstream loop anchor exclusive to neurons and a paternally hypermethylated DMR near the PWAR1 CTCF anchor exclusive to undifferentiated cells, consistent with increases in neuronal transcription. Additionally, DMRs near CTCF loop anchors were observed in both cell types, indicative of allele-specific differences in chromatin loops regulating imprinted transcription. These results provide an integrated view of the 15q11-q13 epigenetic landscape during LUHMES neuronal differentiation, underscoring the complex interplay of transcription, chromatin looping, and DNA methylation. They also provide insights for future therapeutic approaches for AS and PWS.
Assuntos
Fator de Ligação a CCCTC , Diferenciação Celular , Cromossomos Humanos Par 15 , Metilação de DNA , Impressão Genômica , Neurônios , Transcriptoma , Ubiquitina-Proteína Ligases , Humanos , Impressão Genômica/genética , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Cromossomos Humanos Par 15/genética , Neurônios/metabolismo , Metilação de DNA/genética , Transcriptoma/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Diferenciação Celular/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , RNA Longo não Codificante/genética , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/patologia , Síndrome de Prader-Willi/metabolismo , Proteínas Centrais de snRNP/genética , Proteínas Centrais de snRNP/metabolismo , Alelos , Linhagem Celular , EpigenomaRESUMO
Natural killer cells (NK) are the "professional killer" of tumors and play a crucial role in anti-tumor immunotherapy. NK cell desensitization is a key mechanism of tumor immune escape. Dysregulated NKG2D-NKG2DL signaling is a primary driver of this desensitization process. However, the factors that regulate NK cell desensitization remain largely uncharacterized. Here, we present the first report that circular RNA circARAP2 (hsa_circ_0069396) is involved in the soluble MICA (sMICA)-induced NKG2D endocytosis in the NK cell desensitization model. CircARAP2 was upregulated during NK cell desensitization and the loss of circARAP2 alleviated NKG2D endocytosis and NK cell desensitization. Using Chromatin isolation by RNA purification (ChIRP) and RNA pull-down approaches, we identified that RAB5A, a molecular marker of early endosomes, was its downstream target. Notably, transcription factor CTCF was an intermediate functional partner of circARAP2. Mechanistically, we discovered that circARAP2 interacted with CTCF and inhibited the recruitment of CTCF-Polycomb Repressive Complex 2 (PRC2) to the promoter region of RAB5A, thereby erasing histone H3K27 and H3K9 methylation suppression to enhance RAB5A transcription. These data demonstrate that inhibition of circARAP2 effectively alleviates sMICA-induced NKG2D endocytosis and NK cell desensitization, providing a novel target for therapeutic intervention in tumor immune evasion.
Assuntos
Fator de Ligação a CCCTC , Antígenos de Histocompatibilidade Classe I , Células Matadoras Naturais , RNA Circular , Proteínas rab5 de Ligação ao GTP , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Humanos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , RNA Circular/genética , RNA Circular/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Endocitose , Endossomos/metabolismo , Camundongos , AnimaisRESUMO
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/citologiaRESUMO
Chromosome rearrangements may distort 3D chromatin architectures and thus change gene regulation, yet how 3D chromatin structures evolve in insects is largely unknown. Here, we obtain chromosome-level genomes for four butterfly species, Graphium cloanthus, Graphium sarpedon, Graphium eurypylus with 2n = 30, 40, and 60, respectively, and Papilio bianor with 2n = 60. Together with large-scale Hi-C data, we find that inter-chromosome rearrangements very rarely disrupted the pre-existing 3D chromatin structure of ancestral chromosomes. However, some intra-chromosome rearrangements changed 3D chromatin structures compared to the ancestral configuration. We find that new TADs and subTADs have emerged across the rearrangement sites where their adjacent compartments exhibit uniform types. Two intra-chromosome rearrangements altered Rel and lft regulation, potentially contributing to wing patterning differentiation and host plant choice. Notably, butterflies exhibited chromatin loops between Hox gene cluster ANT-C and BX-C, unlike Drosophila. Our CRISPR-Cas9 experiments in butterflies confirm that knocking out the CTCF binding site of the loops in BX-C affected the phenotypes regulated by Antp in ANT-C, resulting in legless larva. Our results reveal evolutionary patterns of insect 3D chromatin structures and provide evidence that 3D chromatin structure changes can play important roles in the evolution of traits.
Assuntos
Borboletas , Cromatina , Evolução Molecular , Genoma de Inseto , Animais , Borboletas/genética , Cromatina/metabolismo , Cromatina/genética , Rearranjo Gênico/genética , Cromossomos de Insetos/genética , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genéticaRESUMO
Transposable elements (TEs) and other repetitive regions have been shown to contain gene regulatory elements, including transcription factor binding sites. However, regulatory elements harbored by repeats have proven difficult to characterize using short-read sequencing assays such as ChIP-seq or ATAC-seq. Most regulatory genomics analysis pipelines discard "multimapped" reads that align equally well to multiple genomic locations. Because multimapped reads arise predominantly from repeats, current analysis pipelines fail to detect a substantial portion of regulatory events that occur in repetitive regions. To address this shortcoming, we developed Allo, a new approach to allocate multimapped reads in an efficient, accurate, and user-friendly manner. Allo combines probabilistic mapping of multimapped reads with a convolutional neural network that recognizes the read distribution features of potential peaks, offering enhanced accuracy in multimapping read assignment. Allo also provides read-level output in the form of a corrected alignment file, making it compatible with existing regulatory genomics analysis pipelines and downstream peak-finders. In a demonstration application on CTCF ChIP-seq data, we show that Allo results in the discovery of thousands of new CTCF peaks. Many of these peaks contain the expected cognate motif and/or serve as TAD boundaries. We additionally apply Allo to a diverse collection of ENCODE ChIP-seq data sets, resulting in multiple previously unidentified interactions between transcription factors and repetitive element families. Finally, we show that Allo may be particularly beneficial in identifying ChIP-seq peaks at centromeres, near segmentally duplicated genes, and in younger TEs, enabling new regulatory analyses in these regions.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Humanos , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Genômica/métodos , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Elementos Reguladores de Transcrição , Elementos de DNA Transponíveis , Análise de Sequência de DNA/métodos , Redes Neurais de ComputaçãoRESUMO
Gene regulatory elements drive complex biological phenomena and their mutations are associated with common human diseases. The impacts of human regulatory variants are often tested using model organisms such as mice. However, mapping human enhancers to conserved elements in mice remains a challenge, due to both rapid enhancer evolution and limitations of current computational methods. We analyze distal enhancers across 45 matched human/mouse cell/tissue pairs from a comprehensive dataset of DNase-seq experiments, and show that while cell-specific regulatory vocabulary is conserved, enhancers evolve more rapidly than promoters and CTCF binding sites. Enhancer conservation rates vary across cell types, in part explainable by tissue specific transposable element activity. We present an improved genome alignment algorithm using gapped-kmer features, called gkm-align, and make genome wide predictions for 1,401,803 orthologous regulatory elements. We show that gkm-align discovers 23,660 novel human/mouse conserved enhancers missed by previous algorithms, with strong evidence of conserved functional activity.
Assuntos
Algoritmos , Sequência Conservada , Elementos Facilitadores Genéticos , Animais , Elementos Facilitadores Genéticos/genética , Humanos , Camundongos , Evolução Molecular , Sítios de Ligação/genética , Mamíferos/genética , Regiões Promotoras Genéticas/genética , Biologia Computacional/métodos , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genéticaRESUMO
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 , TransativadoresRESUMO
The three-dimensional genome structure organized by CTCF is required for development. Clinically identified mutations in CTCF have been linked to adverse developmental outcomes. Nevertheless, the underlying mechanism remains elusive. In this investigation, we explore the regulatory roles of a clinically relevant R567W point mutation, located within the 11th zinc finger of CTCF, by introducing this mutation into both murine models and human embryonic stem cell-derived cortical organoid models. Mice with homozygous CTCFR567W mutation exhibit growth impediments, resulting in postnatal mortality, and deviations in brain, heart, and lung development at the pathological and single-cell transcriptome levels. This mutation induces premature stem-like cell exhaustion, accelerates the maturation of GABAergic neurons, and disrupts neurodevelopmental and synaptic pathways. Additionally, it specifically hinders CTCF binding to peripheral motifs upstream to the core consensus site, causing alterations in local chromatin structure and gene expression, particularly at the clustered protocadherin locus. Comparative analysis using human cortical organoids mirrors the consequences induced by this mutation. In summary, this study elucidates the influence of the CTCFR567W mutation on human neurodevelopmental disorders, paving the way for potential therapeutic interventions.
Assuntos
Fator de Ligação a CCCTC , Transtornos do Neurodesenvolvimento , Organoides , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Animais , Camundongos , Transtornos do Neurodesenvolvimento/genética , Organoides/metabolismo , Mutação , Neurônios GABAérgicos/metabolismo , Neurônios GABAérgicos/patologia , Masculino , Cromatina/metabolismo , Cromatina/genética , Feminino , Encéfalo/metabolismo , Encéfalo/patologia , Mutação Puntual , Células-Tronco Embrionárias Humanas/metabolismoAssuntos
Fator de Ligação a CCCTC , Desenvolvimento Embrionário , Células-Tronco Hematopoéticas , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/embriologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Desenvolvimento Embrionário/genética , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismoRESUMO
OBJECTIVE: The aim of this study was to explore the effect and mechanism of programmed cell death ligand 1 (PD-L1) in promoting the proliferation and osteo/odontogenic-differentiation of human dental pulp stem cells (hDPSCs) by mediating CCCTC-binding factor (CTCF) expression. DESIGN: The interaction between PD-L1 and CTCF was verified through co-immunoprecipitation. hDPSCs transfected with PD-L1 overexpression and CTCF knockdown vectors were treated with lipopolysaccharide or an osteogenic-inducing medium. Inflammatory cytokines and osteo/odontogenic-differentiation related genes were measured. Osteo/odontogenic-differentiation of hDPSCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining. RESULTS: Overexpression of PD-L1 inhibited LPS-induced pro-inflammatory cytokine upregulation, cell proliferation, ALP activity, and calcium deposition in hDPSCs and elevated the expression of osteo/odontogenic-differentiation related genes; however, such expression patterns could be reversed by CTCF knockdown. Co-immunoprecipitation results confirmed the binding of PD-L1 to CTCF, indicating that PD-L1 overexpression in hDPSCs increases CTCF expression, thus inhibiting the inflammatory response and increasing osteo/odontogenic-differentiation of hDPSCs. CONCLUSION: PD-L1 overexpression in hDPSCs enhances the proliferation and osteo/odontogenic-differentiation of hDPSCs and inhibit the inflammatory response by upregulating CTCF expression.