ABSTRACT
The generation of highly diverse antigen receptors in T and B lymphocytes relies on V(D)J recombination. The enhancer Eα has been implicated in regulating the accessibility of Vα and Jα genes through long-range interactions during rearrangements of the T-cell antigen receptor gene Tcra. However, direct evidence for Eα physically mediating the interaction of Vα and Jα genes is still lacking. In this study, we utilized the 3C-HTGTS assay, a chromatin interaction technique based on 3C, to analyze the higher order chromatin structure of the Tcra locus. Our analysis revealed the presence of sufficient information in the 3C-HTGTS data to detect multiway contacts. Three-way contact analysis of the Tcra locus demonstrated the co-occurrence of the proximal Jα genes, Vα genes and Eα in CD4+CD8+ double-positive thymocytes. Notably, the INT2-TEAp loop emerged as a prominent structure likely to be responsible for bringing the proximal Jα genes and the Vα genes into proximity. Moreover, the enhancer Eα utilizes this loop to establish physical proximity with the proximal Vα gene region. This study provides insights into the higher order chromatin structure of the Tcra locus, shedding light on the spatial organization of chromatin and its impact on V(D)J recombination.
Subject(s)
Receptors, Antigen, T-Cell, alpha-beta , Thymocytes , Chromatin/genetics , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , V(D)J Recombination/genetics , Animals , MiceABSTRACT
BACKGROUND: Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation. METHODS: We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS. RESULTS: Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs. CONCLUSIONS: This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.
Subject(s)
Down Syndrome , Transcription Factor AP-1 , Humans , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Chromatin Immunoprecipitation Sequencing , RNA-Seq , Down Syndrome/drug therapy , Down Syndrome/genetics , Chromatin , Myxovirus Resistance Proteins/genetics , Myxovirus Resistance Proteins/metabolismABSTRACT
Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.
Subject(s)
B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Alleles , Animals , Biomarkers/metabolism , Genetic Loci , Mice, Inbred C57BL , Precursor Cells, B-Lymphoid/cytology , Precursor Cells, B-Lymphoid/immunology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproducibility of ResultsABSTRACT
Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation (MCLMR) is an inherited disorder characterized by severe microcephaly and abnormal facial features. Kinesin family member 11 (KIF11) mutations have been reported closely related to microcephaly in different cases, while the pathogenicity was still unclear. Here, we report a de novo heterozygous mutation in exon 20 of the KIF11 (c.2922G>T; p.Pro974=) from a microcephaly patient through whole-exome sequencing. Further studies identified that this variant affected the normal splicing of KIF11 pre-mRNA, thus leading to the c.2815_2922 deletion of exon 20 through PBMC-derived pre-mRNA splicing assay and minigene experiment. Moreover, c.2815_2922 deletion would produce a shortened KIF11 protein, which may competitively bind to the normal KIF11 protein, suggesting a dominant negative effect mechanism in c.2922G>T mutation-induced MCLMR.
Subject(s)
Microcephaly , Retinal Dysplasia , Humans , Kinesins/genetics , Leukocytes, Mononuclear , Microcephaly/genetics , Pedigree , RNA Splicing/genetics , Retinal Dysplasia/geneticsABSTRACT
BACKGROUND: NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS: The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS: NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION: These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.
Subject(s)
Gene Expression Regulation, Neoplastic , Melanoma, Experimental , Animals , Gene Expression Profiling , Humans , Interferons , Mice , Mice, Inbred C57BL , Nitric Oxide Synthase Type IABSTRACT
The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.
Subject(s)
CCCTC-Binding Factor/metabolism , Chromatin/metabolism , Enhancer Elements, Genetic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , Animals , Apoptosis Regulatory Proteins/genetics , Binding Sites , CCCTC-Binding Factor/genetics , Chromatin/genetics , Gene Expression Regulation , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Homeodomain Proteins/genetics , Membrane Proteins/genetics , Mice, Inbred C57BL , Mice, Mutant Strains , Thymus Gland/cytologyABSTRACT
Histone 3 lysine 4 trimethylation (H3K4me3) is associated with promoters of active genes and found at hot spots for DNA recombination. Here we have shown that PAXIP1 (also known as PTIP), a protein associated with MLL3 and MLL4 methyltransferase and the DNA damage response, regulates RAG-mediated cleavage and repair during V(D)J recombination in CD4(+) CD8(+) DP thymocytes. Loss of PAXIP1 in developing thymocytes diminished Jα H3K4me3 and germline transcription, suppressed double strand break formation at 3' Jα segments, but resulted in accumulation of unresolved T cell receptor α-chain gene (Tcra) breaks. Moreover, PAXIP1 was essential for release of mature single positive (SP) αß T cells from the thymus through transcriptional activation of sphingosine-1-phosphate receptor S1pr1 as well as for natural killer T cell development. Thus, in addition to maintaining genome integrity during Tcra rearrangements, PAXIP1 controls distinct transcriptional programs during DP differentiation necessary for Tcra locus accessibility, licensing mature thymocytes for trafficking and natural killer T cell development.
Subject(s)
Carrier Proteins/genetics , Cell Differentiation , Cell Movement , DNA Damage , Gene Expression Regulation , Nuclear Proteins/genetics , Thymocytes/cytology , Thymocytes/immunology , Animals , Carrier Proteins/metabolism , Cell Lineage/genetics , Cell Movement/genetics , DNA-Binding Proteins , Histones/metabolism , Mice , Natural Killer T-Cells/cytology , Natural Killer T-Cells/metabolism , Nuclear Proteins/metabolism , Promoter Regions, Genetic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Lysosphingolipid/genetics , Recombination, Genetic , Sphingosine-1-Phosphate Receptors , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Thymocytes/metabolism , Transcription, GeneticABSTRACT
T cells recognize the universe of foreign antigens with a diverse repertoire of T cell receptors generated by V(D)J recombination. Special AT-rich binding protein 1 (Satb1) is a chromatin organizer that plays an essential role in T cell development. Previous study has shown that Satb1 regulates the re-induction of recombinase Rag1 and Rag2 in CD4+CD8+ thymocytes, affecting the secondary rearrangement of the Tcra gene. Here, we detected the repertoires of four TCR genes, Tcrd, Tcrg, Tcrb, and Tcra, in the adult thymus, and explored the role of the Satb1 in shaping the TCR repertoires. We observed a strong bias in the V and J gene usages of the Tcrd and Tcrg repertoires in WT and Satb1-deleted thymocytes. Satb1 deletion had few effects on the V(D)J rearrangement and repertoire of the Tcrg, Tcrd, and Tcrb genes. The Tcra repertoire was severely impaired in Satb1-deleted thymocytes, while the primary rearrangement was relatively normal. We also found the CDR3 length of TCRα chain was significantly longer in Satb1-deleted thymocytes, which can be explained by the strong bias of the proximal Jα usage. Our results showed that Satb1 plays an essential role in shaping TCR repertoires in αß T cells.
Subject(s)
Chromatin , Matrix Attachment Region Binding Proteins , Receptors, Antigen, T-Cell, alpha-beta , Thymus Gland/cytology , Animals , Matrix Attachment Region Binding Proteins/genetics , Mice , Mice, Knockout , Receptors, Antigen, T-Cell , Receptors, Antigen, T-Cell, alpha-beta/genetics , ThymocytesABSTRACT
The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.
Subject(s)
High-Throughput Nucleotide Sequencing/methods , Immunoglobulins/genetics , Receptors, Antigen, T-Cell/genetics , Animals , CRISPR-Cas Systems , Mice , Sequence Analysis, DNA , V(D)J RecombinationABSTRACT
BACKGROUND: Casticin, an isoflavone compound extracted from the herb Fructus Viticis, has demonstrated anti-inflammatory and anticancer activities and properties. The aim of this study was to investigate the effects and mechanisms of casticin in nasopharyngeal carcinoma (NPC) cells and to determine its potential for targeted use as a medicine. METHODS: NPC cells were used to perform the experiments. The CCK8 assay and colony formation assays were used to assess cell viability. Flow cytometry was used to measure the cell cycle and apoptosis analysis (annexin V/PI assay). A three-dimensional (3D) tumour sphere culture system was used to characterize the effect of casticin on NPC stem cells. In silico molecular docking prediction and high-throughput KINOME scan assays were used to evaluate the binding of casticin to phosphoinositide 3-kinase (PI3K), including wild-type and most of mutants variants. We also used the SelectScreen assay to detect the IC50 of ATP activity in the active site of the target kinase. Western blotting was used to evaluate the changes in key proteins involved cell cycle, apoptosis, stemness, and PI3K/protein kinase B (AKT) signalling. The effect of casticin treatment in vivo was determined by using a xenograft mouse model. RESULTS: Our results indicate that casticin is a new and novel selective PI3K inhibitor that can significantly inhibit NPC proliferation and that it induces G2/GM arrest and apoptosis by upregulating Bax/BCL2 expression. Moreover, casticin was observed to affect the self-renewal ability of the nasopharyngeal carcinoma cell lines, and a combination of casticin with BYL719 was observed to induce a decrease in the level of the phosphorylation of mTORC1 downstream targets in BYL719-insensitive NPC cell lines. CONCLUSION: Casticin is a newly emerging selective PI3K inhibitor with potential for use as a targeted therapeutic treatment for nasopharyngeal carcinoma. Accordingly, casticin might represent a novel and effective agent against NPC and likely has high potential for combined use with pharmacological agents targeting PI3K/AKT.
ABSTRACT
OBJECTIVE: To carry out prenatal diagnosis for a fetus with ultrasonographic abnormality. METHODS: Chromosomal karyotyping and array comparative genomic hybridization (array-CGH) analysis were applied for the diagnosis. Peripheral blood samples were also taken from the parents for chromosome karyotyping analysis. RESULTS: The fetal karyotype showed additional material of unknown-origin attached to Yq. Array CGH analysis confirmed that the material was derived from 3q22.1q29. The father was found to carry a balanced translocation 46, X, t(Y;3)(q12;q23) (which was diagnosed as 46,XY,Y≥18 elsewhere), whilst the mother was found to be normal. CONCLUSION: 3q partial trisomy may present as malformation of multiple systems. Combination of chromosome karyotyping and array-CGH can provide reliable diagnosis for fetuses with abnormalities by ultrasonography.
Subject(s)
Chromosomes, Human, Pair 3/genetics , Prenatal Diagnosis , Trisomy , Comparative Genomic Hybridization , Female , Fetus , Humans , Karyotyping , Male , PregnancyABSTRACT
OBJECTIVE: To explore the genetic basis for a family with non-syndromic autosomal recessive deafness. METHODS: The proband and her parents were subjected to physical and audiological examinations. With genomic DNA extracted from peripheral blood samples, next-generation sequencing was carried out using a panel for deafness genes. Suspected mutation was validated by Sanger sequencing and qPCR analysis of her parents. RESULTS: The proband presented bilateral severe sensorineural hearing loss at three days after birth. Her auditory threshold was 110-120 dBnHL but with absence of vestibular and retinal symptoms. Her brother also had deafness but her parents were normal. No abnormality was found upon physical examination of her family members, while audiological examination showed no middle ear or retrocochlear diseases. Next-generation sequencing identified compound heterozygous mutations of the MYO7A gene, including a previously known c.462C>A (p. Cys154Ter) and a novel EX43_46 Del, which were respectively derived from her mother and father. CONCLUSION: The compound heterozygous mutations of the MYO7A gene probably underlie the disease in this family. Our findings has enriched the mutation spectrum for non-syndromic autosomal recessive deafness 2.
Subject(s)
Hearing Loss, Sensorineural/genetics , Myosins/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Male , Mutation , Myosin VIIa , PedigreeSubject(s)
Genetic Testing , Prenatal Diagnosis , Pregnancy , Female , Humans , Prenatal Diagnosis/ethics , Genetic Testing/ethicsABSTRACT
PURPOSE: Endotoxin-induced uveitis (EIU) is a well-established mouse model for studying human acute inflammatory uveitis. The purpose of this study is to investigate the genome-wide retinal transcriptome profile of EIU. METHODS: The anterior segment of the mice was examined with a slit-lamp, and clinical scores were evaluated simultaneously. The histological changes in the posterior segment of the eyes were evaluated with hematoxylin and eosin (H&E) staining. A high throughput RNA sequencing (RNA-seq) strategy using the Illumina Hiseq 2500 platform was applied to characterize the retinal transcriptome profile from lipopolysaccharide (LPS)-treated and untreated mice. The validation of the differentially expressed genes (DEGs) was analyzed with real-time PCR. RESULTS: At the 24th hour after challenge, the clinical score of the LPS group was significantly higher (3.83±0.75, mean ± standard deviation [SD]) than that of the control group (0.08±0.20, mean ± SD; p<0.001). The histological evaluation showed a large number of inflammatory cells infiltrated into the vitreous cavity in the LPS group compared with the control group. A total of 478 DEGs were identified with RNA-seq. Among these genes, 406 were upregulated and 72 were downregulated in the LPS group. Gene Ontology (GO) enrichment showed three significantly enriched upregulated terms. Twenty-one upregulated and seven downregulated pathways were remarkably enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Eleven inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes were validated to show similar results as the RNA-seq. CONCLUSIONS: We first reported the retinal transcriptome profile of the EIU mouse with RNA-seq. The results indicate that the abnormal changes in the inflammatory response-, complement system-, fibrinolytic system-, and cell stress-related genes occurred concurrently in EIU. These genes may play an important role in the pathogenesis of EIU. This study will lead to a better understanding of the underlying mechanisms and shed light on discovering novel therapeutic targets for ocular inflammation.
Subject(s)
Gene Expression Profiling , Genome , High-Throughput Nucleotide Sequencing , Retina/metabolism , Retina/pathology , Uveitis/genetics , Animals , Anterior Chamber/metabolism , Anterior Chamber/pathology , Cluster Analysis , Down-Regulation/genetics , Endotoxins , Female , Gene Ontology , Inflammation/pathology , Lipopolysaccharides , Mice, Inbred BALB C , Real-Time Polymerase Chain Reaction , Reproducibility of Results , Up-Regulation/genetics , Uveitis/pathologyABSTRACT
Cohesin enables post-replicative DNA repair and chromosome segregation by holding sister chromatids together from the time of DNA replication in S phase until mitosis. There is growing evidence that cohesin also forms long-range chromosomal cis-interactions and may regulate gene expression in association with CTCF, mediator or tissue-specific transcription factors. Human cohesinopathies such as Cornelia de Lange syndrome are thought to result from impaired non-canonical cohesin functions, but a clear distinction between the cell-division-related and cell-division-independent functions of cohesion--as exemplified in Drosophila--has not been demonstrated in vertebrate systems. To address this, here we deleted the cohesin locus Rad21 in mouse thymocytes at a time in development when these cells stop cycling and rearrange their T-cell receptor (TCR) α locus (Tcra). Rad21-deficient thymocytes had a normal lifespan and retained the ability to differentiate, albeit with reduced efficiency. Loss of Rad21 led to defective chromatin architecture at the Tcra locus, where cohesion-binding sites flank the TEA promoter and the Eα enhancer, and demarcate Tcra from interspersed Tcrd elements and neighbouring housekeeping genes. Cohesin was required for long-range promoter-enhancer interactions, Tcra transcription, H3K4me3 histone modifications that recruit the recombination machinery and Tcra rearrangement. Provision of pre-rearranged TCR transgenes largely rescued thymocyte differentiation, demonstrating that among thousands of potential target genes across the genome, defective Tcra rearrangement was limiting for the differentiation of cohesin-deficient thymocytes. These findings firmly establish a cell-division-independent role for cohesin in Tcra locus rearrangement and provide a comprehensive account of the mechanisms by which cohesin enables cellular differentiation in a well-characterized mammalian system.
Subject(s)
Cell Cycle Proteins/metabolism , Cell Differentiation , Chromosomal Proteins, Non-Histone/metabolism , Gene Rearrangement, T-Lymphocyte , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Thymus Gland/cytology , Animals , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/deficiency , Chromosomal Proteins, Non-Histone/genetics , DNA-Binding Proteins , Gene Expression Regulation , Gene Rearrangement, T-Lymphocyte/genetics , Genes, RAG-1/genetics , Mice , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phosphoproteins/deficiency , Phosphoproteins/genetics , Recombinases/metabolism , Thymus Gland/metabolism , Transcription, Genetic , CohesinsABSTRACT
Abnormal cardiac development has been observed in individuals with Cornelia de Lange syndrome (CdLS) due to mutations in genes encoding members of the cohesin complex. However, the precise role of cohesin in heart development remains elusive. In this study, we aimed to elucidate the indispensable role of SMC3, a component of the cohesin complex, in cardiac development and its underlying mechanism. Our investigation revealed that CdLS patients with SMC3 mutations have high rates of congenital heart disease (CHD). We utilized heart-specific Smc3-knockout (SMC3-cKO) mice, which exhibit varying degrees of outflow tract (OFT) abnormalities, to further explore this relationship. Additionally, we identified 16 rare SMC3 variants with potential pathogenicity in individuals with isolated CHD. By employing single-nucleus RNA sequencing and chromosome conformation capture high-throughput genome-wide translocation sequencing, we revealed that Smc3 deletion downregulates the expression of key genes, including Ets2, in OFT cardiac muscle cells by specifically decreasing interactions between super-enhancers (SEs) and promoters. Notably, Ets2-SE-null mice also exhibit delayed OFT development in the heart. Our research revealed a novel role for SMC3 in heart development via the regulation of SE-associated genes, suggesting its potential relevance as a CHD-related gene and providing crucial insights into the molecular basis of cardiac development.
ABSTRACT
Murine Tcra and Tcrd gene segments are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4(-)CD8(-) double-negative (DN) thymocytes to assemble Tcrd genes and in CD4(+)CD8(+) double-positive thymocytes to assemble Tcra genes. Recombination events are regulated by two developmental stage-specific enhancers, E(δ) and E(α). Effects of E(α) on Trca/Tcrd locus chromatin have been well documented, but effects of E(δ) have not. In this regard, E(α) acts over long distances to activate many V(α) and J(α) segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E(δ) functions over long distances to regulate V(δ) gene segments or functions only locally to regulate D(δ) and J(δ) gene segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E(δ)-deficient alleles in adult and fetal thymocytes. We found that E(δ) functions as a local enhancer whose influence is limited to no more than â¼10 kb in either direction (including D(δ), J(δ), and TRDV5 gene segments) in adult DN thymocytes. However, we identified a unique long-distance role for E(δ) promoting accessibility and recombination of fetal V(δ) gene segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.
Subject(s)
Enhancer Elements, Genetic/genetics , Gene Expression Regulation/genetics , Gene Rearrangement, delta-Chain T-Cell Antigen Receptor/genetics , Genes, T-Cell Receptor delta/genetics , Lymphopoiesis/genetics , T-Lymphocytes/immunology , Animals , Blotting, Southern , Chromatin/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation , Fetus , Gene Expression , Genes, T-Cell Receptor alpha/genetics , Histones/genetics , Histones/metabolism , In Situ Hybridization , Mice , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In CD4(-)CD8(-) double-negative thymocytes, the murine Tcrb locus is composed of alternating blocks of active and inactive chromatin containing Tcrb gene segments and trypsinogen genes, respectively. Although chromatin structure is appreciated to be critical for regulated recombination and expression of Tcrb gene segments, the molecular mechanisms that maintain the integrity of these differentially regulated Tcrb locus chromatin domains are not understood. We localized a boundary between active and inactive chromatin by mapping chromatin modifications across the interval extending from Prss2 (the most 3' trypsinogen gene) to D(ß)1. This boundary, located 6 kb upstream of D(ß)1, is characterized by a transition from repressive (histone H3 lysine 9 dimethylation [H3K9me2]) to active (histone H3 acetylation [H3ac]) chromatin and is marked by a peak of histone H3 lysine 4 dimethylation (H3K4me2) that colocalizes with a retroviral long terminal repeat (LTR). Histone H3 lysine 4 dimethylation is retained and histone H3 lysine 9 dimethylation fails to spread past the LTR even on alleles lacking the Tcrb enhancer (E(ß)) suggesting that these features may be determined by the local DNA sequence. Notably, we found that LTR-containing DNA functions as a barrier-type insulator that can protect a transgene from negative chromosomal position effects. We propose that, in vivo, the LTR blocks the spread of heterochromatin, and thereby helps to maintain the integrity of the E(ß)-regulated chromatin domain. We also identified low-abundance, E(ß)-dependent transcripts that initiate at the border of the LTR and an adjacent long interspersed element. We speculate that this transcription, which extends across D(ß), J(ß) and C(ß) gene segments, may play an additional role promoting initial opening of the E(ß)-regulated chromatin domain.
Subject(s)
Genes, T-Cell Receptor beta/immunology , Heterochromatin/metabolism , Insulator Elements/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Transcriptional Activation/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , DNA Methylation/genetics , DNA Methylation/immunology , Enhancer Elements, Genetic/immunology , Heterochromatin/genetics , Histones/genetics , Histones/metabolism , Humans , Insulator Elements/genetics , Jurkat Cells , Mice , Mice, Knockout , Mice, Transgenic , POU Domain Factors/deficiency , POU Domain Factors/genetics , POU Domain Factors/metabolism , Protein Structure, Tertiary/genetics , T-Lymphocyte Subsets/cytology , Trypsinogen/antagonists & inhibitors , Trypsinogen/geneticsABSTRACT
Trisomy 21, or Down syndrome (DS), is the most frequent human autosomal chromosome aneuploidy, which leads to multiple developmental disorders, especially mental retardation in individuals. The presence of an additional human chromosome 21 (HSA21) could account for the pathological manifestations in DS. In this study, we analyzed the mRNA gene expression profile of DS-derived amniocytes compared with normal amniocytes, aiming to evaluate the relationship between candidate dysregulated HSA21 genes and DS developmental phenotypes. Differentially expressed genes (DEGs) included 1794 upregulated genes and 1411 downregulated genes, which are mainly involved in cell adhesion, inflammation, cell proliferation and thus may play an important role in inducing multiple dysplasia during DS fetal development. Furthermore, STRING protein network studies demonstrated 7 candidate HSA21 genes participated Gene Ontology (GO) terms: cell adhesion and extracellular matrix remodeling (COL6A1, COL6A2, COL18A1, ADAMTS5, JAM2, and POFUT2), inflammation and virus infection response (MX1 and MX2), histone modification and chromatin remodeling (NRIP1), glycerolipid and glycerophospholipid metabolism (AGPAT3), mitochondrial function (ATP5PF and ATP5PO), synaptic vesicle endocytosis (ITSN1 and SYNJ1) and amyloid metabolism (APP). Meanwhile, GSEA enrichment identified several transcription factors and miRNAs, which may target gene expression in the DS group. Our study established connections between dysregulated genes, especially HSA21 genes, and DS-associated phenotypes. The alteration of multiple pathways and biological processes may contribute to DS developmental disorders, providing potential pathogenesis and therapeutic targets for DS.