Chromatin Remodeling and Gene Activation
染色质重塑和基因激活
基本信息
- 批准号:10691792
- 负责人:
- 金额:$ 128.34万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:AffectAffinityBase PairingBindingBinding SitesCHD1 geneCell NucleusCellsChIP-seqChildhoodChromatinChromatin ModelingChromatin Remodeling FactorChromatin StructureCodeCollaborationsComplexConsensusConsensus SequenceDNADNA BindingDNA SequenceDataDiseaseEnsureEnzymesEpigenetic ProcessEukaryotic CellEventExcisionFrequenciesGene ActivationGene ExpressionGene Expression RegulationGenesGenetic TranscriptionGenomeGenomic DNAHMGN ProteinsHistonesISWIIn VitroIncubatedLinkMalignant NeoplasmsMapsMeasurementMethodsModelingMusMutateMutationNational Institute of Child Health and Human DevelopmentNucleosomesOrganismPharmaceutical PreparationsPlayPopulationProbabilityPropertyPublishingPurinesPyrimidineRNA Polymerase IIRegulatory ElementRepressionRhabdoid TumorRoleSWI/SNF Family ComplexSaccharomycetalesSequence-Specific DNA Binding ProteinSignal TransductionSiteStructureSystemTestingTranscriptTranscription CoactivatorYeastsattenuationautism spectrum disorderbasechromatin remodelinggenome-widehigh throughput technologyhuman diseasein vivoinsightinterestpreventpromoterrecruitresponsetranscription factorwoundyeast genome
项目摘要
Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II and transcription. These events coincide with the removal of promoter nucleosomes to create a nucleosome-depleted region (NDR). This observation has led to the generally accepted model that promoter nucleosomes physically block transcript initiation, acting as repressors by preventing access to specific transcription factor binding sites. The nucleosome is a very stable structure containing tightly wound DNA that is largely inaccessible to sequence-specific DNA binding proteins. Activation occurs if sequence-specific 'pioneer' transcription factors are present (these proteins bind nucleosomal sites with high affinity), and/or if 'classical' transcription factors, which are normally blocked by nucleosomes, recruit ATP-dependent chromatin remodelers to move or evict promoter nucleosomes, thus facilitating initiation complex formation.
The ATP-dependent chromatin remodelers variously move nucleosomes along DNA, or remove the histones altogether, or form arrays of regularly spaced nucleosomes. Examples include the SWI/SNF and RSC complexes, which remodel nucleosomes on genes and at promoters, and the CHD and ISWI complexes, some of which are involved in determining nucleosome spacing. The INO80C complex is unusual because it appears to have both properties. Human diseases have been linked to chromatin remodeling enzymes. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors, and the CHD remodelers have been linked to cancer and autism. Therapies and drugs aimed at epigenetic targets are being tested. Thus, a full understanding of chromatin structure and the mechanisms by which it is manipulated is vital.
Although the main effort of the lab is focused on the role of the ATP-dependent chromatin remodeling enzymes in gene regulation, with particular emphasis on how they may control the accessibility of genomic DNA, we are also interested in the central role of sequence-specific transcription factors. Transcription factors usually recognize consensus DNA binding sites, mostly containing 4 to 12 base pairs, and in which some bases may be degenerate (e.g., 'Y' for pyrimidine (C or T) or 'R' for purine (A or G)). The probability of finding such a site in the genome is often much higher than the number of actual binding sites detected empirically using methods such as ChIP-seq. Such consensus sites occur not only in regulatory elements, but also inside genes and elsewhere, where they may or may not be functional. The observation that consensus sites often predict far more transcription factor binding sites than are actually bound in vivo has led to the proposal that consensus sites in non-regulatory regions are not bound because they are blocked by chromatin. However, our recent measurements of DNA accessibility in yeast and mouse nuclei imply that all consensus sites are likely to be accessible in some cells within a population. This general but limited accessibility predicts detectable binding at all consensus sites, albeit reduced relative to sites in nucleosome-free DNA. If true, the hypothesis that chromatin prevents binding by transcription factors to consensus sites outside nucleosome-depleted regions is questionable.
We have explored an alternative explanation: that consensus site sequences derived from ChIP-seq data may be too degenerate in some cases, such that only a subset of the predicted sites are true sites. We investigated this possibility using the well-studied yeast Gcn4 transcription factor as a model (1). Previously, we published ChIP-seq data for Gcn4 in a collaboration with the Hinnebusch Lab (NICHD). In that study, we derived a consensus sequence for Gcn4 binding, of which there are 1754 instances in the yeast genome, but only 546 show detectable Gcn4 binding in vivo. To resolve this discrepancy, it is necessary to determine which sites are bound by Gcn4 in the absence of the potential blocking effect of chromatin (i.e., using purified DNA in vitro). Accordingly, we developed a modified SELEX method to identify all of the sites bound by Gcn4 in the yeast genome, which we termed 'G-SELEX'. We used short genomic DNA fragments and purified Gcn4 attached to beads to select DNA fragments containing a Gcn4-bound site. The bound DNA was amplified and incubated with Gcn4 again in a second round of selection; three rounds of selection were performed in total. The final bound product was subjected to paired-end sequencing. The DNA fragments were mapped to the yeast genome to produce a very high quality coverage map. We identified 2,359 Gcn4-bound sites, but most were bound at very low frequency, corresponding to Gcn4 half-sites. In contrast, the major peaks (high-affinity sites) corresponded to the 7-bp sequence TGACTCA. However, of the 1078 instances of this sequence in the yeast genome, less than half are bound in vitro or in vivo. Further analysis revealed that the bound sites conform to a more extensive consensus: RTGACTCAY, such that RTGACTCAR or YTGACTCAY sites are bound only weakly, and YTGACTCAR sites are not bound at all. We conclude that the high-affinity site (RTGACTCAY) essentially accounts for Gcn4 binding in vitro and in vivo, irrespective of whether the site is located in a nucleosome-depleted promoter or inside a gene assembled into nucleosomes.
More generally, we propose that transcription factor binding sites need to be defined more precisely using quantitative data, which should result in more accurate genome-wide prediction of real binding sites and greater insight into gene regulation. Overall, this study, together with our previous studies, suggest that the prevailing model that chromatin is a general block to gene expression unless specific transcription activators are present may be incorrect. Our current studies are aimed at resolving this issue.
基因激活涉及到一系列因子被募集到启动子中,以响应适当的信号,最终通过RNA聚合酶II和转录形成起始复合物。这些事件与启动子核小体的去除产生核小体耗尽区(NDR)相吻合。这一观察结果导致了普遍接受的模型,即启动子核小体物理上阻断转录起始,通过阻止进入特定的转录因子结合位点而起到抑制因子的作用。核小体是一种非常稳定的结构,含有紧密缠绕的DNA,序列特异性DNA结合蛋白在很大程度上是无法接近的。如果序列特异性的“先锋”转录因子存在(这些蛋白质以高亲和力结合核小体位点),和/或通常被核小体阻断的“经典”转录因子招募依赖atp的染色质重塑子移动或驱逐启动子核小体,从而促进起始复合物的形成,就会发生激活。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
david j clark其他文献
david j clark的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('david j clark', 18)}}的其他基金
相似海外基金
Construction of affinity sensors using high-speed oscillation of nanomaterials
利用纳米材料高速振荡构建亲和传感器
- 批准号:
23H01982 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Grant-in-Aid for Scientific Research (B)
Affinity evaluation for development of polymer nanocomposites with high thermal conductivity and interfacial molecular design
高导热率聚合物纳米复合材料开发和界面分子设计的亲和力评估
- 批准号:
23KJ0116 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Grant-in-Aid for JSPS Fellows
Platform for the High Throughput Generation and Validation of Affinity Reagents
用于高通量生成和亲和试剂验证的平台
- 批准号:
10598276 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Development of High-Affinity and Selective Ligands as a Pharmacological Tool for the Dopamine D4 Receptor (D4R) Subtype Variants
开发高亲和力和选择性配体作为多巴胺 D4 受体 (D4R) 亚型变体的药理学工具
- 批准号:
10682794 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
- 批准号:
2233343 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Standard Grant
Collaborative Research: DESIGN: Co-creation of affinity groups to facilitate diverse & inclusive ornithological societies
合作研究:设计:共同创建亲和团体以促进多元化
- 批准号:
2233342 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Standard Grant
Molecular mechanisms underlying high-affinity and isotype switched antibody responses
高亲和力和同种型转换抗体反应的分子机制
- 批准号:
479363 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Operating Grants
Deconstructed T cell antigen recognition: Separation of affinity from bond lifetime
解构 T 细胞抗原识别:亲和力与键寿命的分离
- 批准号:
10681989 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
CAREER: Engineered Affinity-Based Biomaterials for Harnessing the Stem Cell Secretome
职业:基于亲和力的工程生物材料用于利用干细胞分泌组
- 批准号:
2237240 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Continuing Grant
ADVANCE Partnership: Leveraging Intersectionality and Engineering Affinity groups in Industrial Engineering and Operations Research (LINEAGE)
ADVANCE 合作伙伴关系:利用工业工程和运筹学 (LINEAGE) 领域的交叉性和工程亲和力团体
- 批准号:
2305592 - 财政年份:2023
- 资助金额:
$ 128.34万 - 项目类别:
Continuing Grant