Regulation of chromatin dynamics
染色质动力学的调节
基本信息
- 批准号:10172921
- 负责人:
- 金额:$ 94.46万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2017
- 资助国家:美国
- 起止时间:2017-06-01 至 2022-05-31
- 项目状态:已结题
- 来源:
- 关键词:AcetylationAddressAffectAreaBiochemicalBiological AssayBoundary ElementsBudgetsCell divisionCentromereChromatinChromatin FiberChromosome StructuresChromosomesComplementComplexDNADNA Double Strand BreakDNA RepairDNA Sequence AlterationDNA biosynthesisDataDefectDepositionDevelopmentDiseaseDouble Strand Break RepairEnzymesEventFiberFluorescenceGene DeletionGene ExpressionGenesGeneticGenetic TranscriptionGenomeGenome StabilityGenomic InstabilityGoalsHeterochromatinHigher Order Chromatin StructureHistone H3HistonesIn VitroIntercistronic RegionLeadLysineMalignant NeoplasmsMammalsNucleosomesPathway interactionsPlayPrincipal InvestigatorProcessProteinsRNA Polymerase IIReactionRecombinantsRegulationReplication OriginResearchRoleS PhaseStructureSystemTestingUntranslated RNAVariantYeastsbasechromatin remodelingdimerexperimental studygenetic analysisgenome-widehistone acetyltransferasehomologous recombinationin vivomutantnovelpreventprogramspromoterreconstitutionsedimentation velocitystem cell functionstem cellsstoichiometryvirtual
项目摘要
Program Director/Principal Investigator (Last, First, Middle): Peterson, Craig, Lewis
The overall objective of our research is to determine how chromosome structure influences gene
transcription, DNA replication and repair, with special emphasis on identifying and characterizing the chromatin
remodeling machines that control chromosome dynamics. Notably, genetic experiments have revealed ATP-
dependent chromatin remodeling enzymes as essential regulators of virtually every chromosomal process, and
their dysregulation leads to a variety of diseases, including cancer. Our research efforts can be organized
into three inter-related areas: (1) Mechanistic studies of ATP-dependent chromatin remodeling enzymes;; (2)
Role of chromatin dynamics in genome stability pathways;; and (3) Assembly/function of chromatin higher order
structures. A major focus of our mechanistic studies is to continue to dissect the structure and
biochemical mechanisms of the INO80C and SWR1C enzymes. These remodeling enzymes catalyze
novel, ATP-dependent histone exchange events that control the deposition and distribution of the H2A.Z
histone variant within nucleosomes that flank promoters of genes transcribed by RNA polymerase II, as well as
nucleosomes that flank chromatin boundary elements, centromeres, and replication origins. Mammalian
homologs of SWR1C and INO80C, including the p400/Tip60 and hINO80 complexes, are key for proper stem
cell function, genome stability, development, and gene expression. During the past budget period, we identified
a novel regulatory interaction between SWR1C and the acetylation of lysine 56 of histone H3 (H3-K56Ac) that
regulates nucleosome dynamics, noncoding RNA expression, and assembly of large-scale, chromosome
interaction domains (CIDs) that are related to mammalian topologically-associated domains (TADs). These
mechanistic studies will include quantitative, fluorescence-based assays to define steps of the histone dimer
exchange reaction, as well as the reconstitution of these multi-subunit enzymes with recombinant subunits.
Studies from us and others over the past 10 years have demonstrated that chromatin dynamics play a large
role in stabilizing the replisome and in controlling various steps in DNA double strand break repair. Our recent
data suggests that changes in chromatin dynamics can also lead to dysregulation of transcription which
impacts genome stability pathways. Our research will address several key unanswered questions
focused on genome stability pathways: (1) Do Remodelers regulate the homology search step of
homologous recombination and do they function in concert with histone acetylases? (2) How does INO80C
stabilize the replisome and is this role due to the regulation of ncRNA expression? (3) How does INO80C
prevent ncRNA expression from intergenic regions? (4) Does the hyperacetylation of H3-K56Ac lead to
formation of R-loops that disrupt replisome function? (5) Does hypoacetylation of H3-K56Ac and the resulting
defect in nucleosome assembly also lead to aberrant transcription during S phase that leads to genome
instability? We plan to continue to exploit a combination of in vitro and in vivo approaches to address such
questions.
Mechanistic studies of Remodelers have primarily focused on single nucleosome substrates or simple
nucleosomal arrays. In vivo, these enzymes likely target nucleosomes within the context of condensed
chromatin fibers. Recently, we used a combination of sedimentation velocity analyses and AFM to dissect the
stoichiometry and solution dynamics of a simple form of yeast heterochromatin that contains the primary
structural component, Sir3. We plan to extend such studies with chromatin fibers reconstituted with a complete
complement of Sir proteins (Sir2/3/4). The overall goal will be to characterize the structure of yeast
heterochromatin fibers as well as understanding how these structures can be modulated by
Remodelers. A second type of higher order chromosome structure occurs throughout the genome. CIDs
contain strongly self-associating nucleosomes that span ~1-5 genes, separated by distinct boundary regions.
Little is known about what controls CID or TAD assembly or what functional roles they play. One possibility is
that domains of self-associating nucleosomes assemble spontaneously adjacent to nucleosome depleted
regions (NDRs), and mutants that disrupt CIDs either affect the efficiency of NDR formation or increase
transcription through CIDs. One of our goals is to use a genome-wide, nucleosome reconstitution system
to directly test whether formation of promoter-associated, nucleosome-depleted regions is sufficient
for CID assembly in vitro. These in vitro studies will also be complemented by genetic analyses of CID
assembly, where we will either eliminate key factors by gene deletion, or manipulate specific CID boundary
regions by DNA alterations. In the long term, understanding how CIDs are assembled will facilitate studies to
disrupt this process and investigate the functional consequences on both transcription and genome stability.
OMB No. 0925-0001/0002 (Rev. 08/12 Approved Through 8/31/2015) Page Continuation Format Page
项目负责人/主要研究者(最后一名、第一名、中间名):Peterson、克雷格、刘易斯
我们研究的总体目标是确定染色体结构如何影响基因
转录,DNA复制和修复,特别强调识别和表征染色质
重塑控制染色体动力学的机器。值得注意的是,遗传实验已经揭示了ATP酶
依赖性染色质重塑酶作为几乎所有染色体过程的基本调节剂,
它们的失调会导致各种疾病,包括癌症。我们的研究工作可以组织起来,
分为三个相互关联的领域:(1)ATP依赖的染色质重塑酶的机制研究;
染色质动力学在基因组稳定性途径中的作用;(3)染色质高阶的组装/功能
结构。我们机械研究的一个主要重点是继续剖析结构,
INO 80 C和SWR 1C酶的生化机制。这些重塑酶催化
新的ATP依赖性组蛋白交换事件,控制H2A.Z的沉积和分布,
位于RNA聚合酶II转录基因启动子侧翼核小体内的组蛋白变体,以及
位于染色质边界元件、着丝粒和复制起点两侧的核小体。
SWR 1C和INO 80 C的同源物,包括p400/Tip 60和hINO 80复合物,是适当茎的关键,
细胞功能、基因组稳定性、发育和基因表达。在过去的预算期间,我们确定了
SWR 1C和组蛋白H3的赖氨酸56的乙酰化(H3-HPK 56 Ac)之间的一种新的调节相互作用,
调节核小体动力学、非编码RNA表达和大规模染色体组装
这些相互作用结构域(CID)与哺乳动物拓扑结构相关结构域(TADs)相关。
机制研究将包括定量的、基于荧光的分析,以确定组蛋白二聚体的步骤。
交换反应,以及这些多亚基酶与重组亚基的重组。
我们和其他人在过去10年中的研究表明,染色质动力学在细胞凋亡中起着重要作用。
在稳定复制体和控制DNA双链断裂修复的各个步骤中起作用。
数据表明,染色质动力学的变化也可导致转录的失调,
影响基因组稳定性途径。我们的研究将解决几个关键的悬而未决的问题
(1)重塑者是否调节基因组的同源性搜索步骤?
同源重组和他们的功能与组蛋白乙酰化酶? (2)INO 80 C如何
稳定复制体,这种作用是由于ncRNA表达的调节吗? (3)INO 80 C如何
阻止基因间区域的ncRNA表达? (4)H3-PK 56 Ac的超乙酰化是否导致
形成破坏复制体功能的R-β环? (5)H3-PK 56 Ac的低乙酰化和所得的
核小体组装缺陷也导致S期异常转录,导致基因组
不稳定? 我们计划继续利用体外和体内方法的组合来解决这种问题。
问题.
重塑物的机制研究主要集中在单个核小体底物或简单的核小体底物上。
在体内,这些酶可能靶向核小体的背景下浓缩的
最近,我们使用沉降速度分析和AFM的组合来解剖染色质纤维。
化学计量学和溶液动力学的一个简单形式的酵母异染色质,其中包含主要的
我们计划扩展这样的研究与染色质纤维重建与一个完整的
补充爵士蛋白(Sir 2/3/4)。总体目标将是表征酵母的结构
异染色质纤维,以及了解这些结构如何可以调节
第二种更高级的染色体结构出现在整个基因组中。
含有跨越约1- 105个基因的强自相关核小体,由不同的边界区域分开。
关于是什么控制CID或CID组装或它们扮演什么功能角色,我们知之甚少。
自组装核小体的结构域在核小体耗尽的附近自发组装,
破坏CID的突变体影响NDR形成的效率或增加
我们的目标之一是使用全基因组范围的核小体重建系统,
直接测试启动子相关的核小体缺失区的形成是否足够
这些体外研究还将通过CID的遗传分析来补充
组装,其中我们将通过基因缺失消除关键因素,或操纵特定的CID边界
从长远来看,了解CID是如何组装的将有助于研究
破坏这一过程,并研究对转录和基因组稳定性的功能后果。
OMB编号0925- 0001/0002(2012年8月修订版,批准至2015年8月31日)页码 续页格式
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)
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Craig L Peterson其他文献
A ubiquitin crowbar opens chromatin
泛素撬棍打开染色质
- DOI:
10.1038/nchembio.514 - 发表时间:
2011-01-18 - 期刊:
- 影响因子:13.700
- 作者:
Craig L Peterson - 通讯作者:
Craig L Peterson
NPGRJ_NSMB_1413 338..345
NPGRJ_NSMB_1413 338..345
- DOI:
- 发表时间:
2019 - 期刊:
- 影响因子:0
- 作者:
Manolis Papamichos;Craig L Peterson - 通讯作者:
Craig L Peterson
Craig L Peterson的其他文献
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{{ truncateString('Craig L Peterson', 18)}}的其他基金
ROLE OF HISTONE H3 AND H1 PHOSPHORYLATION ON CHROMATIN
组蛋白 H3 和 H1 磷酸化对染色质的作用
- 批准号:
6580356 - 财政年份:2002
- 资助金额:
$ 94.46万 - 项目类别:
SUBUNITS OF YEAST SWI & SNF COMPLEX ARE MEMBERS OF ACTIN RELATED PROTEIN
酵母 SWI 亚基
- 批准号:
6118267 - 财政年份:1998
- 资助金额:
$ 94.46万 - 项目类别:
Analysis of yeast chromatin structure and function
酵母染色质结构和功能分析
- 批准号:
7033550 - 财政年份:1997
- 资助金额:
$ 94.46万 - 项目类别:
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