Nucleosome positioning and transcriptional regulation in Drosophila differentiated cells

果蝇分化细胞中的核小体定位和转录调控

• 批准号: BB/P001564/1
• 负责人: Helen White-Cooper
• 金额: $ 62.24万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FP001564%2F1
• 关键词: Nucleosome; positioning; transcriptional; regulation; Drosophila

Each cell in a multicellular animal expresses (transcribes) only about half of all its genes. Some genes are transcribed in all cells, some in a subset of cell-types, while some are transcribed only in a single cell type. Correct gene expression in cells, both turning on and turning off genes in development and differentiation, is essential for normal cellular function, for normal organism development, and for lifelong health. The gene expression repertoire of each individual cell is determined by its identity, and is established during development and cellular differentiation. 10-20% of all the genes in the genome are transcribed exclusively in testes, in cells destined to differentiate into sperm; 1-2% of all genes are transcribed exclusively in muscle cells. Regulation of gene transcription involves several processes working in concert - notably DNA packaging and sequence-specific DNA-binding factors, which then recruit the transcription machinery. The DNA in the cell is packaged with proteins to form chromatin. The fundamental packing unit is a nucleosome - protein complexes around which the DNA strand is wrapped, forming a beads-on-a-string structure. The precise position of each nucleosome is not determined by the DNA sequence directly and varies between cell-types. On average, in most studied cell-types, nucleosomes are found in a canonical pattern positioned around genes, particularly in the region of DNA where transcription of the gene starts (the transcription start site, TSS). This positioning of nucleosomes is thought important for allowing access of proteins that bind specific DNA sequences and regulate transcription of the adjacent gene. On average, genes that have higher expression levels have more accurately positioned nucleosomes. However, the studies that have led to this view have used tissue culture cells or mixed cell populations. Differentiated cells have not been examined.We have extensive preliminary data to indicate that many genes transcribed at extremely high levels in spermatocytes, the cells that give rise to sperm, do not follow these established rules regarding nucleosome positioning. These data cast doubt on the conclusion that high gene expression and canonical nucleosome positioning are necessarily linked. They also raise the question of how spermatocytes achieve a high level of transcription of these genes, and whether other cell-types also deviate from established rules. These genes rely on a specific transcription factor complex, termed testis meiotic arrest complex (TMAC); we have found that nucleosome positioning at TMAC-target TSSs is altered when this complex is mutated.In this project we have three main aims1) To characterise the nucleosome positioning at TSSs in spermatogonia, the precursors of spermatocytes to reveal how the positioning of nucleosomes changes as genes are turned on and off in this lineage. We will further determine the effect of loss of specific nucleosome positioning factors and transcriptional regulators at these sites. We will map the binding sites of TMAC and other regulators across the genome in these cells.2) To examine whether the unusual pattern of nucleosome position we see for testis-specific gene expression is also seen in other cell-type specific gene expression programmes, or whether the canonical pattern is used in this situation. In either case we will determine how the pattern changes in differentiation. We will analyse nucleosome position around TSSs in adult muscle cells, and their immediate precursor cells.3) To understand the molecular details of how high levels of gene expression are driven in the absence of canonical nucleosome positioning at TSSs in spermatocytes we will characterise the DNA sequences responsible for the testis-specific expression. We will determine how they interact with TMAC and how they influence local nucleosome position in their normal location and when we move them to a new position in the genome.

多细胞动物中的每个细胞只表达(转录)其全部基因的大约一半。有些基因在所有细胞中转录，有些在细胞类型的子集中转录，而有些基因只在一种细胞类型中转录。细胞中正确的基因表达，包括启动和关闭发育和分化过程中的基因，对于正常的细胞功能、正常的生物体发育和终身健康都是必不可少的。每个单个细胞的基因表达谱由其特性决定，并在发育和细胞分化过程中建立。基因组中10%-20%的基因仅在睾丸中转录，在注定要分化为精子的细胞中转录；所有基因中有1%-2%仅在肌肉细胞中转录。基因转录的调控涉及几个协同工作的过程--特别是DNA包装和序列特异性DNA结合因子，然后招募转录机器。细胞中的DNA与蛋白质一起包装，形成染色质。基本的包装单元是核小体-蛋白质复合体，DNA链被包裹在它周围，形成串上珠子的结构。每个核小体的确切位置不是由DNA序列直接决定的，而是随着细胞类型的不同而不同。平均而言，在大多数所研究的细胞类型中，核小体以规范的模式定位在基因周围，特别是在基因转录开始的DNA区域(转录起始点，TSS)。核小体的这种定位被认为是重要的，因为它允许结合特定DNA序列的蛋白质的访问，并调节邻近基因的转录。平均而言，表达水平较高的基因具有更准确的核小体定位。然而，导致这一观点的研究使用了组织培养细胞或混合细胞群体。分化的细胞还没有被检测。我们有大量的初步数据表明，在精母细胞中，许多基因转录水平极高，产生精子的细胞，并不遵循这些关于核小体定位的既定规则。这些数据对高基因表达和核小体定位必然相关的结论提出了质疑。他们还提出了一个问题，即精母细胞如何实现这些基因的高水平转录，以及其他类型的细胞是否也偏离了既定的规则。这些基因依赖于一种特定的转录因子复合体，称为睾丸减数分裂停滞复合体(TMAC)；我们发现，当该复合体发生突变时，TMAC靶标TSS处的核小体定位会发生变化。在这个项目中，我们有三个主要目的1)描述精原细胞中TSS处的核小体定位，精原细胞是精母细胞的前体，以揭示当基因在这一谱系中打开和关闭时，核小体的定位如何变化。我们将进一步确定特定的核小体定位因子和转录调控因子在这些位置丢失的影响。我们将绘制TMAC和其他调控因子在这些细胞基因组中的结合位置图。2)检查我们看到的睾丸特异性基因表达的核小体位置的异常模式是否也在其他细胞类型特异性基因表达程序中看到，或者典型模式是否在这种情况下使用。在任何一种情况下，我们都将确定这种模式在分化中如何变化。我们将分析成年肌肉细胞及其直接前体细胞中TSS周围的核小体位置。3)为了了解在精母细胞中TSS缺乏规范的核小体定位时，基因高水平表达是如何驱动的分子细节，我们将描述负责睾丸特异性表达的DNA序列。我们将确定它们如何与TMAC相互作用，它们如何影响正常位置的局部核小体位置，以及当我们将它们移动到基因组中的新位置时。

项目成果

Emergent dynamics of adult stem cell lineages from single nucleus and single cell RNA-Seq of Drosophila testes.

• DOI: 10.7554/elife.82201
• 发表时间: 2023-02-16
• 期刊: ELIFE
• 影响因子: 7.7
• 作者: Raz, Amelie A.;Vida, Gabriela S.;Stern, Sarah R.;Mahadevaraju, Sharvani;Fingerhut, Jaclyn M.;Viveiros, Jennifer M.;Pal, Soumitra;Grey, Jasmine R.;Grace, Mara R.;Berry, Cameron W.;Li, Hongjie;Janssens, Jasper;Saelens, Wouter;Shao, Zhantao;Hu, Chun;Yamshita, Yukiko M.;Przytycka, Teresa;Oliver, Brian;Brill, Julie A.;Krause, Henry;Matunis, Erika L.;White-Cooper, Helen;DiNardo, Stephen;Fuller, Margaret T.;Buszczak, Michael
• 通讯作者: Buszczak, Michael

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly.

• DOI: 10.1126/science.abk2432
• 发表时间: 2022-03-04
• 期刊: Science (New York, N.Y.)
• 作者: Li H;Janssens J;De Waegeneer M;Kolluru SS;Davie K;Gardeux V;Saelens W;David FPA;Brbić M;Spanier K;Leskovec J;McLaughlin CN;Xie Q;Jones RC;Brueckner K;Shim J;Tattikota SG;Schnorrer F;Rust K;Nystul TG;Carvalho-Santos Z;Ribeiro C;Pal S;Mahadevaraju S;Przytycka TM;Allen AM;Goodwin SF;Berry CW;Fuller MT;White-Cooper H;Matunis EL;DiNardo S;Galenza A;O'Brien LE;Dow JAT;FCA Consortium§;Jasper H;Oliver B;Perrimon N;Deplancke B;Quake SR;Luo L;Aerts S;Agarwal D;Ahmed-Braimah Y;Arbeitman M;Ariss MM;Augsburger J;Ayush K;Baker CC;Banisch T;Birker K;Bodmer R;Bolival B;Brantley SE;Brill JA;Brown NC;Buehner NA;Cai XT;Cardoso-Figueiredo R;Casares F;Chang A;Clandinin TR;Crasta S;Desplan C;Detweiler AM;Dhakan DB;Donà E;Engert S;Floc'hlay S;George N;González-Segarra AJ;Groves AK;Gumbin S;Guo Y;Harris DE;Heifetz Y;Holtz SL;Horns F;Hudry B;Hung RJ;Jan YN;Jaszczak JS;Jefferis GSXE;Karkanias J;Karr TL;Katheder NS;Kezos J;Kim AA;Kim SK;Kockel L;Konstantinides N;Kornberg TB;Krause HM;Labott AT;Laturney M;Lehmann R;Leinwand S;Li J;Li JSS;Li K;Li K;Li L;Li T;Litovchenko M;Liu HH;Liu Y;Lu TC;Manning J;Mase A;Matera-Vatnick M;Matias NR;McDonough-Goldstein CE;McGeever A;McLachlan AD;Moreno-Roman P;Neff N;Neville M;Ngo S;Nielsen T;O'Brien CE;Osumi-Sutherland D;Özel MN;Papatheodorou I;Petkovic M;Pilgrim C;Pisco AO;Reisenman C;Sanders EN;Dos Santos G;Scott K;Sherlekar A;Shiu P;Sims D;Sit RV;Slaidina M;Smith HE;Sterne G;Su YH;Sutton D;Tamayo M;Tan M;Tastekin I;Treiber C;Vacek D;Vogler G;Waddell S;Wang W;Wilson RI;Wolfner MF;Wong YE;Xie A;Xu J;Yamamoto S;Yan J;Yao Z;Yoda K;Zhu R;Zinzen RP
• 通讯作者: Zinzen RP

Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila.

• DOI: 10.1093/g3journal/jkaa046
• 发表时间: 2021-01-18
• 期刊: G3 (Bethesda, Md.)
• 作者: van der Graaf K;Jindrich K;Mitchell R;White-Cooper H
• 通讯作者: White-Cooper H