DP1

KANAZAWA, Japan, Feb. 16, 2024 /PRNewswire/ -- In a study recently published in Nature Genetics, researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University explore chromatin accessibility, i.e., endogenous access pathways to the genomic DNA, and its use as a tool for gene editing. Our DNA is protected from unwanted external modifications by forming structures called nucleosomes that consist of threads of DNA wound around chunks of special proteins known as histones. This unique coiled shape prevents the access of undesirable molecules to a cell's DNA. However, for vital genetic functions—such as DNA repair—the right set of proteins require access to these DNA fragments. This phenomenon known as 'chromatin accessibility' involves a privileged set of protein molecules, many of which are still unknown. Now, researchers from Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, led by Yusuke Miyanari, have used advanced genetic screening methods to unravel chromatin accessibility and its pathways. For the investigation the team used a combination of two technologies—CRISPR screening and ATAC-see. While the former is a method to suppress the function of a desired set of genes, the latter is a means to identify which ones are essential for chromatin accessibility. Thus, using this method all genes playing a crucial role in chromatin accessibility could be pinned down. With the help of these assays, novel pathways and individual players involved in chromatin accessibility were uncovered—some playing a positive role and some negative. Of these, one particular protein, TFDP1, showed a negative effect on chromatin accessibility. When it was suppressed, a significant increase in chromatin accessibility was observed, accompanied by nucleosome reduction. A deeper dive into the mechanism of TFDP1 revealed that it functions by regulating the genes responsible for production of certain histone proteins. The team then focused their study towards exploring biotechnological applications of their findings. After suppressing TFDP1, two different approaches were tried. The first approach involved gene editing using the CRISPR/Cas9 tool. This revealed that deletion of TFDP1 made the gene editing process easier. Now, most chromatin accessibility occurs in nucleosome-depleted regions or NDRs. However, by suppressing TFDP1 chromatin accessibility occurred not only in NDRs but across other regions as well. Secondly, the depletion of TFDP1 aided the process of converting regular cells into stem cells, a massive step forward in cellular transformation. This study identified new chromatin accessibility pathways and channels for exploring its potential even further. "Our study shows the significant potential to manipulate chromatin accessibility as a novel strategy to enhance DNA-templated biological applications, including genome editing and cellular reprogramming," conclude the researchers. Background CRISPR screening: CRISPR screening is a large-scale tool used to find genes involved in a particular function. In this method, genes from the entire genome can be deleted one at a time to investigate which ones play a role in any target pathway. The mechanisms and roles of the genes narrowed down are then understood further using biological assays. In this study the genes identified by CRISPR screening were subjected to ATAC-see to confirm their involvement with chromatin accessibility. ATAC-see: Transposase-Accessible Chromatin with visualization (ATAC-see) is a technique whereby accessible DNA locations can be visualized and quantified. ATAC-see involves using an enzyme to insert fluorescent proteins into the DNA at accessible sites. This technique works within nucleosome-depleted regions but also beyond these regions. Hence, when such sites were visualized after running CRISPR screening, the researchers could understand which genes were affecting chromatin accessibility directly. Reference Satoko Ishii, Taishi Kakizuka, Sung-Joon Park, Ayako Tagawa, Chiaki Sanbo, Hideyuki Tanabe, Yasuyuki Ohkawa, Mahito Nakanishi, Kenta Nakai, Yusuke Miyanari. Genome-wide ATAC-see screening identifies TFDP1 as a modulator of global chromatin accessibility. Nature Genetics, 2024. Published Feb 15, 2024 DOI ：10.1038/s41588-024-01658-1 Related figures Figure 1. a. Screening results of the players involved in chromatin accessibility analyzed by ATAC-see. b. Successful reprogramming to stem cells (indicated by presence of TRA-1-60) after depletion of TFDP1 (right). © 2024 Ishii, et al., Nature Genetics Contact Hiroe Yoneda Senior Specialist in Project Planning and Outreach NanoLSI Administration OfficeNano Life Science Institute (WPI-NanoLSI) Kanazawa University Kakuma-machi, Kanazawa 920-1192, Japan Email: [email protected] Tel: +81 (76) 234-4555 About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University Understanding nanoscale mechanisms of life phenomena by exploring "uncharted nano-realms." Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale. About the World Premier International Research Center Initiative (WPI) The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS). See the latest research news from the centers at the WPI News Portal: Main WPI program site: About Kanazawa University As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities. The University is located on the coast of the Sea of Japan in Kanazawa – a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students including 600 from overseas. SOURCE Kanazawa University

对绝大多数动物而言，睡眠不可或缺，人类一生要花费约1/3的时间用于睡眠，而睡眠不足会导致多种健康问题，引发炎症或慢性免疫系统疾病1,2。动物实验表明，长时间睡眠剥夺会导致其个体死亡3-5。然而，哺乳动物的睡眠如何调控免疫系统，睡眠不足又如何带来负面的健康影响，其机制尚未厘清。2023年11月27日，北京生命科学研究所/清华大学生物医学交叉研究院张二荃实验室在Cell上在线发表了题为Prolonged sleep deprivation induces a cytokine storm-like syndrome in mammals的研究论文。该研究发现小鼠长时间睡眠剥夺将导致严重细胞因子风暴，并且揭示了睡眠剥夺导致外周免疫反应的神经分子机制——脑源PGD2/DP1信号通路。在此研究课题中，张二荃实验室研究人员首创了一套适用于小鼠的定名为“Curling Prevention by Water”(CPW)的睡眠剥夺系统(图1)，通过脑电图/肌电图(EEG/EMG)记录实验组小鼠和对照组小鼠的睡眠/觉醒情况，确认该系统可以持续剥夺小鼠95%以上的睡眠。该系统相较于传统的实验动物转杆睡眠剥夺系统(Stand Alone Sleep Deprivation，SASD)，可以在不引入更多环境刺激和保证实验通量的基础上极大提高睡眠剥夺效率，从而帮助研究人员可以观察到长时间睡眠剥夺后对哺乳动物带来的生理后果。图1Curling Prevention by Water睡眠剥夺系统哈佛医学院的Rogulja实验室于2020年在Cell上发表以果蝇为研究对象，研究睡眠剥夺对动物产生的生理后果。他们发现睡眠剥夺的果蝇在肠道中积累了大量活性氧(ROS)，从而显著缩短了睡眠剥夺果蝇的寿命4。尽管他们也在睡眠剥夺的小鼠中发现，其肠道ROS含量相较于对照组小鼠显著上升，但并未发现睡眠剥夺会导致小鼠死亡，因而高等的如哺乳动物中是否存在类似的现象、其可能的作用机制仍不明确。本实验中，张二荃实验室研究人员发现小鼠肠道ROS的积累与小鼠在睡眠剥夺后免疫反应强度相关，这说明了ROS产生在免疫激活反应的下游，而非上游；更重要的是，为小鼠提供N-乙酰半胱氨酸(NAC)和硫辛酸(lipoic acid)等抗氧化剂4 并不能缓解小鼠睡眠剥夺致死的现象，这些都说明不能把果蝇睡眠剥夺致死的机制简单地推广到小鼠：虽然哺乳动物中长时间完全地睡眠剥夺也会致死，但致死原因与肠道ROS积累无关(既非充分也非必要条件)。研究人员应用CPW系统对小鼠进行长时间睡眠剥夺时发现，对小鼠进行4天连续睡眠剥夺后，高达70%以上小鼠会死于严重的多器官衰竭。在睡眠剥夺过程中，小鼠血液中的促炎性的细胞因子(如IL-6和IL-17A)和趋化因子(如CXCL1和CXCL2)迅速积累，并将中性粒细胞从骨髓招募到循环系统中，从而导致小鼠出现中性粒细胞增多症。随着促炎性的细胞因子与趋化因子的持续性积累，小鼠出现由细胞因子风暴诱发的多器官功能障碍综合征(multiple organ dysregulation syndrome，MODS)，最终导致小鼠的死亡。睡眠不足导致的炎症反应，一般都会被解释为免疫力下降，进而招致的外界细菌或病毒感染。但为了厘清到底是免疫能力减弱，还是反过来的免疫反应过强导致的个体死亡，研究人员做了以下实验：一是在睡眠剥夺过程中，为小鼠提供抗细胞因子风暴药物能够缓和睡眠剥夺导致的免疫反应并延长小鼠的存活时间。二是将野生型小鼠替换为免疫缺陷品系(M-NSG)小鼠。如果是因为免疫力弱而由外界病原体感染导致死亡的话，M-NSG小鼠预期应该会更快地死亡。但事实上，对这种小鼠进行长达一周的睡眠剥夺都不会有个体死亡。以上实验说明了不睡觉导致的死亡真正原因是免疫系统的过度激活(这与引发人体新冠重症的“细胞因子风暴”类似)。除此之外，为了研究睡眠剥夺如何调控免疫反应，研究人员首先探究睡眠剥夺如何影响大脑神经系统的功能。研究人员发现睡眠剥夺导致的免疫反应并非通过大脑向外周的直接神经投射进行调控，而是通过大脑产生的特定分子释放到外周循环系统进而诱发的。睡眠剥夺后，小鼠的血脑屏障的主动外排(active efflux)活动持续显著增强。血脑屏障的主动外排活动由ABC转运受体介导，而这其中，通过药物和抑制ABCC家族转运受体能够缓和睡眠剥夺小鼠体内的免疫反应并且延长睡眠剥夺小鼠的存活时间。因而，研究人员推断睡眠剥夺导致的免疫反应由ABCC转运受体的某种底物介导；在睡眠剥夺过程中，随着血脑屏障主动外排活动从大脑转运到外周，进而引起免疫反应。应用北京大学李毓龙实验室最新开发的GRAB(GPCR activation-based)探针，研究人员发现，睡眠剥夺后PGD2(前列腺素D2)在大脑内积累显著增加。同时，研究人员还应用UPLC-MS质谱检测到循环系统中PGD2相对稳定的代谢物Tetranor-PGDM含量也显著增加。为进一步验证PGD2在睡眠剥夺后的转运情况，研究人员使用3H标记的PGD2进行了小鼠活体血脑屏障主动外排指数BEI(BBB Efflux Index)检测，发现睡眠剥夺后PGD2转运到外周循环系统的速度明显提高。研究人员使用遗传学手段验证PGD2在睡眠剥夺导致的免疫反应中发挥的核心作用：分别对脑内PGD2合成酶(包括全身敲除的Ptgds-/-、和应用脑内嵌合基因敲除技术6 获得的PtgdsBKO)，以及将由脑内PGD2 efflux出来的特异性转运受体Abcc4-/-小鼠，进行睡眠剥夺，发现这些遗传突变鼠都能够显著降低诱发的炎症反应和延长小鼠存活时间。最后，研究人员发现静脉注射PGD2受体(DP1)激动剂能够诱发正常小鼠出现与睡眠剥夺小鼠类似的中性粒细胞增多症；同时，在睡眠剥夺过程中，如果为小鼠提供DP1拮抗剂，则能显著缓和小鼠因睡眠剥夺导致的炎症反应。这些结果也印证了睡眠剥夺导致的免疫激活反应依赖于从脑源的PGD2到外周器官DP1的信号通路(图二)。该研究不仅为解释和解决睡眠不足导致的健康问题奠定了基础，而且有助于深入理解睡眠如何与其他生物学过程相互作用，从而进一步阐释睡眠的功能与意义。图2 睡眠剥夺导致细胞因子风暴的通路模型原文链接：https://doi.org/10.1016/j.cell.2023.10.025本文仅用于学术分享，转载请注明出处。若有侵权，请联系微信：bioonSir 删除或修改！

脊髓损伤是世界医学难题之一，迄今仍无临床可用的有效治疗手段。中国科学院遗传与发育生物学研究所戴建武团队20年来专注脊髓损伤修复研究，成功研发了神经再生胶原支架 脊髓损伤是世界医学难题之一，迄今仍无临床可用的有效治疗手段。中国科学院遗传与发育生物学研究所戴建武团队20年来专注脊髓损伤修复研究，成功研发了神经再生胶原支架（NeuroRegen Scaffold）。8年、100余例患者的临床研究表明神经再生胶原支架修复脊髓损伤安全有效。该产品实现了神经修复的医疗器械产品的重要突破。为了更好地利用神经再生胶原支架产品提升脊髓再生修复效果，研究脊髓发育过程至关重要，但目前关于该过程的调控机制仍缺乏系统认识。单细胞多组学技术能帮助科学家在单细胞水平探究脊髓神经细胞在不同尺度上的异质性和复杂性，从而探索脊髓神经发育过程的调控机制。 戴建武团队和陆发隆团队联合厦门大学黄佳良团队，在Developmental Cell上，在线发表了题为Single-cell chromatin accessibility identifies enhancer networks driving gene expression during spinal cord development in mouse的研究论文。该研究系统绘制了小鼠脊髓发育过程中的单细胞染色质开放性图谱，鉴定出影响脊髓神经细胞发育的关键调控因子及其增强子调控网络。最后，以dp1/dIl细胞重要调控因子Atoh1基因的增强子调控网络为例，科研人员利用增强子报告基因转基因小鼠和增强子敲除小鼠，在体内验证了该增强子网络对Atoh1基因表达的影响。 该研究揭示了增强子网络在发育关键基因表达调控中的作用和机制，为剖析脊髓发育过程的表观遗传调控机制提供了新视角。同时，该工作为脊髓损伤修复和相关疾病治疗提供了潜在的新靶点和新思路。 研究工作得到国家自然科学基金、中科院战略性先导科技专项“器官重建与制造”等的支持。中科院脑科学与智能技术卓越创新中心（神经科学研究所）刘志勇团队与遗传发育所鲍时来团队对本工作亦有贡献。