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May 19, 2023

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Oct 10, 2023

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Oct 23, 2023

NPAS4

자연 614권, 페이지

Nature 614권, 732~741페이지(2023)이 기사 인용

29,000회 액세스

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측정항목 세부정보

신경 활동은 적응 회로 리모델링에 중요하지만 유사분열 후 뉴런의 긴 수명에 걸쳐 게놈의 안정성에 내재된 위험을 초래합니다. 뉴런이 활동이 활발한 기간 동안 잠재적으로 손상을 줄 수 있는 수십 년의 자극을 견딜 수 있는 특수한 게놈 보호 메커니즘을 획득했는지 여부는 알려져 있지 않습니다. 여기에서 우리는 새로운 형태의 NuA4-TIP60 염색질 변형자가 유도성 신경 세포 특이 전사 인자인 NPAS4 주변의 활성화된 뉴런에서 조립되는 활동 의존적 DNA 복구 메커니즘을 식별합니다. 우리는 뇌에서 이 복합체를 정화하고 신경 전사체 및 회로에 대한 활동 의존적 변화를 유도하는 기능을 보여줍니다. 활동으로 인해 유발되는 뇌의 DNA 이중 가닥 파손 환경을 특성화함으로써 우리는 NPAS4-NuA4가 반복적으로 손상된 조절 요소에 결합하고 추가 DNA 복구 기계를 동원하여 복구를 자극한다는 것을 보여줍니다. NPAS4-NuA4에 의해 결합된 유전자 조절 요소는 연령에 따른 체세포 돌연변이 축적으로부터 부분적으로 보호됩니다. 손상된 NPAS4-NuA4 신호 전달은 조절 장애가 있는 활성 의존성 전사 반응, 신경 억제에 대한 통제력 상실 및 게놈 불안정성을 비롯한 일련의 세포 결함을 초래하며, 이는 모두 유기체 수명을 단축시킵니다. 또한, NuA4 복합체의 여러 구성 요소의 돌연변이가 신경발달 및 자폐 스펙트럼 장애를 유발하는 것으로 보고되었습니다. 함께, 이러한 발견은 신경 활동을 게놈 보존과 직접 연결하는 신경 특이적 복합체를 식별하며, 그 중단은 발달 장애, 신경 퇴행 및 노화에 기여할 수 있습니다.

적절한 신경 성숙과 회로 가소성을 위해서는 감각 경험이 필수적입니다1. 경험에 기반한 신경 활동에 의해 시작된 신호 전달 계통은 수상돌기 및 시냅스 성장, 시냅스 제거, 억제성 신경전달 동원 및 적응성 수초화와 같은 다양한 과정을 제어하는 ​​유전자 프로그램의 유도로 최고조에 이릅니다. 그러나 신경 활동은 또한 유기체의 수명 동안 생존해야 하는 유사분열 후 뉴런의 게놈 무결성을 위협합니다. 예를 들어, 활동이 증가하는 기간 동안 대사 요구가 높아지면 게놈의 활발하게 전사되는 영역에 대한 산화 손상이 증가할 수 있습니다8. 활동 유도 전사 자체는 자극 유도 유전자의 프로모터와 같은 조절 요소에서 반복적인 DNA 이중 가닥 절단(DSB)의 유도와 연결되어 있기 때문에 게놈 안정성에 추가 위협을 제기합니다2,3,4,5 ,9,10. 전사와 DNA 절단의 결합은 세포 유형 전반에 걸쳐 관찰되지만, 이 과정은 복제 의존적 DNA 복구 경로를 사용할 수 없고 손상된 세포를 대체하기 위한 제한된 재생 메커니즘을 보유하는 수명이 긴 뉴런에 특정한 문제를 제기합니다. 신경 게놈에 축적되는 DNA 손상은 신경퇴행성 장애와 유기체 노화의 주요 특징입니다12,13. 따라서 손상을 예방하고 복구하기 위해 뉴런이 사용하는 전략을 이해하면 인간의 장수 및 노화 치료법으로 직접적으로 해석될 수 있습니다. 지금까지 활동이 증가하는 동안 게놈 불안정성의 위험을 완화하는 신경세포별 복구 기계의 예는 없습니다. 뉴런에 특이적인 활동 의존적 전사 프로그램의 특징을 조사함으로써 우리는 유도성 뉴런 특이적 전사 인자 주위에 조립되는 이전에 알려지지 않은 형태의 NuA4 염색질 리모델러-DNA 복구 복합체를 통해 DNA 복구에 대한 뉴런 활동의 생화학적 결합을 발견했습니다. NPAS4.

다양한 자극에 의해 광범위하게 발현되고 유도되는 대부분의 활성 유도성 전사 인자와 달리 NPAS4는 막 탈분극 유도 칼슘 신호 전달에 따라 뉴런에서 선택적으로 발현됩니다. 신경 활동에 특별히 맞춰진 이 요소의 기능을 이해하기 위해 우리는 성체 마우스 뇌에서 NPAS4 함유 단백질 복합체를 정제하려고 했습니다. 우리는 NPAS4가 활성화된 뉴런에서 생화학적 활동을 확장하는 다중 하위 단위 복합체로 조립될 수 있다고 추론했습니다. 크기 배제 크로마토그래피와 비변성 겔 전기영동을 사용하여 우리는 NPAS4가 약 1 MDa의 고분자량 복합체에 존재한다는 것을 관찰했습니다. 이종이량체 파트너(ARNT1 및 ARNT2) 중 하나를 포함하는 NPAS4의 예상 크기는 약 175kDa이므로 이 발견은 NPAS4가 알려지지 않은 여러 단백질 파트너와 상호 작용한다는 것을 나타냅니다(확장 데이터 그림 1a).

 0, adjusted P < 0.1 and that are within Q4 of NPAS4 IgG-normalized CUT&RUN signals are indicated in blue. Right inset, IGV tracks of aggregate sBLISS-seq (n = 8 individual mice) and NPAS4 CUT&RUN (n = 5 pools of 3–5 mice) at Cgref1 and Rgs7bp promoters. Coloured bars represent statistically defined peaks. f, Aggregate plots showing sBLISS-seq coverage (fragment depth per bp per peak) at activity-inducible regulatory elements, subset by quartiles of NPAS4 binding. Signals are mean ± s.e.m. (n = 8 individual mice). ***P < 2.2 × 10−16. P values were calculated using the average signal extracted in a 500 bp window around the element centre using unpaired, two-tailed Wilcoxon rank-sum tests./p> Cre). b, Line plot depicting average ± s.e.m. of sBLISS-seq normalized counts in Cre-infected or ΔCre-infected hippocampi of Npas4fl/fl mice, subset by quartiles of NPAS4 binding. Q1 = 1,017 sites, Q4 = 764 sites. Data from n = 5 each for Cre-infected and ΔCre-infected mice. ***P < 2.2 × 10–16. P values were calculated using unpaired, one-tailed Wilcoxon rank-sum tests (Cre > ΔCre). See Extended Data Fig. 12a for boxplot distribution of data. c, Genome-wide breaks in hippocampal nuclei isolated from Npas4fl/fl (n = 5) or wild-type (n = 3) mice infected with Cre or ΔCre virus. Data are mean ± s.e.m. Npas4fl/fl: 0 h, P = 5.24 × 10–6; 2 h, P = 0.044; 10 h, P = 0.022. Wild-type: 2 h, P = 0.906. P values were calculated using two-tailed, unpaired t-tests. d, Collection of hippocampal neuronal nuclei across lifespan. e,Normalized mutation frequency at NPAS4-bound and unbound sites in wild-type mice. Mutation frequency (base changes and insertions and deletions) with age was calculated per site and normalized to the median frequency for that site in young animals. Points represent the normalized rate for one site sampled from one mouse. Data are mean ± s.e.m. Data are from n = 4 (young), 4 (middle aged) and 3 (old) mice. No NPAS4 sites: mid–young, *P = 0.03; old–young, *P = 0.016. NPAS4-bound sites: mid–young, NS = 0.18; old–young, **P = 0.0095. P values calculated using one-tailed, unpaired t-tests. f, Survival of Npas4 wild-type (n = 53; 28 females, 25 males) and Npas4 knockout (n = 64; 37 females, 27 males) mice, showing abbreviated lifespan of Npas4 knockout mice. P = 4.09 × 10–13 by a two-tailed Mantel–Cox test, P = 3.63 × 10–11 by a two-tailed Gehan–Breslow–Wilcoxon test./p>/J (Jackson Laboratory, 021039)53; and B6.Cg-Tg(Camk2a-cre)T29-1Stl/J (Jackson Laboratory, 005329)54. Mice were housed in a temperature and humidity-controlled environment using ventilated microisolator cages. Mice were kept under a standard 12 h light–dark cycle, with food and water provided ad libitum. Male and female littermate mice were used in similar proportions and divided between control and experimental groups for all experiments conducted. No statistical methods were used to predetermine sample sizes. For biochemistry and genomic experiments, animals were collected at 4–6 weeks of age throughout the manuscript. For physiology experiments, animals were dissected and patched at postnatal day 24 (P24) to P28. For ageing experiments, animals were collected at 3-4 months, 12 months and 23–27 months of age. Details of animal age and sex are detailed within each protocol./p>30% infected CA1 pyramidal neurons were not used for recordings. Slices were also discarded if Cre-mCherry expression was observed in the CA3 or dentate gyrus regions./p>25 KU (1 µl per 10 million nuclei)) followed by the addition of NaCl to a final concentration of 300 mM. Following high-speed centrifugation to remove insoluble material, lysates were diluted to achieve a final salt concentration of 200 mM. To preclear nonspecific interactions, lysates were incubated for 30 min at 4 °C with 50 µl of ms-IgG-coated agarose beads (Sigma-Aldrich, A0919). Following preclear, lysates were incubated for 1.5 h with 60 µl of anti-M2-Flag resin (Sigma-Aldrich, A2220) per 1 ml of diluted lysate. Samples were washed 4× in NE1 buffer containing 250 mM NaCl for 5 min with rotation at 4 °C. NPAS4-interacting or ARNT2-interacting proteins were competitively eluted off M2 resin by incubation in 50–100 µl of 500 µg ml–1 3× Flag peptide (Sigma-Aldrich, F4799) diluted in NE1 buffer for 30 min at room temperature. For mass spectrometry analysis, eluted proteins were precipitated with trichloroacetic acid. Replicates shown in Fig. 1c consisted of 3 independent pools of 8–12 mice collected from wild-type or Npas4–FH lines and processed on separate days. Validation immunoprecipitation assays using either Npas4–Flag-HA mice or Tip60-H3F mice followed by immunoblotting analysis were conducted under the same conditions as described above. For validation experiments in mouse visual cortex following light stimulation, 20 hippocampi were isolated from the V1 cortices of Npas4–Flag-HA or wild-type controls. Mice were housed in the dark for 1 week followed by 2 h of light stimulation. Data are shown in Extended Data Fig. 1f./p> 1 were removed from further analysis. The voom and limma (edgeR_3.28.1;limma_3.42.2) analysis software packages were used to quantify differential gene expression (requiring FDR-corrected q < 0.01). For analysis of the paired RNA-seq samples that matched sBLISS-seq, the DeSeq2 package was used to generate normalized counts. After running a standard DeSeq2 pipeline, normalized counts were generated with the function counts(dds, normalized=TRUE)59./p>100. For information on primer pooling and the amplicons included in the final analysis, see Supplementary Table 5./p>A/(G>T)) divided by the total number of the given base included in the amplicon. Because it is not possible to know on which strand a mutation occurred, complementary base changes were collapsed into a single category (for example, C>T was combined with G>A). Insertion and deletion frequency was also calculated as a separate category. We also calculated a per amplicon normalized mutation rate in which we divided the total mutation rate for each animal by the median total mutation frequency in young mice. For ageing gradient samples, wild-type mice aged 3 months old were considered young, 12 months old were considered middle aged and 23–27 months old were considered old. Extreme outlier points of both normalized mutation frequency and non-normalized frequency were removed across all samples using a ROUT's test at 0.1% confidence (Fig. 5e and Extended Data Fig. 13g). Outlier removal and statistical tests on mutational samples were performed in Prism (v.8.4.2)./p>

 0 h)./p>

 Cre). g. Boxplots of average EP400 CUT&RUN normalized counts in Cre or ΔCre-infected hippocampi of Npas4fl/fl mice at activity-inducible sites, subset by quartiles of NPAS4 binding. Q1 = 44,864 sites, Q4 = 7,378 sites. Boxplot shows median (line), IQR (box), 1.5x IQR (whiskers), notches indicate median ± 1.58× IQR/sqrt(n). EP400 ΔCre (n = 3), EP400 Cre (n = 4). 2-3 mice pooled per replicate. EP400 Q1: P = 1, Q4: ***P < 2.2e-16. P values by unpaired, one-tailed Wilcoxon rank-sum tests (ΔCre > Cre). h. Integrative Genomics Viewer browser image displaying CUT&RUN signal for MRE11 and EP400 in Npas4-cKO (Cre) or Control (ΔCre) in 2 h KA-stimulated nuclei at the Rgs7bp gene. MRE11 CRE (n = 3), MRE11 ΔCre (n = 3), EP400 ΔCre (n = 3), EP400 Cre (n = 4). 2-3 mice pooled per replicate./p> ΔCre). b. Boxplots of sBLISS-seq normalized counts in nuclei isolated from wild-type mice injected with Cre-mCherry or ΔCre-GFP virus at activity-inducible regulatory elements, subset by quartiles of NPAS4 CUT&RUN signal. Q1 = 1,017 sites, Q4 = 764 sites. Data plotted includes all datapoints coming from 3 replicates per genotype; no averaging across replicates was performed. Boxplot shows median (line), IQR (box), 1.5x IQR (whiskers), notches indicate median ± 1.58× IQR/sqrt(n). Q1: P = 1; Q4 P = 0.995. P values by unpaired, one-tailed Wilcoxon rank-sum tests (Cre > ΔCre). c. Boxplots of sBLISS-seq normalized counts in nuclei isolated from Npas4fl/fl mice injected with Cre-mCherry (Npas4-cKO) or ΔCre-GFP virus (Control) at all regulatory elements, subset by quartiles of NPAS4 CUT&RUN signal (see Methods for regulatory site definition). Q1 = 44,864 sites, Q4 = 7,378 sites. Data plotted includes all datapoints coming from 5 replicates per genotype. Boxes represent the interquartile range with line at the median. Boxplot shows median (line), IQR (box), 1.5x IQR (whiskers), notches indicate median ± 1.58× IQR/sqrt(n). ***P<2.2e-16. P values by unpaired, one-tailed Wilcoxon rank-sum tests (Cre > ΔCre). d. Boxplots of sBLISS-seq DeSeq2 normalized counts in nuclei isolated from wild-type injected with Cre-mCherry or ΔCre-GFP virus at all regulatory elements, subset by quartiles of NPAS4 CUT&RUN signal (see Methods for regulatory site definition). Data plotted includes all datapoints coming from 3 replicates per genotype. Boxplot shows median (line), IQR (box), 1.5x IQR (whiskers), notches indicate median ± 1.58× IQR/sqrt(n). Q1: P = 1; Q4 P = 0.1092. P values by unpaired, one-tailed Wilcoxon rank-sum tests (Cre > ΔCre). e. Average ± s.e.m. of genome-wide breaks in hippocampal nuclei isolated from Tip60fl/fl mice (0 and 2 h; n = 3, 10 h; n = 4) infected with Cre or ΔCre virus. To compare across samples, input reads were downsampled to the lowest value among all conditions, and numbers of unique DNA breaks are shown. Tip60fl/fl: 0 h: P = 0.0017, 2 h: P = 0.013, 10 h: P = 0.158. P values by two-tailed, unpaired t-tests. f. Cleaved caspase 3 (apoptosis marker) staining in Npas4fl/fl mice injected with AAV to express Cre-mCherry. Representative image from 3 animals. Scale bar = 300 μm. g. Cleaved caspase 3 staining in Tip60fl/fl mice injected with AAV to express Cre-mCherry. Representative image from 3 animals. Scale bar = 300 μm./p>

A P = 0.99, T>G P = 0.99, T>C P = 0.0049, C>A P = 0.027, C>G P = 0.99, C>T P = 1.85e-09. P values by a one-way ANOVA with Holm-Sidak's correction for multiple hypothesis testing. Right panel: Average Insertion/Deletion frequency ± s.e.m. in NPAS4-Bound and No NPAS4 sites in young (3-month-old) animals. Each point represents a single site sampled from a mouse and data from 4 mice are shown. ***P = 2.96e-05. P value by an unpaired, two-tailed Wilcoxon rank-sum test. h. Lifespan analysis on Npas4 wild-type (n = 25) vs Npas4 KO (n = 27) male littermates. Median lifespan KO = 12 months; Median lifespan of wild-type not determined. P = 8.67e-06 by two-tailed Gehan-Breslow-Wilcoxon test; P = 1.37e-06 by two-tailed log-rank Mantel-Cox test. i. Lifespan analysis on Npas4 wild-type (n = 28) vs Npas4 KO (n = 37) female littermates. Median lifespan KO = 11 months; Median lifespan of wild-type not determined. P = 1.01e-06 by two-tailed Gehan-Breslow-Wilcoxon test; P = 8.48e-08 by two-tailed log-rank Mantel-Cox test. j. Lifespan analysis on Npas4 wild-type (n = 16, Npas4+/+; Camk2a-Cre+; Sun1fl/+) vs Npas4 cKO (n = 9, Npas4fl/fl; Camk2a-Cre+; Sun1fl/+). Median lifespan cKO = 21.46 months; Median lifespan of wild-type = 29 months. P = 0.049 by two-tailed Gehan-Breslow-Wilcoxon test; P = 0.19 by a two-tailed log-rank Mantel-Cox test./p>