Nature Genetics volume 54, páginas 1919–1932 (2022)Cite este artigo
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Ainda não está claro por que a depleção aguda de CTCF (fator de ligação ao CCCTC) e da coesina afeta apenas marginalmente a expressão da maioria dos genes, apesar de perturbar substancialmente o dobramento do genoma tridimensional (3D) no nível dos domínios e das alças estruturais. Para resolver esse enigma, usamos Micro-C de alta resolução e perfil de transcrição nascente em células-tronco embrionárias de camundongos. Descobrimos que as interações intensificador-promotor (E – P) são amplamente insensíveis à depleção aguda (3 horas) de CTCF, coesina ou WAPL. O YY1 foi proposto como um regulador estrutural das alças EP, mas a depleção aguda de YY1 também teve efeitos mínimos nas alças EP, na transcrição e no dobramento do genoma 3D. Surpreendentemente, imagens de células vivas e de molécula única revelaram que a depleção de coesina reduziu a ligação do fator de transcrição (TF) à cromatina. Assim, embora CTCF, coesina, WAPL ou YY1 não sejam necessários para a manutenção a curto prazo da maioria das interações E – P e expressão gênica, nossos resultados sugerem que a coesina pode facilitar os TFs a procurar e ligar seus alvos de forma mais eficiente.
Ensaios baseados em captura de conformação cromossômica de alto rendimento (Hi-C) transformaram nossa compreensão do dobramento do genoma 3D . Com base em tais estudos, podemos distinguir pelo menos três níveis de dobramento do genoma 3D. Primeiro, o genoma é segregado nos compartimentos A e B, que correspondem em grande parte aos segmentos de cromatina ativos e inativos, respectivamente, e aparecem como um padrão xadrez nos mapas de contato Hi-C . Em segundo lugar, as proteínas CTCF e coesina ajudam a dobrar o genoma em domínios de associação topológica (TADs)4,5 e alças estruturais de cromatina6, provavelmente através da extrusão de alça de DNA7,8. Terceiro, em uma escala muito mais refinada, os elementos transcricionais envolvem-se em interações de cromatina de longo alcance, como interações E – P e promotor-promotor (P – P) para formar domínios locais .
Experimentos elegantes combinando depleção proteica aguda de CTCF, coesina e proteínas reguladoras de coesina com Hi-C ou abordagens de imagem revelaram o papel de CTCF e coesina na regulação dos dois primeiros níveis: TADs e compartimentos . No entanto, o Hi-C é ineficaz para capturar o terceiro nível de dobramento do genoma 3D: as interações E – P / P – P transcricionalmente importantes em escala fina . Nossa compreensão do papel do CTCF e da coesina na regulação da expressão gênica veio principalmente de experimentos genéticos com foco em alguns loci de desenvolvimento. Assim, não ficou claro se, quando, onde e como o CTCF / coesina regula as interações E – P / P – P e a expressão gênica.
Recentemente, relatamos que o Micro-C pode resolver efetivamente o dobramento ultrafino do genoma 3D na resolução do nucleossomo, incluindo interações E – P / P – P . No presente estudo, usamos Micro-C, sequenciamento de imunoprecipitação da cromatina (ChIP-seq), sequenciamento de RNA total (RNA-seq) e RNA-seq24 nascente para investigar sistematicamente quão depletante agudamente CTCF, RAD21 (subunidade de coesina), WAPL ( descarregador de coesina) ou YY1 (uma suposta proteína estrutural25) afeta as interações e a transcrição da cromatina reguladora do gene em células-tronco embrionárias de camundongo (mESCs). Finalmente, focar na dinâmica do YY1 revelou um papel inesperado para a coesina na facilitação da ligação ao TF.
Nosso estudo anterior usou Micro-C para revelar que a estrutura do genoma 3D em escala fina se correlaciona bem com a atividade transcricional, formando 'pontos' ou 'loops' (ver Métodos para terminologia) nas interseções EP e P – P . No presente estudo, identificamos mais de 75.000 loops estatisticamente significativos em mESCs usando o recém-desenvolvido chamador de loop Mustache26 (Fig. 1a) ou Chromosight27 (Extended Data Fig. 1a), aproximadamente 2,5x mais do que em nosso relatório anterior9,26 e cerca de 4 × mais que Hi-C26,28 (Dados Estendidos Fig. 1b). Através da análise do estado local da cromatina nas âncoras de loop (Extended Data Fig. 1c, d), subclassificamos esses loops em loops de coesina (~ 13.735), loops E – P (~ 20.369), loops P – P (~ 7.433) e polycomb -contatos associados (~ 700) (Fig. 1a, b), com um tamanho médio de ~ 160 kb para loops de coesina e ~ 100 kb para loops E – P / P – P (Extended Data Fig. 1e).
75,190 chromatin dots/loops, subclassified into four primary types (Mustache loop caller26; see Methods and Supplementary Note). b, Probability distribution of loop strength for cohesin, E–P, P–P and random loops. Chromatin loop numbers are shown on the left. The box plot indicates the quartiles for the loop strength score distribution (min. = lower end of line, Q1 = lower bound of box, Q2 = line in box, Q3 = higher bound of box and max. = higher end of line). Genome-wide averaged contact signals (aggregate peak analysis (APA)) are plotted on the right. The contact map was normalized by matrix balancing and distance (Obs/Exp), with positive enrichment in red and negative signal in blue, shown as the diverging color map with the gradient of normalized contact enrichment in log10. The ratio of contact enrichment for the center pixels is annotated within each plot. This color scheme and normalization method are used for normalized matrices throughout the manuscript unless otherwise mentioned. Loop anchors are annotated as ‘C’ for CTCF/cohesin, ‘P’ for promoter and ‘E’ for enhancer. Asterisks denote a P < 10−16 using two-sided Wilcoxon’s signed-rank test. The data are presented in the same format and color scheme throughout the manuscript unless otherwise indicated (n = 37 biological replicates)9. c, Genome-wide averaged transcript counts for nascent transcript profiling. Genes are grouped into high, medium and low expression levels based on nascent RNA-seq data (gene body) and rescaled to the same length from TSS (transcription start site) to poly(adenylation) cleavage site (PAS) or TES (transcription end site) on the x axis. d, Rank-ordered distribution of loop strength against gene expression for cohesin, E–P and P–P loops. Gene expression levels for the corresponding chromatin loop were calculated by averaging the genes with TSSs located ±5 kb around the loop anchors. Loop strength was obtained from the same analysis shown in b. The distribution for each loop type was fitted and smoothed by LOESS (locally estimated scatterplot smoothing) regression. Error bands indicate fitted curve ± s.e.m. with 95% confidence interval (CI). e, APAs are plotted by paired E–P/P–P loops and sorted by the level of nascent transcription into high, mid and low levels./p>90% of CTCF peaks and 60% of cohesin peaks are significantly decreased on loss of CTCF (Padj < 0.05; Fig. 3e and Extended Data Fig. 3g). Despite the substantial loss of cohesin peaks, biochemical fractionation experiments show that the fraction of RAD21 associated with chromatin remains fairly constant 3 h after CTCF degradation (Extended Data Fig. 2f, green box). Thus, our results are in line with the widely accepted conclusion that CTCF positions cohesin43. On the other hand, loss of cohesin affects a subset of CTCF binding (Fig. 3c,d)13, resulting in ~20% reduction in the number of CTCF peaks (Fig. 3e) and a slight decrease in its global chromatin association (Extended Data Fig. 2f, blue box)./p> 0.1 µm2 s−1), which can be separated further into slow (Dslow ~0.1–2 µm2 s−1) and fast moving (Dfast > 2 µm2 s−1). Scale bar, 1 μm. f, Aggregate likelihood of diffusive YY1 molecules. Top, bar graph showing fractions of YY1 binned into bound, slow- and fast-diffusing subpopulations. Bottom, YY1 diffusion coefficient estimation by regular Brownian motion with marginalized localization errors. g, Western blots of cytoplasmic (Cyt) and nuclear proteins dissociating from chromatin at increasing salt concentrations (Extended Data Fig. 2b). A subpopulation (~30%) of YY1 stays on chromatin, resisting 1 M washes. Ins, insoluble pellet after sonication; Son, sonicated, solubilized chromatin. Percentage of total shows the signal intensity of the indicated fractions divided by the total signal intensity. Anti-histone 2B controls for chromatin integrity during fractionation. h, FRAP analysis of YY1 bleached with a square spot. Error bars are fitted curve ± s.e.m. with 95% CI. i, Slow-SPT measuring YY1 residence time. Individual molecules were tracked at 100-ms exposure time to blur fast-moving molecules into the background and capture stable binding. The unbinding rate is obtained by fitting a model to the molecules’ survival curve. Each datapoint indicates the unbinding rate of YY1 molecules in a single cell. The box plot shows quartiles of data. Error bars are mean ± s.d. j. Slow-SPT measures YY1’s residence time at multiple exposure times./p>90% depletion after 3 h of IAA treatment (Fig. 7a and Extended Data Fig. 9a). Despite the high degradation efficiency, neither YY1’s nuclear distribution nor its clustering was strongly affected after acute loss of CTCF and cohesin in either live or fixed cells (Fig. 7b,c and Extended Data Fig. 9b). This suggests that the maintenance of YY1 hubs is independent of CTCF and cohesin./p>82% of these loci were associated with promoter regions (Fig. 7f and Extended Data Fig. 9d,e). In contrast, both CTCF and WAPL depletion had a negligible effect on YY1 occupancy (Fig. 7f and Extended Data Fig. 9d,e). In biochemical fractionation analysis, we also observed a similar, though less pronounced, reduction in YY1 chromatin association after RAD21 depletion (Extended Data Fig. 9f). To test whether cohesin facilitates the target search of TFs in general, we performed spaSPT on additional TFs. We thus generated RAD21–AID cell lines stably expressing either HaloTag-conjugated SOX2 or KLF4 and found that the bound fraction of both TFs was reduced by ~20% after 3-h cohesin degradation (Extended Data Fig. 9g). These results suggest that cohesin probably facilitates chromatin binding of TFs in general./p>20% of E–P/P–P loops can cross TAD boundaries and retain high contact probability and transcriptional activity (Fig. 2)18,35; (2) only a very small handful of genes showed altered expression levels after CTCF, cohesin or WAPL depletion (Fig. 3)12,13,14,15,16; (3) CTCF and cohesin loops are both rare (~5% of the time) and dynamic (median lifetime ~10–30 min)34; (4) most of the E–P/P–P loops persist after depletion of these structural proteins (Fig. 4)39,63; (5) CTCF/cohesin generally does not colocalize with transcription loci67; and (6) E–P loops and transcription can be established before CTCF/cohesin interactions on mitotic exit71, in some cases even with no CTCF/cohesin expression36,65,66. Second, YY1 was proposed to be a master structural regulator of E–P interactions25 (Fig. 8, Model 2). However, our Micro-C data are inconsistent with this model, because acute YY1 depletion has little effect on E–P/P–P interactions or gene expression. It is still possible that YY1 specifically connects development-related chromatin loops during neural lineage commitment47, but is less important in the pluripotent state. In summary, we conclude that, in mESCs, CTCF, cohesin, WAPL or YY1 is not generally required for the short-term maintenance of most E–P interactions and the subsequent expression of most genes after acute depletion and loss of function./p>2. Full lists of DEGs are available in Supplementary Table 11./p>2). Full lists of DEGs are available in Supplementary Table 12./p> 100 & intensity > 100 & sigma < 220 & uncertainty_xy < 50; (2) merge: Max distance = 10 & Max frame off = 1 & Max frames = 0; and (3) remove duplicates enabled. This setting combines the blinking molecules into one and removes the multiple localizations in a frame./p>
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