MNaseseq 是 micrococcal nuclease digestion with deep sequencing（微球菌核酸酶消化結合深度測序）的縮寫，是 2008 年以來用於檢測人類基因組上核小體佔用情況的分子生物學技術。在 2009 年才定名 MNaseseq。簡而言之，該技術依賴於來自金黃色葡萄球菌的非特異性核酸內外切酶微球菌核酸酶，用其結合和切割染色質上未結合蛋白質的 DNA 區域。與此同時，此種酶不消化與組蛋白或其他染色質結合蛋白（例如轉錄因子）結合的 DNA。然後從蛋白質中純化未切割的 DNA，並透過一種或多種不同的下一代測序方法進行測序。 MNaseseq 是用於透過分析染色質可及性來評估表觀基因組狀態的四類方法之一。其他三種技術是 DNaseseq、FAIREseq 和 ATACseq。MNaseseq 主要用於對由組蛋白或其他染色質結合蛋白結合的 DNA 區域進行測序，其他三種測序的物件通常分別是：脫氧核糖核酸酶 I 過敏位點（DHS）、測未結合的 DNA 的染色質蛋白質和鬆散包裹的染色質區域（透過標記物的轉座測定）。 歷史 1956 年，首次在金黃色葡萄球菌中發現微球菌核酸酶，蛋白質於 1966 年結晶檢測，特徵於 1967 年被闡明。染色質的 MNase 消化是染色質結構早期研究的關鍵；用於確定染色質的每個核小體單位由大約 200bp 的 DNA 組成。這與奧林斯和奧林斯的 串珠 模型一起，證實了科恩伯格關於基本染色質結構的設想。進一步研究發現，MNase 無法降解短於～140bp 的與組蛋白結合的 DNA，而 DNase I 和 II 可以將結合的 DNA 降解至低至 10bp。這最終闡明瞭核小體核心由～146bp 的 DNA 包裹著，50bp 的接頭 DNA 連線每個核小體，並且 10 個連續的 DNA 鹼基對間隔地與核小體的核心緊密結合。 微球菌核酸酶消化自特徵於 1967 年被闡明以來，除了用於研究染色質結構外，一直用於寡核苷酸測序實驗。由於 MNase 優先消化腺嘌呤和胸腺嘧啶豐富的區域，此技術用來分析無染色質序列，例如酵母（釀酒酵母）線粒體 DNA 以及噬菌體 DNA。在 1980 年代初期，MNase 消化被用於確定成熟 SV40、果蠅（黑腹果蠅）、酵母、和猴子等染色體的核小體定相和相關 DNA。1985 年，第一次使用這種消化來研究染色質可及性與人類基因表達的相關性。在這項研究中，核酸酶用於檢測某些致癌序列與染色質和核蛋白的關聯。在沒有測序或陣列資訊的情況下，利用 MNase 消化來確定核小體定位的研究一直持續到 2000 年代初期。 隨著 1990 年代末和 2000 年代初全基因組測序的出現，將純化的 DNA 序列與釀酒酵母、秀麗隱杆線蟲、黑腹果蠅、擬南芥、小鼠和人類的真核基因組進行比較成為可能。MNase 消化首先被應用於釀酒酵母和秀麗隱杆線蟲的全基因組核小體佔據研究。MNase 消化處理後，透過微陣列進行分析，確定哪些 DNA 區域富含 MNase 抗性核小體。基於 MNase 的微陣列分析通常用於酵母的全基因組範圍和人類的有限基因組區域以確定核小體定位，這可用作轉錄失活的推斷。 2008 年開發出下一代測序時，MNase 消化與高通量測序（即 SolexaIllumina 測序）相結合以研究人類全基因組範圍內的核小體定位。一年後，術語 MNaseSeq 和 MNaseChIP 最終被創造出來，用於染色質免疫沉澱的微球菌核酸酶消化。自 2008 年首次應用以來，MNaseseq 已被用於對與核小體佔據和跨真核生物表觀基因組學相關的 DNA 進行深度測序。截至 2020 年 2 月，MNaseseq 仍用於測定染色質可及性。 Description Chromatin is dynamic and the positioning of nucleosomes on DNA changes through the activity of various transcription factors and remodeling complexes approximately reflecting transcriptional activity at these sites DNA wrapped around nucleosomes are generally inaccessible to transcription factors Hence MNaseseq can be used to indirectly determine which regions of DNA are transcriptionally inaccessible by directly determining which regions are bound to nucleosomes In a typical MNaseseq experiment eukaryotic cell nuclei are first isolated from a tissue of interest Then MNaseseq uses the endoexonuclease micrococcal nuclease to bind and cleave proteinunbound regions of DNA of eukaryotic chromatin first cleaving and resecting one strand then cleaving the antiparallel strand as well The chromatin can be optionally crosslinked with formaldehyde MNase requires Ca2 as a cofactor typically with a final concentration of 1mM If a region of DNA is bound by the nucleosome core ie histones or other chromatinbound proteins eg transcription factors then MNase is unable to bind and cleave the DNA Nucleosomes or the DNAprotein complexes can be purified from the sample and the bound DNA can be subsequently purified via gel electrophoresis and extraction The purified DNA is typically 150bp if purified from nucleosomes or shorter if from another protein eg transcription factors This makes shortread highthroughput sequencing ideal for MNaseseq as reads for these technologies are highly accurate but can only cover a couple hundred continuous basepairs in length Once sequenced the reads can be aligned to a reference genome to determine which DNA regions are bound by nucleosomes or proteins of interest with tools such as Bowtie The positioning of nucleosomes elucidated through MNaseseq can then be used to predict genomic expression and regulation at the time of digestion Extended Techniques MNaseChIPCUTRUN sequencing Recently MNaseseq has also been implemented in determining where transcription factors bind on the DNA Classical ChIPseq displays issues with resolution quality stringency in experimental protocol and DNA fragmentation Classical ChIPseq typically uses sonication to fragment chromatin which biases heterochromatic regions due to the condensed and tight binding of chromatin regions to each other Unlike histones transcription factors only transiently bind DNA Other methods such as sonication in ChIPseq requiring the use of increased temperatures and detergents can lead to the loss of the factor CUTRUN sequencing is a novel form of an MNasebased immunoprecipitation Briefly it uses an MNase tagged with an antibody to specifically bind DNAbound proteins that present the epitope recognized by that antibody Digestion then specifically occurs at regions surrounding that transcription factor allowing for this complex to diffuse out of the nucleus and be obtained without having to worry about significant background nor the complications of sonication The use of this technique does not require high temperatures or high concentrations of detergent Furthermore MNase improves chromatin digestion due to its exonuclease and endonuclease activity Cells are lysed in an SDSTriton X100 solution Then the MNaseantibody complex is added And finally the proteinDNA complex can be isolated with the DNA being subsequently purified and sequenced The resulting soluble extract contains a 25fold enrichment in fragments under 50bp This increased enrichment results in costeffective highresolution data Singlecell MNaseseq Singlecell micrococcal nuclease sequencing scMNaseseq is a novel technique that is used to analyze nucleosome positioning and to infer chromatin accessibility with the use of only a singlecell input First cells are sorted into single aliquots using fluorescenceactivated cell sorting FACS The cells are then lysed and digested with micrococcal nuclease The isolated DNA is subjected to PCR amplification and then the desired sequence is isolated and analyzed The use of MNase in singlecell assays results in increased detection of regions such as DNase I hypersensitive sites as well as transcription factor binding sites Comparison to other Chromatin Accessibility Assays MNaseseq is one of four major methods DNaseseq MNaseseq FAIREseq and ATACseq for more direct determination of chromatin accessibility and the subsequent consequences for gene expression All four techniques are contrasted with ChIPseq which relies on the inference that certain marks on histone tails are indicative of gene activation or repression not directly assessing nucleosome positioning but instead being valuable for the assessment of histone modifier enzymatic function DNaseseq As with MNaseseq DNaseseq was developed by combining an existing DNA endonuclease with NextGeneration sequencing technology to assay chromatin accessibility Both techniques have been used across several eukaryotes to ascertain information on nucleosome positioning in the respective organisms and both rely on the same principle of digesting open DNA to isolate 140bp bands of DNA from nucleosomes or shorter bands if ascertaining transcription factor information Both techniques have recently been optimized for singlecell sequencing which corrects for one of the major disadvantages of both techniques that being the requirement for high cell input At sufficient concentrations DNase I is capable of digesting nucleosomebound DNA to 10bp whereas micrococcal nuclease cannot Additionally DNaseseq is used to identify DHSs which are regions of DNA that are hypersensitive to DNase treatment and are often indicative of regulatory regions eg promoters or enhancers An equivalent effect is not found with MNase As a result of this distinction DNaseseq is primarily utilized to directly identify regulatory regions whereas MNaseseq is used to identify transcription factor and nucleosomal occupancy to indirectly infer effects on gene expression FAIREseq FAIREseq differs more from MNaseseq than does DNaseseq FAIREseq was developed in 2007 and combined with NextGeneration sequencing three years later to study DHSs FAIREseq relies on the use of formaldehyde to crosslink target proteins with DNA and then subsequent sonication and phenolchloroform extraction to separate noncrosslinked DNA and crosslinked DNA The noncrosslinked DNA is sequenced and analyzed allowing for direct observation of open chromatin MNaseseq does not measure chromatin accessibility as directly as FAIREseq However unlike FAIREseq it does not necessarily require crosslinking nor does it rely on sonication but it may require phenol and chloroform extraction Two major disadvantages of FAIREseq relative to the other three classes are the minimum required input of 100000 cells and the reliance on crosslinking Crosslinking may bind other chromatinbound proteins that transiently interact with DNA hence limiting the amount of noncrosslinked DNA that can be recovered and assayed from the aqueous phase Thus the overall resolution obtained from FAIREseq can be relatively lower than that of DNaseseq or MNaseseq and with the 100000 cell requirement the singlecell equivalents of DNaseseq or MNaseseq make them far more appealing alternatives ATACseq ATACseq is the most recently developed class of chromatin accessibility assays ATACseq uses a hyperactive transposase to insert transposable markers with specific adapters capable of binding primers for sequencing into open regions of chromatin PCR can then be used to amplify sequences adjacent to the inserted transposons allowing for determination of open chromatin sequences without causing a shift in chromatin structure ATACseq has been proven effective in humans amongst other eukaryotes including in frozen samples As with DNaseseq and MNaseseq a successful singlecell version of ATACseq has also been developed ATACseq has several advantages over MNaseseq in assessing chromatin accessibility ATACseq does not rely on the variable digestion of the micrococcal nuclease nor crosslinking or phenolchloroform extraction It generally maintains chromatin structure so results from ATACseq can be used to directly assess chromatin accessibility rather than indirectly via MNaseseq ATACseq can also be completed within a few hours whereas the other three techniques typically require overnight incubation periods The two major disadvantages to ATACseq in comparison to MNaseseq are the requirement for higher sequencing coverage and the prevalence of mitochondrial contamination due to nonspecific insertion of DNA into both mitochondrial DNA and nuclear DNA Despite these minor disadvantages use of ATACseq over the alternatives is becoming more prevalent 參考資料 CategoryMolecular biology techniques