MNase-seq

MNase-seq是micrococcal nuclease digestion with deep sequencing（微球菌核酸酶消化結合深度測序）的縮寫^[1][2][3]，是2008年以來用於檢測人類基因組上核小體占用情況的分子生物學技術^[1]。在2009年才定名「MNase-seq」^[2]。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，並通過一種或多種不同的下一代測序方法進行測序^[4]。

MNase digestion with sequencing

MNase-seq是用於通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DNase-seq、FAIRE-seq和ATAC-seq^[3]。MNase-seq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^[1]序，其他三種測序的對象通常分別是：脫氧核糖核酸酶I過敏位點（DHS）^[5]、測未結合的DNA的染色質蛋白質^[6]和鬆散包裹的染色質區域（通過標記物的轉座測定）^[7][8]。^[3]

歷史

1956年，首次在金黃色葡萄球菌中發現微球菌核酸酶^[9]，蛋白質於1966年結晶檢測^[10]，特徵於1967年被闡明^[11]。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^[12]。這與奧林斯和奧林斯的「串珠」模型一起^[13]，證實了科恩伯格關於基本染色質結構的設想^[14]。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^[15][16]。這最終闡明了核小體核心由~146bp的DNA包裹着^[17]，~50bp的接頭DNA連接每個核小體^[18]，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^[16]。

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^[19]。由於MNase優先消化腺嘌呤和胸腺嘧啶豐富的區域^[20]，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^[21]以及噬菌體DNA^[22][23]。在1980年代初期，MNase消化被用於確定成熟SV40^[24]、果蠅（黑腹果蠅）^[25]、酵母^[26]、和猴子^[27]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^[28]。在沒有測序或陣列信息的情況下，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^[29]。

隨着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^[30]、秀麗隱杆線蟲^[31]、黑腹果蠅^[32]、擬南芥^[33]、小鼠^[34]和人類^[35]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^[36]和秀麗隱杆線蟲^[37]的全基因組核小體占據研究。MNase消化處理後，通過微陣列進行分析，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^[38][39]的全基因組範圍和人類的有限基因組區域^[40][41]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Solexa/Illumina測序）相結合以研究人類全基因組範圍內的核小體定位^[1]。一年後，術語「MNase-Seq」和「MNase-ChIP」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^[2]。自2008年首次應用以來^[1]，MNase-seq已被用於對與核小體占據和跨真核生物表觀基因組學相關的DNA進行深度測序^[4]。截至2020年2月，MNase-seq仍用於測定染色質可及性^[42]。

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.^[43] Hence, MNase-seq can be used to indirectly determine which regions of DNA are transcriptionally inaccessible by directly determining which regions are bound to nucleosomes.^[4]

In a typical MNase-seq experiment, eukaryotic cell nuclei are first isolated from a tissue of interest. Then, MNase-seq uses the endo-exonuclease micrococcal nuclease to bind and cleave protein-unbound regions of DNA of eukaryotic chromatin, first cleaving and resecting one strand, then cleaving the antiparallel strand as well.^[2] The chromatin can be optionally crosslinked with formaldehyde.^[44] MNase requires Ca²⁺ as a cofactor, typically with a final concentration of 1mM.^[4][11] If a region of DNA is bound by the nucleosome core (i.e. histones) or other chromatin-bound proteins (e.g. transcription factors), then MNase is unable to bind and cleave the DNA. Nucleosomes or the DNA-protein 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,^[1] or shorter, if from another protein (e.g. transcription factors).^[45] This makes short-read, high-throughput sequencing ideal for MNase-seq as reads for these technologies are highly accurate but can only cover a couple hundred continuous base-pairs in length.^[46] 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.^[3] The positioning of nucleosomes elucidated, through MNase-seq, can then be used to predict genomic expression^[47] and regulation^[48] at the time of digestion.

Extended Techniques

Technical applications of MNase in sequencing

MNase-ChIP/CUT&RUN sequencing

Recently, MNase-seq has also been implemented in determining where transcription factors bind on the DNA.^[49][50] Classical ChIP-seq displays issues with resolution quality, stringency in experimental protocol, and DNA fragmentation.^[50] Classical ChIP-seq typically uses sonication to fragment chromatin, which biases heterochromatic regions due to the condensed and tight binding of chromatin regions to each other.^[50] Unlike histones, transcription factors only transiently bind DNA. Other methods, such as sonication in ChIP-seq, requiring the use of increased temperatures and detergents, can lead to the loss of the factor. CUT&RUN sequencing is a novel form of an MNase-based immunoprecipitation. Briefly, it uses an MNase tagged with an antibody to specifically bind DNA-bound 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 SDS/Triton X-100 solution. Then, the MNase-antibody complex is added. And finally, the protein-DNA complex can be isolated, with the DNA being subsequently purified and sequenced. The resulting soluble extract contains a 25-fold enrichment in fragments under 50bp. This increased enrichment results in cost-effective high-resolution data.^[50]

Single-cell MNase-seq

Single-cell micrococcal nuclease sequencing (scMNase-seq) is a novel technique that is used to analyze nucleosome positioning and to infer chromatin accessibility with the use of only a single-cell input.^[51] First, cells are sorted into single aliquots using fluorescence-activated cell sorting (FACS).^[51] 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.^[51] The use of MNase in single-cell assays results in increased detection of regions such as DNase I hypersensitive sites as well as transcription factor binding sites.^[51]

Comparison to other Chromatin Accessibility Assays

MNase-seq is one of four major methods (DNase-seq, MNase-seq, FAIRE-seq, and ATAC-seq) for more direct determination of chromatin accessibility and the subsequent consequences for gene expression.^[52] All four techniques are contrasted with ChIP-seq, which relies on the inference that certain marks on histone tails are indicative of gene activation or repression,^[53] not directly assessing nucleosome positioning, but instead being valuable for the assessment of histone modifier enzymatic function.^[3]

DNase-seq

As with MNase-seq,^[1] DNase-seq was developed by combining an existing DNA endonuclease^[5] with Next-Generation sequencing technology to assay chromatin accessibility.^[54] Both techniques have been used across several eukaryotes to ascertain information on nucleosome positioning in the respective organisms^[3] and both rely on the same principle of digesting open DNA to isolate ~140bp bands of DNA from nucleosomes^[1][55] or shorter bands if ascertaining transcription factor information.^[45][55] Both techniques have recently been optimized for single-cell sequencing, which corrects for one of the major disadvantages of both techniques; that being the requirement for high cell input.^[56][51]

At sufficient concentrations, DNase I is capable of digesting nucleosome-bound DNA to 10bp, whereas micrococcal nuclease cannot.^[16] Additionally, DNase-seq is used to identify DHSs, which are regions of DNA that are hypersensitive to DNase treatment and are often indicative of regulatory regions (e.g. promoters or enhancers).^[57] An equivalent effect is not found with MNase. As a result of this distinction, DNase-seq is primarily utilized to directly identify regulatory regions, whereas MNase-seq is used to identify transcription factor and nucleosomal occupancy to indirectly infer effects on gene expression.^[3]

FAIRE-seq

FAIRE-seq differs more from MNase-seq than does DNase-seq.^[3] FAIRE-seq was developed in 2007^[6] and combined with Next-Generation sequencing three years later to study DHSs.^[58] FAIRE-seq relies on the use of formaldehyde to crosslink target proteins with DNA and then subsequent sonication and phenol-chloroform extraction to separate non-crosslinked DNA and crosslinked DNA. The non-crosslinked DNA is sequenced and analyzed, allowing for direct observation of open chromatin.^[59]

MNase-seq does not measure chromatin accessibility as directly as FAIRE-seq. However, unlike FAIRE-seq, it does not necessarily require crosslinking,^[4] nor does it rely on sonication,^[3] but it may require phenol and chloroform extraction.^[4] Two major disadvantages of FAIRE-seq, relative to the other three classes, are the minimum required input of 100,000 cells and the reliance on crosslinking.^[6] Crosslinking may bind other chromatin-bound proteins that transiently interact with DNA, hence limiting the amount of non-crosslinked DNA that can be recovered and assayed from the aqueous phase.^[52] Thus, the overall resolution obtained from FAIRE-seq can be relatively lower than that of DNase-seq or MNase-seq^[52] and with the 100,000 cell requirement,^[6] the single-cell equivalents of DNase-seq^[56] or MNase-seq^[51] make them far more appealing alternatives.^[3]

ATAC-seq

ATAC-seq is the most recently developed class of chromatin accessibility assays.^[7] ATAC-seq 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.^[7][8] ATAC-seq has been proven effective in humans, amongst other eukaryotes, including in frozen samples.^[60] As with DNase-seq^[56] and MNase-seq,^[51] a successful single-cell version of ATAC-seq has also been developed.^[61]

ATAC-seq has several advantages over MNase-seq in assessing chromatin accessibility. ATAC-seq does not rely on the variable digestion of the micrococcal nuclease, nor crosslinking or phenol-chloroform extraction.^[4][8] It generally maintains chromatin structure, so results from ATAC-seq can be used to directly assess chromatin accessibility, rather than indirectly via MNase-seq. ATAC-seq can also be completed within a few hours,^[8] whereas the other three techniques typically require overnight incubation periods.^[4][5][6] The two major disadvantages to ATAC-seq, in comparison to MNase-seq, are the requirement for higher sequencing coverage and the prevalence of mitochondrial contamination due to non-specific insertion of DNA into both mitochondrial DNA and nuclear DNA.^[7][8] Despite these minor disadvantages, use of ATAC-seq over the alternatives is becoming more prevalent.^[3]