Patent Application
20210163900
The present disclosure provides, inter alia, various methods, kits and compositions for modifying nucleic acid using MTA1c or any components thereof. Such embodiments may be used to treat disease and as research tools.
This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file “CU19015-PCT-seq.txt”, file size of 478 KB, created on Aug. 28, 2019. The aforementioned sequence listing is hereby incorporated by reference in its entirety.
Covalent modifications on DNA have long been recognized as a hallmark of epigenetic regulation. DNA N6-methyladenine (6 mA) has recently come under scrutiny in eukaryotic systems, with proposed roles in retrotransposon or gene regulation, transgenerational epigenetic inheritance, and chromatin organization (Luo et al., 2015). 6 mA exists at low levels in Arabidopsis thaliana (0.006%-0.138% 6 mA/dA), rice (0.2%), C. elegans (0.01%-0.4%), Drosophila (0.001%-0.07%), Xenopus laevis (0.00009%), mouse embryonic stem cells (ESCs) (0.0006-0.007%), human cells (Greer et al., 2015; Koziol et al., 2016; Liang et al., 2018; Wu et al., 2016; Xiao et al., 2018; Zhang et al., 2015; Zhou et al., 2018), and the mouse brain (Yao et al., 2017), although it accumulates in abundance (0.1%-0.2%) during vertebrate embryogenesis (Liu et al., 2016). Disruption of DMAD, a 6 mA demethylase, in the Drosophila brain leads to the accumulation of 6 mA and Polycomb-mediated silencing (Yao et al., 2018). The existence of 6 mA in mammals remains a subject of debate. Quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of HeLa and mouse ESCs failed to detect 6 mA above background levels (Schiffers et al., 2017). A recent study, however, reported that loss of 6 mA in human cells promotes tumor formation (Xiao et al., 2018), suggesting that 6 mA is a biologically relevant epigenetic mark.
In contrast to metazoa, 6 mA is abundant in various unicellular eukaryotes, including ciliates (0.18%-2.5%) (Ammermann et al., 1981; Cummings et al., 1974; Gorovsky et al., 1973; Rae and Spear, 1978), and the green algae Chlamydomonas (0.3%-0.5%) (Fu et al., 2015; Hattman et al., 1978). High levels of 6 mA (up to 2.8%) were also recently reported in basal fungi (Mondo et al., 2017). Ciliates have long served as powerful models for the study of chromatin modifications (Brownell et al., 1996; Liu et al., 2007; Strahl et al., 1999; Taverna et al., 2002; Wei et al., 1998). They possess two structurally and functionally distinct nuclei within each cell (Bracht et al., 2013; Yerlici and Landweber, 2014). In the ciliate Oxytricha trifallax, the germline micronucleus is transcriptionally silent and contains ˜100 megabase-sized chromosomes (Chen et al., 2014). In contrast, the somatic macronucleus is transcriptionally active, being the sole locus of Pol II-dependent RNA production in non-developing cells (Khurana et al., 2014). The Oxytricha macronuclear genome is extraordinarily fragmented, consisting of ˜16,000 unique chromosomes with a mean length of ˜3.2 kb, most encoding a single gene. Macronuclear chromatin yields a characteristic ˜200 bp ladder upon digestion with micrococcal nuclease, indicative of regularly spaced nucleosomes (Gottschling and Cech, 1984; Lawn et al., 1978; Wada and Spear, 1980). Yet it remains unknown how and where nucleosomes are organized within these miniature chromosomes and if this in turn regulates (or is regulated by) 6 mA deposition.
The ciliate Oxytricha is a natural source of tools for RNA-guided genome reorganization and other nucleic acid modification. Long template RNAs instruct new linkages between pieces of DNA (Nowacki et al. 2008), and small RNAs instruct which DNA segments to keep (Fang et al. 2012) or eliminate. Foreseeable uses of these or other machinery derived from the Oxytricha genome include in vitro and/or in vivo modification of nucleic acids.
Intriguingly, in green algae, basal yeast, and ciliates, 6 mA is enriched in ApT dinucleotide motifs within nucleosome linker regions near promoters (Fu et al., 2015; Hattman et al., 1978; Karrer and VanNuland, 1999; Mondo et al., 2017; Pratt and Hattman, 1981; Wang et al., 2017). In the present disclosure, four ciliate proteins-named MTA1, MTA9, p1, and p2—have been identified as being necessary for 6 mA methylation in a complex form termed MTA1c. MTA1 and MTA9 contain divergent MT-A70 domains, while p1 and p2 are homeobox-like proteins that likely function in DNA binding. The present disclosure delineates key biochemical properties of this methyltransferase and dissects the function of 6 mA in vitro and in vivo.
The present disclosure provides a novel ciliate enzyme “MTA1” effective for N6-methyladenine (m6dA) methylation of DNA (see, e.g., Appendix 4). MTA1 has been identified in a ciliate, Tetrahymena thermophila, and its functional role validated in m6dA methylation in Oxytricha. (See, Genbank ID: XP 001032074.3 [Tetrahymena MTA1] and EJY79437.1 [Oxytricha MTA1]). MTA1 is evolutionarily distinct from all known m6dA methyltransferases. Evolutionary analysis reveals that it is present in ciliates (including Oxytricha and Tetrahymena), algae, and basal fungi, but not multicellular eukaryotes. MTA1 exhibits a unique substrate specificity in vivo, being essential for the deposition of dimethylated AT (5′-A*T-3′/3′-TA*-5′), as well as a wide range of other motifs in vivo (
The present disclosure provides that MTA1c or any components thereof presents immediate commercial applications in: 1) generation of DNA substrates containing m6dA at locations distinct from known m6dA methyltransferases, circumventing the need for slow, expensive synthesis of methylated DNA; and 2) rational design of N6-adenine methylating enzymes with novel substrate specificities.
Accordingly, one embodiment of the present disclosure is a method of modifying a nucleic acid from a cell, the cell derived from a multicellular eukaryote. This method comprises the steps of: (a) obtaining the nucleic acid from the cell; and (b) contacting the nucleic acid with MTA1c or any components thereof under conditions effective to methylate the nucleic acid.
The modified base, m6dA, has been discovered in a wide range of eukaryotes, including humans. m6dA levels are significantly reduced in gastric and liver cancer tissues, and disruption of m6dA promotes tumor formation (Xiao et al. 2018). As disclosed herein, MTA1 is a novel m6dA “writer”, paving the way for cost-effective methods to understand mechanisms of m6dA function in biomedically relevant models.
Accordingly, another embodiment of the present disclosure is a method of treating or ameliorating the effects of a disease characterized by an abnormal level of m6dA in a subject. This method comprises administering to the subject an amount of MTA1c or any components thereof effective to modulate m6dA levels in the subject. In some embodiments, the modulation comprises restoring m6dA levels to normal or near-normal ranges in the subject.
Another embodiment of the present disclosure is a pharmaceutical composition comprising MTA1c or any components thereof that is effective to modulate m6dA levels in a subject in need thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
Yet another embodiment of the present disclosure is a kit for treating or ameliorating the effects of a disease characterized by an abnormal level of m6dA in a subject, such as, e.g., cancer, comprising an effective amount of MTA1c or any components thereof, packaged together with instructions for its use.
Another embodiment of the present disclosure is a cell line obtained from a multicellular eukaryote comprising a nucleic acid encoding MTA1c or any components thereof and/or an MTA1c protein complex or any components thereof. As used herein, a “cell line” refers to all types of cell lines such as, e.g., immortalized cell lines and primary cell lines. In certain embodiments, the nucleic acid encoding MTA1c or any components thereof is operably linked to a recombinant expression vector.
Another embodiment of the present disclosure is a recombinant expression vector comprising a polynucleotide encoding MTA1c or any components thereof.
Still another embodiment of the present disclosure is a transgenic organism whose genome comprises a transgene comprising a nucleotide sequence encoding MTA1c or any components thereof. Non-limiting examples of possible organism include an archaea, a bacterium, a eukaryotic single-cell organism, algae, a plant, an animal, an invertebrate, a fly, a worm, a cnidarian, a vertebrate, a fish, a frog, a bird, a mammal, an ungulate, a rodent, a rat, a mouse, and a non-human primate.
The present disclosure also provides a method of identifying protein binding sites on DNA. This method comprises the steps of: (a) providing DNA; (b) contacting the DNA with MTA1c or any components thereof under conditions effective to methylate the DNA; (c) contacting the DNA with one or more proteins; (d) contacting the DNA with an enzyme effective to hydrolize the DNA in positions where no protein binding occurs; (e) removing the DNA bound protein; and (f) isolating and sequencing the DNA fragments. In certain embodiments, the one or more proteins in step (c) comprise histone octamers.
Another embodiment of the present disclosure is a method of mediating DNA N6-adenine methylation. This method comprises the steps of: (a) providing DNA; and (b) contacting the DNA with MTA1c or any components thereof under conditions effective to methylate the DNA.
Another embodiment of the present disclosure is a method of modulating nucleosome organization and/or transcription in a cell, comprising providing to the cell an agent that is effective to modulate the expression of MTA1c or any components thereof.
The present disclosure also provides a method of generating a synthetic chromosome. This method comprises the steps of: (a) generating chromosome segments containing terminal restriction sites, wherein the chromosome segments comprise one or more m6dA bases; (b) digesting the chromosome segments with a restriction enzyme; and (c) purifying and ligating the digested chromosome segments to form a synthetic chromosome. In some embodiments, the method further comprises enriching the synthetic chromosome. A synthetic chromosome made by the method above is also provided.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
DNA N6-adenine methylation (6 mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. In the present disclosure, it's reported a DNA 6 mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6 mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. The present disclosure investigates the impact of 6 mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6 mA in native or ectopic positions. It's shown that 6 mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. The present disclosure identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6 mA in chromatin organization.
One embodiment of the present disclosure is a method of modifying a nucleic acid from a cell, the cell derived from a multicellular eukaryote. This method comprises the steps of: (a) obtaining the nucleic acid from the cell; and (b) contacting the nucleic acid with MTA1c or any components thereof under conditions effective to methylate the nucleic acid.
In some embodiments, the nucleic acid is RNA or DNA. In some embodiments, the eukaryotic cell is mammalian. In some embodiments, the multicellular eukaryote is a human. In some embodiments, the modification is a DNA N6-adenine methylation including one of more of the following motifs: dimethylated AT (5′-A*T-3′/3′-TA*-5′), dim ethylated TA (5′-TA*-3′/3′-A*T-5′), dim ethylated AA (5′-A*A*-3′/3′-TT-5′), methylated AT (5′-A*T-3′/3′-TA-5′), methylated AA (5′-A*A-3′/3′-TT-5′), methylated AC (5′-A*C-3′/3′-TG-5′), methylated AG (5′-A*G-3′/3′-TC-5′), methylated TA (5′-TA*-3′/3′-AT-5′), methylated AA (5′-AA*-3′/3′-TT-5′), methylated CA (5′-CA*-3′/3′-GT-5′), and methylated GA (5′-GA*-3′/3′-CT-5′). In certain embodiments, the MTA1 or an ortholog thereof comprises a mutation effective to abrogate dimethylation of the nucleic acid. Preferably, the mutation comprises loss of a C-terminal methyltransferase domain. In some embodiments, the MTA1c or any components thereof is obtained from ciliates, algae, or basal fungi. Preferably, the MTA1c or any components thereof is obtained from Oxytricha or Tetrahymena.
As used herein, an “ortholog,” or orthologous gene, is a gene with a sequence that has a portion with similarity to a portion of the sequence of a known gene, but found in a different species than the known gene. An ortholog and the known gene originated by vertical descent from a single gene of a common ancestor. As used herein an ortholog encodes a protein that has a portion of at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% or at least about 80% of the total length of the sequence of the encoded protein that is similar to a portion of a length of at least about 50%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% or at least about 80% of a known protein. The respective portion of the ortholog and the respective portion of the known protein to which it is similar may be a continuous sequence or be fragmented a number, for example, into 1 to about 3, including 2, individual regions within the sequence of the respective protein. For example, the 1 to about 3 regions are arranged in the same order in the amino acid sequence of the ortholog and the amino acid sequence of the known protein. Such a portion of an ortholog has an amino acid sequence that has at least about 40%, at least about 45%, such as at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75% or at least about 80% sequence identity to the amino acid sequence of the known protein encoded by a MTA1 gene.
As used herein, an asterisk “*” indicates the presence of a methylated base. For example, “A*” represents a methylated adenine.
The modified base, m6dA, has been discovered in a wide range of eukaryotes, including humans. m6dA levels are significantly reduced in gastric and liver cancer tissues, and disruption of m6dA promotes tumor formation (Xiao et al. 2018). As disclosed herein, MTA1 is a novel m6dA “writer”, paving the way for cost-effective methods to understand mechanisms of m6dA function in biomedically relevant models.
Accordingly, another embodiment of the present disclosure is a method of treating or ameliorating the effects of a disease characterized by an abnormal level of m6dA in a subject. This method comprises administering to the subject an amount of MTA1c or any components thereof effective to modulate m6dA levels in the subject. In some embodiments, the modulation comprises restoring m6dA levels to normal or near-normal ranges in the subject.
In some embodiments, the subject is a mammal that can be selected from the group consisting of humans, veterinary animals, and agricultural animals. Preferably, the subject is a human.
In some embodiments, the disease is a cancer, e.g., gastric cancer or liver cancer. In certain embodiments, the method further comprises administering to the subject one or more of anti-gastric cancer and anti-liver cancer drugs. Non-limiting examples of anti-liver cancer drugs include Nexavar™ (Sorafenib Tosylate) and Stivarga™ (Regorafenib). Non-limiting examples of anti-gastric cancer drugs include Cyramza™ (Ramucirumab), Doxorubicin Hydrochloride, 5-FU (Fluorouracil Injection), Fluorouracil Injection, Herceptin™ (Trastuzumab), Mitomycin C, Taxotere™ (Docetaxel), Trastuzumab, Afinitor™ (Everolimus), Somatuline Depot™ (Lanreotide Acetate), FU-LV, TPF, and XELIRI.
In some embodiments, the method furthering comprises co-administering to the subject an epigenetic agent that is selected from the group consisting of methylation inhibiting drugs, Bromodomain inhibitors, histone acetylase (HAT) inhibitors, protein methyltransferase inhibitors, histone methylation inhibitors, histone deacetlyase (HDAC) inhibitors, histone acetylases, histone deacetlyases, and combinations thereof.
Another embodiment of the present disclosure is a pharmaceutical composition comprising MTA1c or any components thereof that is effective to modulate m6dA levels in a subject in need thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
Yet another embodiment of the present disclosure is a kit for treating or ameliorating the effects of a disease characterized by an abnormal level of m6dA in a subject, such as, e.g., cancer, comprising an effective amount of MTA1c or any components thereof, packaged together with instructions for its use.
Another embodiment of the present disclosure is a cell line obtained from a multicellular eukaryote comprising a nucleic acid encoding MTA1c or any components thereof and/or an MTA1c protein complex or any components thereof. As used herein, a “cell line” refers to all types of cell lines such as, e.g., immortalized cell lines and primary cell lines. In certain embodiments, the nucleic acid encoding MTA1c or any components thereof is operably linked to a recombinant expression vector.
Another embodiment of the present disclosure is a recombinant expression vector comprising a polynucleotide encoding MTA1c or any components thereof.
Still another embodiment of the present disclosure is a transgenic organism whose genome comprises a transgene comprising a nucleotide sequence encoding MTA1c or any components thereof. Non-limiting examples of possible organism include an archaea, a bacterium, a eukaryotic single-cell organism, algae, a plant, an animal, an invertebrate, a fly, a worm, a cnidarian, a vertebrate, a fish, a frog, a bird, a mammal, an ungulate, a rodent, a rat, a mouse, and a non-human primate.
The present disclosure also provides a method of identifying protein binding sites on DNA. This method comprises the steps of: (a) providing DNA; (b) contacting the DNA with MTA1c or any components thereof under conditions effective to methylate the DNA; (c) contacting the DNA with one or more proteins; (d) contacting the DNA with an enzyme effective to hydrolize the DNA in positions where no protein binding occurs; (e) removing the DNA bound protein; and (f) isolating and sequencing the DNA fragments. In certain embodiments, the one or more proteins in step (c) comprise histone octamers.
Another embodiment of the present disclosure is a method of mediating DNA N6-adenine methylation. This method comprises the steps of: (a) providing DNA; and (b) contacting the DNA with MTA1c or any components thereof under conditions effective to methylate the DNA.
Another embodiment of the present disclosure is a method of modulating nucleosome organization and/or transcription in a cell, comprising providing to the cell an agent that is effective to modulate the expression of MTA1c or any components thereof.
The present disclosure also provides a method of generating a synthetic chromosome. This method comprises the steps of: (a) generating chromosome segments containing terminal restriction sites, wherein the chromosome segments comprise one or more m6dA bases; (b) digesting the chromosome segments with a restriction enzyme; and (c) purifying and ligating the digested chromosome segments to form a synthetic chromosome. In some embodiments, the method further comprises enriching the synthetic chromosome. A synthetic chromosome made by the method above is also provided.
The following examples are provided to further illustrate certain aspects of the present disclosure. These examples are illustrative only and are not intended to limit the scope of the disclosure in any way.
E. coli
15N5-dA nucleoside
Oxytricha trifallax
Vegetative Oxytricha trifallax strain J RB310 was cultured at a density of 1.5×107 cells/L to 2.5×107 cells/L in Pringsheim media (0.11 mM Na2HPO4, 0.08 mM MgSO4, 0.85 mM Ca(NO3)2, 0.35 mM KCl, pH 7.0) and fed daily with Chlamydomonas reinhardtii. Cells were filtered through cheesecloth to remove debris and collected on a 10 pm Nitex mesh for subsequent experiments.
Tetrahymena thermophila
Stock cultures of vegetative Tetrahymena thermophila strain SB210 were maintained in Neff medium (0.25% w/v proteose peptone, 0.25% w/v yeast extract, 0.5% glucose, 33.3 pM FeCl3). These cultures were inoculated into SSP medium (2% w/v proteose peptone, 0.1% w/v yeast extract, 0.2% w/v glucose, 33 pM FeCl3) and grown to log-phase (˜3.5×105 cells/mL) through constant shaking at 125 rpm/30° C.
3×105 vegetative Oxytricha cells were fixed in 1% w/v formaldehyde for 10 min at room temperature with gentle shaking, and then quenched with 125 mM glycine. Cells were lysed by dounce homogenization in lysis buffer (20 mM Tris pH 6.8, 3% w/v sucrose, 0.2% v/v Triton X-100, 0.01% w/v spermidine trihydrochloride) and centrifuged in a 10%-40% discontinuous sucrose gradient (Lauth et al., 1976) to purify macronuclei. The resulting macronuclear preparation was pelleted by centrifugation at 4000×g, washed in 50 ml TMS buffer (10 mM Tris pH 7.5, 10 mM MgCl2, 3 mM CaCl2), 0.25M sucrose), resuspended in a final volume of 300 μL, and equilibriated at 37° C. for 5 min. Chromatin was then digested with MNase (New England Biolabs) at a final concentration of 15.7 Kunitz Units/μL at 37° C. for 1 min 15 s, 3 min, 5 min, 7 min 30 sec, 10 min 30 s, and 15 min respectively. Reactions were stopped by adding ½ volume of PK buffer (300 mM NaCl, 30 mM Tris pH 8, 75 mM EDTA pH 8, 1.5% w/v SDS, 0.5 mg/mL Proteinase K). Each sample was incubated at 65° C. overnight to reverse crosslinks and deproteinate samples. Subsequently, nucleosomal DNA was purified through phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. Each sample was loaded on a 2% agarose-TAE gel to check the extent of MNase digestion. The sample exhibiting −80% mononucleosomal species was selected for MNase-seq analysis, in accordance with previous guidelines (Zhang and Pugh, 2011). Mononucleosome-sized DNA was gel-purified using a QIAquick gel extraction kit (QIAGEN). Illumina libraries were prepared using an NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) and subjected to paired-end sequencing on an Illumina HiSeq 2500 according to manufacturer's instructions. All vecietative Tetrahymena MNase-sea data were obtained from (Beh et al., 2015).
Oxytricha cells were lysed in TRIzol reagent (Thermo Fisher Scientific) for total RNA isolation according to manufacturer's instructions. Poly(A)+ RNA was then purified using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). Oxytricha poly(A)+ RNA was prepared for RNA-seq using the ScriptSeq v2 RNA-Seq Library Preparation Kit (Illumina). Tetrahymena poly(A)+ RNA-seq data was obtained from (Xiong et al., 2012). The 5′ ends of capped RNAs were enriched from vegetative Oxytricha total RNA using the RAMPAGE protocol (Batut et al., 2013), and used for library preparation, Illumina sequencing and subsequent transcription start site determination (ie. “TSS-seq”). These data were used to plot the distribution of Oxytricha TSS positions in
Genomic DNA was isolated from vegetative Oxytricha cells using the Nucleospin Tissue Kit (Takara Bio USA, Inc.). DNA was sheared into 150 bp fragments using a Covaris LE220 ultra-sonicator (Covaris). Samples were gel-purified on a 2% agarose-TAE gel, blunted with DNA polymerase I (New England Biolabs), and purified using MinElute spin columns (QIAGEN). The fragmented DNA was dA-tailed using Klenow Fragment (3′->5′ exo-) (New England Biolabs) and ligated to Illumina adaptors following manufacturer's instructions. Subsequently, 2.2 μg of adaptor-ligated DNA containing 6 mA was immunoprecipitated using an anti-N6-methyladenosine antibody (Cedarlane Labs) conjugated to Dynabeads Protein A (Invitrogen). The anti-6 mA antibody is commonly used for RNA applications, but has also been demonstrated to recognize 6 mA in DNA (Fioravanti et al., 2013; Xiao and Moore, 2011). The immunoprecipitated and input libraries were treated with proteinase K, extracted with phenol:chloroform, and ethanol precipitated. Finally, they were PCR-amplified using Phusion Hot Start polymerase (New England Biolabs) and used for Illumina sequencing.
Vegetative Oxytricha macronuclei were isolated as described in the subheading “in vivo MNase-seq” of this study. Vegetative Tetrahymena macronuclei were isolated by differential centrifugation (Beh et al., 2015). Oxytricha and Tetrahymena cells were not fixed prior to nuclear isolation. Genomic DNA was isolated from Oxytricha and Tetrahymena macronuclei using the Nucleospin Tissue Kit (Macherey-Nagel). Alternatively, whole Oxytricha cells instead of macronuclei were used. SMRT-seq according to manufacturer's instructions, using P5-C3 and P6-C4 chemistry, as in (Chen et al., 2014). Oxytricha and Tetrahymena macronuclear DNA were used for SMRT-seq in
Reads from all biological replicates were merged before downstream processing. All Illumina sequencing data were quality trimmed (minimum quality score=20) and length-filtered (minimum read length=40nt) using Galaxy (Blankenberg et al., 2010; Giardine et al., 2005; Goecks et al., 2010). MNase-seq and 6 mA IP-seq reads were mapped to complete chromosomes in the Oxytricha trifallax JRB310 (August 2013 build) or Tetrahymena thermophila SB210 macronuclear reference genomes (June 2014 build) using Bowtie2 (Langmead and Salzberg, 2012) with default settings, while poly(A). RNA-seq and TSS-seq reads were mapped using TopHat2 (Mortazavi et al., 2008) with August 2013 Oxytricha gene models or June 2014 Tetrahymena gene models, with default settings.
MNase-seq datasets were generated by paired-end sequencing. Within each MNase-seq dataset, the read pair length of highest frequency was identified. All read pairs with length±25 bp from this maximum were used for downstream analysis. On the other hand, 6 mA IP-seq datasets were generated by single-read sequencing. 6 mA IP-seq single-end reads were extended to the mean fragment size, computed using cross-correlation analysis (Kharchenko et al., 2008). The per-basepair coverage of Oxytricha MNase-seq read pair centers and extended 6 mA IP-seq reads were respectively computed across the genome. Subsequently, the per-basepair coverage values were normalized by the average coverage within each chromosome to account for differences in DNA copy number (and hence, read depth) between Oxytricha chromosomes (Swart et al., 2013). The per-basepair coverage values were then smoothed using a Gaussian filter of standard deviation=15. This smoothed data is denoted as “normalized coverage” or “nucleosome occupancy.” Tetrahymena MNase-seq data were processed similarly to Oxytricha, except that DNA copy number normalization was omitted as Tetrahymena chromosomes have uniform copy number (Eisen et al., 2006).
For the MNase-seq analysis in
Nucleosome positions were iteratively called as local maxima in normalized MNase-seq coverage, as previously described (Beh et al., 2015). “Consensus”+1, +2, +3 nucleosome positions downstream of the TSS were inferred from aggregate MNase-seq profiles across the genome (
RNA-seq and TSS-seq read coverage were calculated without normalization by DNA copy number since there is no correlation between Oxytricha DNA and transcript levels (Swart et al., 2013).
Oxytricha TSSs were called from TSS-seq data using CAGEr (Haberle et al., 2015); with clusterCTSS parameters (threshold=1.6, thresholdlsTpm=TRUE, nrPassThreshold=1, method=“paraclu,” removeSingletons=TRUE, keepSingletonsAbove=5). Only TSSs with tags per million counts>0.1 were used for downstream analysis. Tetrahymena TSSs were obtained from (Beh et al., 2015).
We processed SMRT-seq data with SMRTPipe v1.87.139483 in the SMRT Analysis 2.3.0 environment using, in order, the P Fetch, P Filter (with minLength=50, minSubreadLength=50, readScore=0.75, and artifact=−1000), P FilterReports, P Mapping (with gff2Bed=True, pulsemetrics=DeletionQV, IPD, InsertionQV, PulseWidth, QualityValue, MergeQV, SubstitutionQV, DeletionTag, and load PulseOpts=byread), P_MappingReports, P_GenomicConsensus (with algorithm=quiver, outputConsensus=True, and enableMapQVFilter=True), P_ConsensusReports, and P Mod ificationDetection (with identifyModifcations=True, enableMapQVFilter=False, and mapQvThreshold=10) modules. All other parameters were set to the default. The Oxytricha August 2013 reference genome build was used for mapping Oxytricha SMRT-seq reads, with Contig10040.0.1, Contig1527.0.1, Contig4330.0.1, and Contig54.0.1 removed, as they are perfect duplicates of other Contigs in the assembly. Tetrahymena SMRT-seq reads were mapped to the June 2014 reference genome build. Only chromosomes with high SMRT-seq coverage (>=80× for Oxytricha; >=100× for Tetrahymena) were used for all 6 mA-related analyses.
Synthetic Contig1781.0 chromosomes were constructed from “building blocks” of native chromosome sequence (
All chromosomes were dA-tailed using Klenow Fragment (3′->5′ exo-) (New England Biolabs), cloned using a TOPO TA cloning kit (Thermo Fisher) or StrataClone PCR Cloning Kit (Agilent Technologies), transformed into One Shot TOP10 chemically competent E. coli, and sequenced using flanking T7, T3, M13F, or M13R primers.
Vegetative Oxytricha trifallax strain JRB310 was cultured as described in the subheading: “Experimental model and subject details” of this study. Cells were starved for 14 hr and subsequently harvested for macronuclear isolation as described in the subheading: “in vivo MNase-seq” of this study. However, formaldehyde fixation was omitted. Purified nuclei were pelleted by centrifugation at 4000×g, resuspended in 0.421 mL 0.4N H2SO4 per 106 input cells, and nutated for 3 hr at 4° C. to extract histones. Subsequently, the acid-extracted mixture was centrifuged at 21,000× a for 15 min to remove debris. Proteins were precipitated from the cleared supernatant using trichloroacetic acid (TCA), washed with cold acetone, then dried and resuspended in 2.5% v/v acetic acid. Individual core histone fractions were purified from crude acid-extracts using semi-preparative RP-HPLC (Vydac C18, 12 micron, 10 mM×250 mm) with 40%-65% HPLC solvent B over 50 min (
All RP-HPLC analyses were performed using 0.1% TFA in water (HPLC solvent A), and 90% acetonitrile, 0.1% TFA in water (HPLC solvent B) as the mobile phases. Wild-type Xenopus H4, H3 C110A, H2B and H2A proteins were expressed in BL21(DE3) pLysS E. coli and purified from inclusion bodies through ion exchange chromatography (Debelouchina et al., 2017). Purified histones were characterized by ESI-MS using a MicrOTOF-Q II ESI-Qq-TOF mass spectrometer (Bruker Daltonics). H4: calculated 11,236 Da, observed 11,236.1 Da; H3 C110A: calculated 15,239 Da, observed 15,238.7 Da; H2A: calculated 13,950 Da, observed 13,949.8 Da; H2B: calculated 13,817 Da, observed 13,816.8 Da.
Oxytricha and Xenopus histone octamers were respectively refolded from core histones using established protocols (Beh et al., 2015; Debelouchina et al., 2017). Briefly, lyophilized histone proteins (Xenopus modified or wild-type; Oxytricha acid-extracted) were combined in equimolar amounts in 6 M guanidine hydrochloride, 20 mM Tris pH 7.5 and the final concentration was adjusted to 1 mg/mL. The solution was dialyzed against 2M NaCl, 10 mM Tris, 1 mM EDTA, and the octamers were purified from tetramer and dimer species using size-exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare Life Sciences). The purity of each fraction was analyzed by SDS-PAGE. Pure fractions were combined, concentrated and stored in 50% v/v glycerol at −20° C.
98 full-length chromosomes were individually amplified from Oxytricha trifallax strain JRB310 genomic DNA using Phusion DNA polymerase (New England Biolabs). Primer pairs are listed in Table 2. Amplified chromosomes were separately purified using a MinElute PCR purification kit (QIAGEN), and then mixed in equimolar ratios to obtain “mini-genome” DNA. The sample was concentrated by ethanol precipitation and adjusted to a final concentration of ˜1.6 mg/mL.
Macronuclei were isolated from vegetative Oxytricha trifallax strain JRB310 as described in the subheading “in vivo MNase-seq” of this study. However, cells were not fixed prior to nuclear isolation. Genomic DNA was purified using the Nucleospin Tissue kit (Macherey-Nagel). Approximately 200 μg of genomic DNA was loaded on a 15%-40% linear sucrose gradient and centrifuged in a SW 40 Ti rotor (Beckman Coulter) at 160,070×g for 22.5 hr at 20° C. Sucrose solutions were in 1M NaCl, 20 mM Tris pH 7.5, 5 mM EDTA. Individual fractions from the sucrose gradient were analyzed on 0.9% agarose-TAE gels. Fractions containing high molecular weight DNA that migrated at the mobility limit were discarded as such DNA species were found to interfere with downstream chromatin assembly. All other fractions were pooled, ethanol precipitated, and adjusted to 0.5 mg/mL DNA.
Chromatin assemblies were prepared by salt gradient dialysis as previously described (Beh et al., 2015; Luger et al., 1999), or using mouse NAP1 histone chaperone and Drosophila ACF chromatin remodeler as previously described (An and Roeder, 2004; Fyodorov and Kadonaga, 2003). Details of each chromatin assembly procedure are listed below. To reduce sample requirements while maintaining adequate DNA concentrations for chromatin assembly, synthetic chromosomes were first mixed with a hundred-fold excess of “buffer” DNA (PCR-amplified Oxytricha Contig17535.0). We verified that nucleosome occupancy in the methylated region (qPCR primer pairs 6 and 7) of the synthetic chromosome is unaffected by the presence of buffer DNA (
For chromatin assembly through salt dialysis: histone octamers and (synthetic chromosome+buffer) DNA were mixed in a 0.8:1 mass ratio, while histone octamers and (native or mini-genome) DNA were mixed in a 1.3:1 mass ratio, each in a 50 μL total volume. Samples were first dialyzed into start buffer (10 mM Tris pH 7.5, 1.4M KCl, 0.1 mM EDTA pH 7.5, 1 mM DTT) for 1 hr at 4° C. Then, 350 mL end buffer (10 mM Tris pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT) was added at a rate of 1mUmin with stirring. The assembled chromatin was dialyzed overnight at 4° C. into 200 mL end buffer, followed by a final round of dialysis in fresh 200 mL end buffer for 1 hr at 4° C. The assembled chromatin was then adjusted to 50 mM Tris pH 7.9, 5 mM CaCl2) and digested with MNase (New England Biolabs) to mainly mononucleosomal DNA as previously described (Beh et al., 2015).
For chromatin assembly using mouse NAP1 and Drosophila ACF: NAP1 was recombinantly expressed and purified as described in (An and Roeder, 2004). ACF was purchased from Active Motif. 0.49 μM NAP1 and 58 nM histone octamer were first mixed in a 302p1 reaction volume containing 62 mM KCl, 1.2% w/v polyvinyl alcohol (Sigma Aldrich), 1.2% w/v polyethylene glycol 8000 (Sigma Aldrich), 25 mM HEPES-KOH pH 7.5, 0.1 mM EDTA-KOH, 10% v/v glycerol, and 0.01% v/v NP-40. The NAP1-histone mix was incubated on ice for 30 min. Meanwhile, “AM” mix was prepared, consisting of 20 mM ATP (Sigma Aldrich), 200 mM creatine phosphate (Sigma Aldrich). 33.3 mM MgCl2, 33.3 μg/μl creatine kinase (Sigma Aldrich) in a 56u1 reaction volume. After the 30 min incubation. 5.29 μl of 1.7 μM ACF complex (Active Motif) and the “AM” mix were sequentially added to the NAP1-histone mix. Then, 10.63 μl of native or mini-genome DNA (2.66 μg) was added, resulting in a 374 μl reaction volume. The final mixture was incubated at 27° C. for 2.5 hr to allow for chromatin assembly. Subsequently, CaCl2 was added to a final concentration of 5 mM, and the chromatin was digested with MNase (New England Biolabs) to mainly mononucleosomal DNA as previously described (Beh et al., 2015).
Mononucleosome-sized DNA from MNase-digested chromatin was gel-purified and used for tiling qPCR on a Viia 7 Real-Time PCR System with Power SYBR Green PCR master mix (Thermo Fisher), or in vitro MNase-seq on an Illumina HiSeq 2500, according to the manufacturer's instructions. qPCR primer sequences are listed in Table 2.
Tiling qPCR Analysis of Nucleosome Occupancy
qPCR data were analyzed using the ΔΔCt method (Livak and Schmittgen, 2001). At each locus along the synthetic chromosome, ΔCt=(Ct at locus of interest)−(Ct at qPCR primer pair 22, far from the methylated region). See
ATP-dependent nucleosome spacing was performed in accordance with a previous study (Lieleg et al., 2015). Chromatin was assembled by salt gradient dialysis as described above, and then adjusted to 20 mM HEPES-KOH pH 7.5, 80 mM KCl, 0.5 mM EGTA, 12% v/v glycerol, 10 mM (NH4)2SO4, 2.5 mM DTT. Samples were then incubated for 2.5 hr at 27° C. with 3 mM ATP, 30 mM creatine phosphate, 4 mM MgCl2, 5 ng/0 creatine kinase, and 11 ng/μL ACF complex (Active Motif). Remodeled chromatin was then adjusted to 5 mM CaCl2) and subjected to MNase digestion, mononucleosomal DNA purification, and qPCR analysis as described above.
The MTA1 amino acid sequence (UniProt ID: J9IF92 9SPIT) was queried against the NCBI nr database using PSI-BLAST (Altschul et al., 1997; Schaffer et al., 2001) (maximum e-value=1e−4; enable short queries and filtering of low complexity regions). Retrieved hits were collapsed using CD-HIT (Huang et al., 2010) with minimum sequence identity=0.97 to remove redundant sequences. The resulting sequences were added to existing MT-A70 alignments from (Greer et al., 2015) using MAFFT (-add) (Katoh et al., 2017; Kuraku et al., 2013). Gaps and duplicate sequences were removed from the merged alignment. Only sequences corresponding to the taxa in
The above procedure was also used for constructing phylogenetic trees from p1 (UniProt ID: Q22VV9 TETTS) and p2 (UniProt ID: I7M8B9 TETTS). However, protein sequences were aligned using MAFFT without adding to an existing alignment.
Preparation of Nuclear Extracts with DNA Methyltransferase Activity
Vegetative Tetrahymena cells were grown in SSP medium to log-phase (˜3.5×106 cells/mL) and collected by centrifugation at 2,300×g for 5 min in an SLA-3000 rotor. The supernatant was discarded, and cells were resuspended in medium B (10 mM Tris pH 6.75, 2 mM MgCl2, 0.1M sucrose, 0.05% w/v spermidine trihydrochloride, 4% w/v gum Arabic, 0.63% w/v 1-octanol, and 1 mM PMSF). Gum arabic (Sigma Aldrich) is prepared as a 20% w/v stock and centrifuged at 7,000×g for 30 min to remove undissolved clumps. For each volume of cell culture, one-third volume of medium B was added to the Tetrahymena cell pellet. Cells were resuspended and homogenized in a chilled Waring Blender (Waring PBB212) at high speed for 40 s. The resulting lysate was subsequently centrifuged at 2,750×g for 5 min in an SLA-3000 rotor to pellet macronuclei. The nuclear pellet was washed twice with medium B and then five times in MM medium (10 mM Tris-HCl pH 7.8, 0.25M sucrose, 15 mM MgCl2, 0.1% w/v spermidine trihydrochloride, 1 mM DTT, 1 mM PMSF). Macronuclei were pelleted between wash steps by centrifuging at 2,500×g for 5 min in an SLA-3000 rotor. Finally, the total number of washed macronuclei was counted with a hemocytometer using a Zeiss ID03 microscope. Nuclear proteins were extracted by vigorously resuspending the pellet in M M salt buffer (10 mM Tris-HCl pH 7.8, 0.25M sucrose, 15 mM MgCl2, 350 mM NaCl, 0.1% w/v spermidine trihydrochloride, 1 mM DTT, 1 mM PMSF). 1 mL M M salt buffer was added per 2.33×108 macronuclei. The viscous mixture was nutated for 45 min at 4° C., and then cleared at 175,000×g for 30 min at 4° C. in a SW 41 Ti rotor. Following this, the supernatant was dialyzed in a Slide-A-Lyzer 3.5K MWCO cassette (Thermo Fisher) overnight at 4° C. against two changes of MM minus medium (10 mM Tris-HCl pH 7.8, 15 mM MgCl2, 1 mM DTT, 0.5 mM PMSF). The dialysate was then centrifuged at 7,197×g for 1 hr at 4″C to remove precipitates, and dialyzed overnight in a Slide-A-Lyzer 3.5K MWCO cassette (Thermo Fisher) at 4° C. against two changes of MN3 buffer (30 mM Tris-HCl pH 7.8, 1 mM EDTA, 15 mM NaCl, 20% v/v glycerol, 1 mM DTT, 0.5 mM PMSF). The final dialysate was cleared by centrifugation at 7,197 g for 1.5 hr at 4° C., flash frozen, and stored at −80° C. This nuclear extract was used for all subsequent biochemical fractionation and 6 mA methylation assays.
Partial Purification of MTA1c from Nuclear Extracts
Tetrahymena nuclear extracts were passed through a HiTrap O HP column (GE Healthcare) and eluted using a linear aradient of 15 mM to 650 mM NaCl in 30 mM Tris-HCl pH 7.8, 1 mM EDTA, 20% v/v glycerol, 1 mM DTT, 0.5 mM PMSF, over 30 column volumes. Each fraction was assayed for DNA methyltransferase activity using radiolabeled SAM as described in the next section. The DNA methyltransferase activity eluted in two peaks, at ˜60 mM and ˜365 mM NaCl, termed the “low salt sample” and “high salt sample.” Fractions corresponding to each peak were pooled and passed through a HiTrap Heparin HP column (GE Healthcare). Bound proteins were eluted using a linear gradient of 60 mM to 1M NaCl (for the low salt sample) or 350 mM to 1M NaCl (for the high salt sample) over 30 column volumes. Fractions with DNA methyltransferase activity were respectively pooled and dialyzed into 10 mM sodium phosphate pH 6.8, 100 mM NaCl, 10% v/v glycerol, 0.3 mM CaCl2), 0.5 mM DTT (for the low salt sample); or 30 mM Tris-HCl pH 7.8, 1 mM EDTA, 200 mM NaCl, 10% v/v glycerol, 1 mM DTT, 0.2 mM PMSF (for the high salt sample). The dialyzed low salt sample was passed through a Nuvia cPrime column (Bio-Rad) and eluted using a linear gradient of 100 mM to 1M NaCl in 50 mM sodium phosphate pH 6.8, 10% v/v glycerol, 0.5 mM DTT. Separately, the dialyzed high salt sample was fractionated using a Superdex 200 10/300 GL column (GE Healthcare) in 30 mM Tris-HCl pH 7.8, 1 mM EDTA, 200 mM NaCl, 10% v/v glycerol, 1 mM DTT. Fractions from the Nuvia cPrime and Superdex 200 columns were dialyzed into 30 mM Tris-HCl pH 7.8, 1 mM EDTA, 15 mM NaCl, 20% v/v glycerol, 1 mM DTT, 0.5 mM PMSF and assayed for DNA methyltransferase activity. Those with qualitatively low, medium, and high activity were subjected to mass spectrometry to identify candidate methyltransferase proteins (
Full length MTA1, MTA9, p1, and p2 open reading frames were codon-optimized for bacterial expression and cloned into a pET-His6-SUMO vector using ligation independent cloning. Protein sequences are listed in Table 3. The vector was a gift from Scott Gradia (Addgene plasmid #29659; http://addgene.org/29659; RRID: Addgene 29659). Mutations in the MTA1 open reading frame was introduced using the OS® Site-Directed Mutagenesis Kit (New England Biolabs). For recombinant expression, pET-His6-SUMO-MTA1 (wild-type and mutant) was transformed into SHuffle T7 competent E. co/i (New England Biolabs); pET-His6-SUMO-MTA9 was transformed into Lemo (DE3) competent E. coli (New England Biolabs); pET-His6-SUMO-p1 and pET-His6-SUMO-p2 were transformed into BL21(DE3) competent E. coli (New England Biolabs). IPTG induction was performed at 16′C overnight. Induced cells were resuspended in 25 ml of lysis buffer B (50 mM Tris pH 7.8, 300 mM NaCl, 5% v/v glycerol, 10 mM imidazole, 5 mM BME, 1 mM PMSF, 0.5× ProBlock Gold Bacterial protease inhibitor cocktail [GoldBio]). The cells were sonicated at 35% amplitude for a total of 4 minutes, with a 10 s off, 10 s cycle using a Model 505 Sonic Dismembrator (Fisherbrand). Lysates were cleared by centrifugation at 30,000 g for 30 min at 4° C., mixed with pre-washed Ni-NTA agarose (Invitrogen), and nutated for 45 min at 4° C. The resin was subsequently washed with lysis buffer and eluted in 50 mM Tris pH 7.8, 300 mM NaCl, 5% v/v glycerol, 400 mM glycerol, 5 mM BME, lx ProBlock Gold bacterial protease inhibitor cocktail [GoldBio]). Eluates were dialyzed into lysis buffer B and then digested with TEV protease (gift from S.H. Sternberg) at 4° C. overnight. The resulting mixture was passed through a fresh batch of Ni-NTA agarose (Invitrogen) to remove cleaved affinity tags. The flow-through containing each recombinant protein was flash frozen and used for all downstream methyltransferase assays.
A 954 bp dsDNA PCR product was used in all assays involving Tetrahymena nuclear extract. This substrate was amplified by PCR from Tetrahymena thermophila strain SB210 macronuclear SB210 genomic DNA using PCR primers metGATC F2 and metGATC_R2 (Table 2). The resulting product was purified using Ampure XP beads (Beckman Coulter). This 954 bp region of the genome contains a high level of 6 mA in vivo. Thus, the underlying DNA sequence may be intrinsically amenable to methylation by Tetrahymena MTA1. Note that the amplified 954 bp product is devoid of DNA methylation as unmodified dNTPs were used for PCR. Separately, a 350 bp dsDNA PCR product was used in all assays involving recombinant MTA1, MTA9, p1 and p2. This sequence lacks 5′-NATC-3′ motifs, and was used to reduce background DNA methylation from contaminating Dam methyltransferase in recombinant protein preparations. The 350 bp dsDNA PCR product was amplified from Tetrahymena thermophila strain SB210 macronuclear SB210 genomic DNA using the PCR primers noGATC2 F and noGATC2_R (Table 2), and purified using Ampure XP beads (Beckman Coulter).
For short DNA substrates (<50 bp), oligonucleotides were purchased from Integrated DNA Technologies and either directly used as ssDNA, or annealed with its complementary sequence to obtain dsDNA. To prepare hemimethylated 27 bp dsDNA in
To generate ˜350nt ssRNA and −350 bp dsRNA, the aforementioned 350 bp dsDNA was first PCR-amplified using primers containing T7 overhangs (primer pairs T7noGATC2_F2/noGATC2_R and T7noGATC2_F2/T7noGATC2_R2 respectively; see Table 2 for primer sequences). Each PCR product was used as a template for in vitro transcription using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs). The synthesized RNA was rigorously treated with DNase (ThermoFisher) purified using acid phenol:chloroform extraction, followed by two rounds of chloroform extraction. Each sample was subsequently ethanol precipitated and resuspended in water for use in methyltransferase assays.
For experiments involving nuclear extract, 2.18 μg of 954 bp dsDNA substrate was mixed with 4-8 μl nuclear extract and 0.64 μM 3H-labeled S-adenosyl-L-methionine ([3H]SAM) in 33 mM Tris-HCl pH 7.5. 6 mM EDTA. 4.3 mM BME. in a 15p1 reaction volume. For experiments involving recombinant MTA1c protein components (ie. MTA1, MTA9, p1, and/or p2), ˜3 μM oligonucleotide ssDNA/annealed dsDNA is used. Alternatively, 1.3 μg of 350 bp dsDNA substrate (or an equimolar amount ˜350nt ssRNA, or ˜350 bp dsDNA) was used in place of DNA oligonucleotide substrates. ssRNA was heated at 90° C. for 2 min and snap cooled to minimize secondary structures before mixing with other components of the methyltransferase assay. All samples were incubated overnight at 37° C., and subsequently spotted onto 1 cm×1 cm squares of Hybond-XL membrane (GE Healthcare). Membranes were then washed thrice with 0.2M ammonium bicarbonate, once with distilled water, twice with 100% ethanol, and finally air-dried for 1 hr. Each membrane was immersed in 5 mL Ultima Gold (PerkinElmer) and used for scintillation counting on a TriCarb 2910 TR (Perkin Elmer).
For assays involving nuclear extract: 5.5 pg of 954 bp DNA substrate was mixed with 20 nuclear extract and 0.2 mM S-adenosyl-L methionine (NEB) in 33 mM Tris-HCl pH 7.5, 6 mM EDTA, 4.3 mM BME in a 15p1 reaction volume. For assays involving recombinant MTA1c protein components (ie. MTA1, MTA9, p1, and/or p2), 2.6 μg of 350 bp DNA substrate was mixed with 540 nM MTA1, 90 nM MTA9, 1.5 μM p1, 1.0 μM p2 proteins. The band of expected size in each recombinant protein preparation was compared against a series of BSA standards to calculate protein concentration. All methylation reactions were incubated at 37° C. overnight, then purified using a MinElute purification kit (QIAGEN), denatured at 95° C. for 10 min, and snap cooled in an ice water bath. Samples were spotted on a Hybond N+ membrane (GE Healthcare), air-dried for 5 min and UV-cross-linked with 120,000 μJ/cm2 exposure using an Ultra-Lum UVC-515 Ultraviolet Multilinker. The cross-linked membrane was blocked in 5% milk in TBST (containing 0.1% v/v Tween) and incubated with 1:1,000 anti-N6-methyladenosine antibody (Synaptic Systems) at 4° C. overnight. The membrane was then washed three times with TBST, incubated with 1:3,000 Goat anti-rabbit HRP antibody (Bio-Rad) at room temperature for 1 hr, washed another three times with 1×TBST, and developed using Amersham ECL Western Blotting Detection Kit (GE Healthcare). This dot blot assay was used to measure 6 mA levels in
10.5 μg Oxytricha or Tetrahymena macronuclear genomic DNA was first digested to nucleosides by mixing with 14p1 DNA degradase plus enzyme (Zymo Research) in a 262.5 μl reaction volume. Samples were incubated at 37° C. overnight, then 70° C. for 20 min to deactivate the enzyme.
The internal nucleoside standards 15N5-dA and D3-6 mA were used to quantify endogenous dA and 6 mA levels in ciliate DNA. 15N5-dA was purchased from Cambridge Isotope Laboratories, while D3-6 mA was synthesized as described in the following section. Nucleoside samples were spiked with 1 ng/μl 15N5-dA and 200 pg/μl D3-6 mA in an autosampler vial. Samples were loaded onto a 1 mm×100 mm C18 column (Ace C18-AR, Mac-Mod) using a Shimadzu HPLC system and PAL auto-sampler (20 μl/injection) at a flow rate of 70 μl/min. The column was connected inline to an electrospray source couple to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher). Caffeine (2 pmol/μl in 50% Acetonitrile with 0.1% FA) was injected as a lock mass through a tee at the column outlet using a syringe pump at 0.5p1/min (Harvard PHD 2000). Chromatographic separation was achieved with a linear gradient from 10% to 99% B (A: 0.1% Formic Acid, B: 0.1% Formic Acid in Acetonitrile) in 5 min, followed by 5 min wash at 100% B and equilibration for 10 min with 1% B (total 20 min program). Electrospray ionization was achieved using a spray voltage of 4.50 kV aided by sheath gas (Nitrogen) flow rate of 18 (arbitrary units) and auxiliary gas (Nitrogen) flow rate of 2 (arbitrary units). Full scan MS data were acquired in the Orbitrap at a resolution of 60,000 in profile mode from the m/z range of 190-290. A parent mass list was utilized to acquire MS/MS spectra at a resolution of 7500 in the Orbitrap. LC-MS data were manually interpreted in Xcalibur's Qual browser (Thermo, Version 2.1) to visualize nucleoside mass spectra and to generate extracted ion chromatograms by using the theoretical [M+H] within a range of ±2 ppm. Peak areas were extracted in Skyline (Ver. 3.5.0.9319).
2′-Deoxyadenosine and CD3I were purchased from Sigma Aldrich. Flash chromatography was performed on a Biotage Isolera using silica columns (Biotage SNAP Ultra, HP-Sphere 25 pm). Semi-preparative RP-HPLC was performed on a Hewlett-Packard 1200 series instrument equipped with a Waters XBridge BEH C18 column (5 μm, 10×250 mm) at a flow rate of 4 mL/min, eluting using A (0.1% formic acid in H2O) and B (0.1% formic acid in 9:1 MeCN/H2O). 1H NMR spectra were recorded on a Bruker UltraShield Plus 500 MHz instrument. Data for 1H NMR are reported as follows: chemical shift (8 ppm), multiplicity (s=singlet, br=broad signal, d=doublet, dd=doublet of doublets) and coupling constant (Hz) where possible. 13C NMR spectra were recorded on a Bruker UltraShield Plus 500 MHz.
D3-6 mA (2′Deoxy-6-[D3]-methyladenosine) were synthesized and purified according to (Schiffers et al., 2017). After an initial purification by flash column chromatography, the methylated compounds were further purified by semipreparative RP-HPLC (linear gradient of 0% to 20% B over 30 min) affording the desired compounds in 14% and 10% yields respectively after lyophilization.
1H NMR (500 MHz, D2O) δ 7.98 (s, 1H), 7.77 (s, 1H), 6.17 (m, 1H), 4.54 (m, 1H), 4.10 (m, 1H), 3.79 (dd, J=12.7, 3.2 Hz, 1H), 3.71 (dd, J=12.7, 4.3 Hz, 1H), 2.60 (m, 1H), 2.44 (ddd, J=14.0, 6.3, 3.3 Hz, 1H).
13C NMR (126 MHz, D2O) δ 154.0, 151.5, 146.1, 138.9, 118.4, 87.3, 84.3, 71.1, 61.6, 39.2, 26.4 ppm. (Peak at 26.4 ppm appears as a broad signal. C-D coupling is not resolved).
HR-MS (ESI+): m/z calculated for [C11H13D3N5O3]+ ([M+Hr): 269.1436. found 269.1421.
Samples where topped up to 200p1 with 50 mM ammonium bicarbonate pH 8. TCEP was added to 5 mM final concentration and left to incubate at 60° C. for 10 min. 15 mM chloroacetamide was then added and left to incubate in the dark at room temperature for 30 min. 1 μg of Trypsin Gold (Promega) was added to each sample and incubated end-over-end at 37° C. for 16 hr. An additional 0.25 μg of Trypsin Gold was added and incubated end-over-end at 37° C. for 3 hr. Samples were acidified by adding TFA to 0.2% final concentration, and desalted using SDB stage-tips (Rappsilber et al., 2007). Samples were dried completely in a speedvac and resuspended in 20p1 of 0.1% formic acid pH 3.5 μl was injected per run using an Easy-nLC 1200 UPLC system. Samples were loaded directly onto a 45 cm long 75 pm inner diameter nano capillary column packed with 1.9 μm C18-AQ (Dr. Maisch, Germany) mated to metal emitter in-line with an Orbitrap Fusion Lumos (Thermo Scientific, USA). The mass spectrometer was operated in data dependent mode with the 120,000 resolution MS1 scan (AGC 4e5, Max IT 50 ms, 400-1500 m/z) in the Orbitrap followed by up to 20 MS/MS scans with CID fragmentation in the ion trap. Dynamic exclusion list was invoked to exclude previously sequenced peptides for 60 s if sequenced within the last 30 s, and maximum cycle time of 3 s was used. Peptides were isolated for fragmentation using the quadrupole (1.6 Da window). Ns was utilized. Ion-trap was operated in Rapid mode with AGC 2e3, maximum IT of 300 msec and minimum of 5000 ions.
Raw files were searched using Byonic (Bern et al., 2012) and Sequest HT algorithms (Eng et al., 1994) within the Proteome Discoverer 2.1 suite (Thermo Scientific, USA). 1 Oppm MS1 and 0.4 Da MS2 mass tolerances were specified. Caramidomethylation of cysteine was used as fixed modification, while oxidation of methionine, pyro-Glu from Gln and deamidation of asparagine were specified as dynamic modifications. Trypsin digestion with maximum of 2 missed cleavages were allowed. Files were searched against the Tetrahymena themophila macronuclear reference proteome (June 2014 build), supplemented with common contaminants (27,099 total entries).
Scaffold (version Scaffold 4.8.7, Proteome Software Inc., Portland, Oreg.) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 93.0% probability. Peptide Probabilities from Sequest and Byonic were assigned by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 3 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
A frameshift mutation in the MTA1 gene was created by inserting a small non-coding DNA segment immediately downstream of the MTA1 start codon (
ssRNA was generated by in vitro transcription using a Hi-Scribe T7 High Yield RNA Synthesis Kit (New England Biolabs). The DNA template for in vitro transcription consists of the ectopic DNA segment flanked by 100-200 bp cognate MTA1 sequence. Following DNase treatment, ssRNA was acid-phenol:chloroform extracted and ethanol precipitated. After precipitation, ssRNA was resuspended in nuclease-free water (Ambion) to a final concentration of 1 to 3 mg/mL for injection.
ssRNA Microinjections
Oxytricha cells were mated by mixing 3 mL of each mating type, JRB310 and JRB510, along with 6 mL of fresh Pringsheim media. At 10 to 12 hr post mixing, pairs were isolated and placed in Volvic water with 0.2% bovine serum albumin (Jackson ImmunoResearch Laboratories) (Fang et al., 2012). ssRNA constructs were injected into the macronuclei of paired cells under a light microscope as previously described with DNA constructs (Nowacki et al., 2008). After injection, cells were pooled in Volvic water. At 60 to 72 hr post mixing, the pooled cells were singled out to grow clonal injected cell lines. As clonal population size grew, lines were transferred to 10 cm Petri dishes and grown in Pringsheim media. Only water from the “Volvic” brand has been empirically tested in our laboratory to support Oxytricha growth. Similar products from other vendors have not been tested.
This experiment was performed in
All statistical tests were performed in Python (v2.7.10) or R (v3.2.5), and described in the respective Figure and Table legends.
Oxytricha SMRT-seq data are deposited in SRA under the accession numbers SRA: SRX2335608 and SRX2335607, and GEO: GSE94421. Tetrahymena SMRT-seq and all Oxytricha Illumina data are deposited in NCBI GEO under accession number GEO: GSE94421.
Sequences were manually curated by mapping RNaseq reads to reference gene annotations and verifying the accuracy of predicted exon boundaries.
We generated genome-wide in vivo maps of nucleosome positioning, transcription, and 6 mA in the macronuclei of asexually growing (vegetative) Oxytricha trifallax cells using Mnase sequencing (MNase-seq), poly(Ar RNA sequencing (RNA-seq), transcriptional start site sequencing (TSS-seq), and single-molecule real-time sequencing (SMRT-seq) (
Properties of 6 mA distribution in nucleosome linkers. In Oxytricha, methyl cluster 1=between 5′ chromosome end and +1 nucleosome; methyl cluster 2=between +1 and +2 nucleosome; methyl cluster 3=between +2 and +3 nucleosome. In Tetrahymena, methyl cluster 1=between +1 and +2 nucleosome; methyl cluster 2=between +2 and +3 nucleosome; methyl cluster 3=between +3 and +4 nucleosome. Consensus +1/+2/+3/+4 nucleosome positions: 193, 402, 618, 837 bp downstream of Oxytricha 5′ chromosome ends; 112, 304, 497, 698 bp downstream of Tetrahymena TSSs.
To uncover the functions of 6 mA in vivo, we set out to identify and disrupt putative 6 mA methytransferases (MTases). The Oxytricha genome encodes a large number of candidate methyltransferases (Table 5), rendering it impractical to test gene function, one at a time or in combination. To identify the ciliate 6 mA MTase, we undertook a biochemical approach by fractionating nuclear extracts and identifying candidate proteins that co-purified with DNA methylase activity. The organism of choice for this experiment was Tetrahymena thermophila, a ciliate that divides significantly faster than Oxytricha (˜2 h versus 18 h; Cassidy-Hanley, 2012; Laughlin et al., 1983). This faster growth time rendered it feasible to culture large amounts of Tetrahymena cells for nuclear extract preparation. Tetrahymena and Oxytricha exhibit similar genomic localization and 6 mA abundance (
We prepared nuclear extracts from log-phase Tetrahymena cells, since 6 mA could be readily detected at this developmental stage through quantitative MS and PacBio sequencing (
We next investigated the phylogenetic relationship of MTA1 and MTA9 to other eukaryotic MT-A70 domain-containing proteins. Two widely studied mammalian MT-A70 proteins—METTL3 and METTL14 (Ime4 and Kar4 in yeast)-form a heterodimeric complex that is responsible for m6A methylation on mRNA. METTL3 is the catalytically active subunit, while METTL14 functions as an RNA-binding scaffold protein (Sledi arid Jinek, 2016; Wang et al., 2016a, 2016b). MTA1 and MTA9 derive from distinct monophyletic clades, outside of those that contain mammalian METTL3, METTL14, and C. elegans DAMT-1 (METTL4) (
We then sought to determine whether MTA1 and/or MTA9 are bona fide 6 mA methyltransferases. MTA1, but not MTA9, contains a catalytic DPPW motif (
Purification of the MTA1c proteins from an E. coli overexpression system raises the possibility of methyltransferase activity arising from contaminating Dam methylase; however, we exclude this possibility for three reasons. (1) The DNA substrate used in this assay does not contain 5′-NATC-3′ sites, which are recognized and methylated by Dam methylase (Horton et al., 2006). (2) Methyltransferase activity was only observed when all four recombinant proteins were incubated with DNA. If contaminating Dam methylase were present in one or more of these protein preparations, then background activity should be observed when subsets of these proteins are used in the assay. 3) Mutation of MTA1 catalytic residues leads to loss of methylation, which is also inconsistent with contaminating methyltransferase activity.
The Uniprot ID of each gene is listed. The Oxytricha macronuclear genome encodes five genes belonging to the MT-A70 family (Iyer et al., 2016; Swart et al., 2013). Such genes commonly function as RNA m6 A MTases in eukaryotes, having evolved from m.MunI-like MTases in bacterial restriction-modification systems (Iyer et al., 2016). An MT-A70 gene belonging to the METTL4 subclade, DAMT1, is a putative 6 mA methyltransferase in C. elegans (Greer et al., 2015). However, none of the Oxytricha MT-A70 genes in this Table cluster together with METTL4 on a phylogenetic tree (
Percentage of each polypeptide that is covered by peptide data is calculated. “Low Salt Sample” and “High Salt Sample” correspond to partially purified nuclear extracts that elute as two distinct peaks of activity from a Q sepharose anion exchange column (
MTA1c Preferentially Methylates ApT Dinucleotides in dsDNA
We next investigated the substrate preferences of MTA1c. First, in vitro transcription was performed to generate doublestranded RNA (dsRNA) and single-stranded RNA (ssRNA) from the input dsDNA substrate. We found that MTA1c methylates dsDNA but not dsRNA or ssRNA of the same sequence, indicating that it is selective for DNA over RNA (
Since 6 mA methylation mainly lies in ApT dinucleotides in vivo (
Given that 6 mA occurs on both strands of genomic DNA in vivo (
We then asked whether MTA1c activity is modulated not only by the dinucleotide motif sequence per se, but also by flanking sequences. This may manifest as the wide variation in frequency of DNA 4-mer containing a methylated ApT dinucleotide 5′-NA*TN-3′ in vivo (
Having established that MTA1c is a 6 mA methyltransferase, we tested the role of MTA1c in mediating 6 mA methylation in vivo in Oxytricha, for which we have ease of generating mutants. The genome-wide localization of 6 mA is conserved between Oxytricha and Tetrahymena (
What are the phenotypic consequences of 6 mA loss in vivo? It has been proposed that DNA methylation—including 6 mA and cytosine methylation—is involved in nucleosome organization (Fu et al., 2015; Huff and Zilberman, 2014). We thus asked whether nucleosome organization is altered in mta1 mutants. We quantified nucleosome “fuzziness,” defined as the SD of MNase-seq read locations surrounding the called nucleosome peak (Lai and Pugh, 2017; Mavrich et al., 2008). A poorly positioned nucleosome consists of a shallow and wide peak of MNase-seq reads, manifested by a high fuzziness score. Nucleosomes were first grouped according to the change in flanking 6 mA between wild-type and mta1 mutant cells (
Multiple factors, including 6 mA, DNA sequence, and chromatin remodeling complexes, may collectively contribute to nucleosome organization in vivo. The effect of 6 mA could therefore be masked by these elements. We next sought to determine whether 6 mA directly impacts nucleosome organization. To this end, we assembled chromatin in vitro using Oxytricha gDNA, which contains cognate 6 mA. To obtain a matched negative control lacking DNA methylation, 98 complete chromosomes were amplified using PCR (
We then directly compared the impact of 6 mA on nucleosome occupancy in vitro and in vivo. Loss of 6 mA in vitro is achieved by mini-genome construction, while loss in vivo is achieved by the mta1 mutation. For each overlapping DNA window, we calculated the difference in nucleosome occupancy: (1) between native genome and mini-genome DNA in vitro, and (2) between wild-type and mta1 mutants in vivo (
Properties of Oxytricha chromosomes in native genomic DNA and mini-genome DNA. “+/−” indicates one standard deviation above or below the mean.
The above experiments used kinetic signatures from SMRT-seq data to infer the presence of 6 mA marks in genomic DNA. We next sought to confirm that 6 mA is directly responsible for disfavoring nucleosomes in vitro, and to understand how this effect could be overcome by cellular factors. 6 mA-containing oligonucleotides were annealed and subsequently ligated with DNA building blocks to form full-length chromosomes. Importantly, these chromosomes contain 6 mA at all locations identified by SMRT-seq in vivo. The representative chromosome, Contig1781.0, is 1.3 kb, contains a clearly defined TSS, and encodes a single highly transcribed gene with a predicted RING finger domain. The length and gene structure are characteristic of typical Oxytricha chromosomes (
Four chromosome variants were synthesized, with cognate 6 mA sites on neither, one, or both DNA strands (chromosomes 1-4 in
Nucleosome occupancy in vivo is influenced not only by DNA sequences but also by trans-acting factors such as ATP-dependent chromatin remodeling factors (Struhl and Segal, 2013). We used synthetic, methylated chromosomes to test how the well-studied chromatin remodeler ACF responds to 6 mA in native DNA. ACF generates regularly spaced nucleosome arrays in vitro and in vivo (Clapier and Cairns, 2009; Ito et al., 1997). Its catalytic subunit ISWI is conserved across eukaryotes, including Oxytricha and Tetrahymena (Table 5). Synthetic chromosomes were assembled into chromatin by salt dialysis as before and then incubated with ACF in the presence of ATP (
Since mta1 mutants exhibit genome-wide loss of 6 mA, we assayed these cells for transcriptional changes by poly(A)+ RNAseq. Only a small minority of genes show significant changes in gene expression (10% false discovery rate [FDR];
Because the aforementioned phenotypic changes were assayed in vegetative Oxytricha cells, we asked whether MTA1 may play roles outside of this developmental state. MTA1 transcript levels are markedly upregulated in the sexual cycle, as assayed by poly(A). RNA-seq (
The present disclosure has identified MTA1c as a conserved, hitherto undescribed 6 mA methyltransferase. It consists of two MT-A70 proteins (MTA1/MTA9) and two homeobox-like proteins (p1/p2). The composition of MTA1c provides immediate insights into how it specifically methylates DNA (
The observation that MTA1c is more active in the presence of pre-methylated DNA templates is reminiscent of the CpG methyltransferase DNMT1. Yet, MTA1c and DNMT1 exhibit distinct protein domain architectures. Further biochemical studies are required to elucidate the molecular basis of this property. A distinct MT-A70 protein, named TAMT-1, was recently reported to act as a 6 mA methyltransferase in Tetrahymena, (Luo et al., 2018), suggesting that multiple enzymes mediate 6 mA deposition. It remains to be determined how MTA1c and TAMT-1 collectively mediate DNA methylation at various developmental stages, and whether cross-talk occurs between these enzymes.
In addition to identifying the ciliate 6 mA methyltransferase, we investigated the function of 6 mA in vitro by building epigenetically defined chromosomes. We show that 6 mA directly disfavors nucleosome occupancy in a local, quantitative manner, independent of DNA sequence (
Intriguingly, nucleosome organization exhibits only subtle changes after genome-wide loss of 6 mA (
More broadly, our work showcases the utility of Oxytricha chromosomes for advancing chromatin biology. By extending current technologies (Muller et al., 2016), it should be feasible to introduce both modified nucleosomes and DNA methylation in a site-specific manner on full-length chromosomes. Such “designer” chromosomes will serve as powerful tools for studying DNA-templated processes such as transcription within a fully native DNA environment.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
The embodiments described in this disclosure can be combined in various ways. Any aspect or feature that is described for one embodiment can be incorporated into any other embodiment mentioned in this disclosure. While various novel features of the inventive principles have been shown, described and pointed out as applied to particular embodiments thereof, it should be understood that various omissions and substitutions and changes may be made by those skilled in the art without departing from the spirit of this disclosure. Those skilled in the art will appreciate that the inventive principles can be practiced in other than the described embodiments, which are presented for purposes of illustration and not limitation.
The present application is a continuation of PCT international application no. PCT/US2019/042625, filed on Jul. 19, 2019, which claims benefit of claims benefit of U.S. Provisional patent Application Ser. No. 62/701,536, filed on Jul. 20, 2018, and U.S. Provisional patent Application Ser. No. 62/848,414, filed on May 15, 2019. The entire contents of the aforementioned applications are incorporated by reference as if recited in full herein.
This invention was made with government support under GM059708 and GM122555 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62701536 | Jul 2018 | US | |
| 62848414 | May 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/042625 | Jul 2019 | US |
| Child | 17153761 | US |
