Part:BBa_K2881007
mMBD-eGFP-avi-His (XbaI)
MBD stands for Methyl-CpG-binding proteins. This family of proteins contain MBD domains specifically binding to methylated CpG sites in vitro. Our part aims to utilize MBD domain to quantify methylation levels of circulating tumor DNA (ctDNA); it is also useful for characterizing other methylated DNA substrates. A single mMBD domain is fused to eGFP to provide a way to visualize the signal; eGFP also significantly enhances expression of this part in BL-21(DE3). His-tag enables the protein to be purified by Nickel column. Avi-tag serves as a versatile platform for other signal detection/amplification possibilities: it could be specifically recognized by BirA enzyme in vitro to attach a biotin.
Biology
First reported in 1998, Methyl-CpG-binding domain proteins are a family of DNA binding proteins specifically recognizing symmetrically methylated CpG sites on DNA. Multiple variants exist in this family including MBDs 1-4 and MeCP2. These proteins all share an analogous Methyl-CpG-binding domain (MBD), which is the functional part to recognize methylated CpG sites (1). MBD proteins have been studied extensively, revealing that many of them are involved in gene expression regulation (2, 3). One important way for cells to control gene expression is to control methylation levels of CpG sites in the promotor regions. CpG sites are dinucleotides in DNA when a cytosine (C) is followed by a guanine (G) in 5’ to 3’ fashion. Many of them are co-incident with gene promotors and could be modified by methyltransferase/demethylase to add/remove methylation (methyl group at 5’ cytosine) (4). Altered methylation status of promotor CpG sites have been shown to involved in multiple cancers (5). MBD proteins serve as the “epigenome reader” to read the methylation status of CpG sites by MBD domain (6); in vivo, their functions such as protein interactions with HDAC1 (7) could then be achieved by other domains.
Design
In our project, we aim to build a general platform to detect and quantify methylation levels of promotor CpG sites in circulating cellular tumor DNAs (ctDNA). MBD domains have been cloned and expressed in the absence of other domains in MBD proteins; research has also demonstrated that MBD domains alone retain their methylated CpG binding affinity in vitro (8). This piece of evidence lays the foundation of our effort to repurpose MBD domain to detect CpG methylation in in vitro assay contexts. In addition, our design of the MBD parts are advised by the following research:
MBD domain from MBD2 protein
MBD proteins not only vary in their functions, but also has significant differences in their MBD domains. Sequences alignment has been reported to demonstrate the variances in the amino acid sequences. Unsurprisingly, MBD domains in different MBD proteins have also been shown to exhibit different affinities toward methylated CpG sites (9). MBD2 protein was shown to have highest affinity in the protein family; among different species human MBD2 protein gives the highest affinity of 5.9nM (10). Another key distinction among MBD domains is their sequence specificity. MeCP2, for example, selectively associates with sequence-specific CpG sites when naturally coupled with an A/T rich sequence. Its binding activity is lost when the sequence is deleted. This largely limits its potential for a general assay to characterize CpG sites in different sequences. On the other hand, MBD2 shows little sequence specificity (11). Coupled with the advantage of high affinity MBD2 is the best candidate for our DNA methylation quantification project.
MBD-eGFP fusion
One of the earliest proof-of-concept MBD methylation detection platform was reported by Yu in 2010 (8). They fused a single MBD domain with eGFP protein and found out the recombinant protein had higher yield than expressing MBD domain alone in E.Coli. This construct is one of the basic constructs in our project, possessing the advantage of high protein yield and easy quantification with eGFP.
Note
Although this construct does not meet RFC10 standard because of the presence of a single XbaI site in the coding region, XbaI and SpeI were removed from the vector during assembly. As a result, one could digest the construct with EcoRI and PstI to get the part without interferences.
Characterization
MBD recombinant Protein expression and quantification
The constructed plasmids with their sequences confirmed was transformed into E.coli strain BL-21 (DE3) for protein expression. A starter culture containing 6mL autoclaved LB broth and 50ug/mL Kanamycin was grown for each construct. The incubation at 37 degree generally took around 3 hours for the culture to become cloudy. Then, 1.5 mL starter culture was used to grow a 750mL large culture (1:500 dilution). The incubation at 37 degree was stopped when OD600 was around 0.8. The culture was then cooled on ice and 100uL of it was taken as the reference before IPTG induction. The protein expression was induced with 0.5mM IPTG at 18 degree for 16 hours with shaking. 100uL of culture was taken after the induction to assess the expression of the protein. The E.coli cells were harvested by centrifuging the culture at 8000rpm, 4 degree for 10min each time. Each pellet was resuspended in 12mL resuspension buffer (300mM NaCl + 20mM Tris (pH =8)). The E.coli cells were then lysed by adding ~5mg lysozyme and fast freezing in liquid nitrogen for 15min.
5uL DNAseI, 15uL 1M MgCl2 and 3uL 1M CaCl2 were added into thawed E.coli cell lysate to digest the DNA. The lysate was left in room-temperature water bath for 1 hour until it became less viscous. 5uL lysate was taken as the reference before centrifuging for separation. The soluble and insoluble fractions of the lysate was separated by centrifuging at 14500rpm, 4 degree for 1h, and 5uL supernatant was taken to assess the solubility of the protein.
Purification of the recombinant protein generally took use of the His-tag at the end. 1mL Ni-NTA agarose equilibrated with resuspension buffer (300mM NaCl + 20mM Tris (pH = 8)) was used for each construct. The supernatant after centrifugation was loaded, the column was then washed with 3 column volumes of resuspension buffer. 5 4mL elutions containing imidazole gradient was performed (50mM, 100mM, 150mM, 200mM and 250mM). TGX stain-free gel was ran to check the expression, solubility, and Ni column purification For mMBD-eGFP, the expression of the protein is not significant according to the gel and its solubility is also hard to determine, but the band of expected size was observed in Ni elution 2 and 3 (Figure 1). From the gel, Ni elution 3 is purer, so it is used for EMSA later. To get rid of the possible effect of imidazole on later experiment, the mMBD-eGFP Ni elution 3 was dialyzed in 3000 MWCO with 500mL 150mM NaCl + 20mM Tris (7.3) overnight. The concentration of the sample after dialysis was determined by Nanodrop to be 7.88uM.
Electrophoretic gel mobility shrift (EMSA)
MBD constructs were characterized by electrophoretic mobility shrift assay (EMSA) (10). In this method, protein and DNA will be pre-incubated and run on a native polyacrylamide gel. If protein binds to DNA, DNA will be slowed while running; at the same time, a “retardation band” will form when the gel is visualized with DNA-specific dye. DNA not incubated with the proteins was run along the same gel as standards.
mMBD-eGFP were characterized in this project. 24 bp primers containing two methylation sites in close proximity were chosen as the target to test protein-DNA binding, in order to characterize specificity of different protein constructs. To be more clinically relevant, one of the methylation sites on the primer was cg10428836, which was shown to have significant methylation level change in hepatocellular carcinoma (HCC) (14). The other methylation site, by contrast, has little known clinal relevance. Forward and reverse primers were pre-hybridized to dsDNA before the assay; in the figures, primers were specially labeled as like “F 0_0”, which indicates that it was a forward primer with no methylation on both sites “0”. Another example will be “R M_0”, which stands for reverse primer with a site methylated near 5’ end “M” (this methylation site is cg10428836).
Pre-hybridization was performed for: no methylation (both 0_0), hemi-methylation (0_0 + M_0), symmetrical methylation (M_0, M_0), and unsymmetrically methylation (0_M + M_0). A good protein construct will only bind to symmetrically methylated DNA but not hemi-methylated nor unsymmetrically methylated ones; latter will be the noises for our device.
mMBD-eGFP showed hints of protein-DNA interaction while it was not decisively conclusive for a confirmed binding. Although no retardation band was observed, DNA-MBD binding was likely happen because: 1. DNA band became thinner in symmetrically methylated substrate. 2. More “streaks” were present in the same lane. A more well crafted gel may be necessary to visualize mMBD-eGFP/DNA binding events; internal DNA control could also be used in the future to confirm that the DNA band becomes thinner (Figure 1).
Complementary Platforms
MBD domain is good at detecting methylated CpG sites, therefore it is suitable for methylation detection; but two challenges are present when a sensitive and quantitative MBD assay is required, such as characterization of ctDNAs. First of all, quantitative methylation assay typically has an output of methylation percentage such as beta value, which requires knowledge of total DNA content. This remains unknown when the assay only captures MBD-DNA binding activity. Secondly, MBD2 binding is sequence-nonspecific, meaning that it could not differentiate between different methylation sites. This will become a crucial problem when multiple methylation sites are present in the DNA substrates, common for most methylated DNA. In our project, we introduced and characterized a graphene oxide (GO) platform to overcome these two challenges. Details of the platform could be referred to 2018 UC San Diego Wiki page and original reference of the platform were provided (12). In brief, we coupled MBD binding assay with a total DNA quantification fluorescent assay, utilizing GO’s fluorescent quenching property. Differentiation of specific sites are achieved by using complementary fluorescent ssDNA probes. We further improved our design by introducing exonuclease III signal amplification strategy. This platform aims to complement our fusion MBD parts to fully quantify methylation levels of specific CpG sites on ctDNA. We also anticipate MBD parts coupled with this platform will be useful for future projects to quantify other methylated DNA substrates.
references
1. Hendrich B, Bird A (November 1998). "Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins". Mol Cell Biol. 18 (11): 6538–47. PMC 109239. PMID 9774669
2. E. Li, T. H. Bestor, R. Jaenisch, Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915-926 (1992).
3. P. W. Laird, R. Jaenisch, THE ROLE OF DNA METHYLATION IN CANCER GENETICS AND EPIGENETICS. Annual Review of Genetics 30, 441-464 (1996).
4. D. Macleod, R. R. Ali, A. Bird, An Alternative Promoter in the Mouse Major Histocompatibility Complex Class 11 I-A3 Gene: Implications for the Origin of CpG Islands. Molecular and Cellular Biology 18, 4433-4443 (1998).
5. Sun, Chang, Laura L. Reimers, and Robert D. Burk. “Methylation of HPV16 Genome CpG Sites Is Associated with Cervix Precancer and Cancer.” Gynecologic oncology 121.1 (2011): 59–63. PMC. Web. 18 Oct. 2018.
6. Epigenomics. 2015;7(6):1051-73. doi: 10.2217/epi.15.39. Epub 2015 Apr 30.
7. Guezennec, Xavier (2006). "MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties". Molecular and Cellular Biology. 26 (3): 843–851. doi:10.1128/MCB.26.3.843-851.2006. PMC 1347035. PMID 16428440. Retrieved 2018-09-05.
8. Y. Yu, S. Blair, D. Gillespie, R. Jensen, D. Myszka, A. H. Badran, I. Ghosh, A. Chagovetz, Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins. Analytical Chemistry 82, 50125019 (2010).
9. M. F. Fraga, E. Ballestar, G. Montoya, P. Taysavang, P. A. Wade, M. Esteller, The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties. Nucleic Acids Research 31, 1765-1774 (2003).
10. Heimer, Brandon Walter, Methylated DNA detection using a high-affinity methyl-CpG binding protein and photopolymerization-based amplification. http://hdl.handle.net/1721.1/98796
11. Robert J. Klose, Shireen A. Sarraf, Lars Schmiedeberg, Suzanne M. McDermott, Irina Stancheva, Adrian P. Bird, DNA Binding Selectivity of MeCP2 Due to a Requirement for A/T Sequences Adjacent to Methyl-CpG, Molecular Cell, Volume 19, Issue 5, 2005, Pages 667-678, ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2005.07.021. (http://www.sciencedirect.com/science/article/pii/S1097276505015091)
12. Lu Chun-Hua, et al. A Graphene Platform for Sensing Biomolecules. Angewandte Chemie International Edition. Vol 48, Issue 26. Jun2009. https://doi.org/10.1002/anie.200901479
13. Shu-yan Niu, Quan-yi Li, Rui Ren, Shu-sheng Zhang, Enzyme-enhanced fluorescence detection of DNA on etched optical fibers, Biosensors and Bioelectronics, Volume 24, Issue 9, 2009, Pages 2943-2946, ISSN 0956-5663, https://doi.org/10.1016/j.bios.2009.02.022. (http://www.sciencedirect.com/science/article/pii/S0956566309001067)
14. Zhang K, et al. Circulating tumor DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nature material. Oct 2017. DOI: 10.1038/NMAT4997.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 220
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1009
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 220
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 220
- 1000COMPATIBLE WITH RFC[1000]
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