COMPOSITIONS AND METHODS FOR PROMOTING PROLIFERATION IN CARDIOMYOCYTES
20230348858 · 2023-11-02
Inventors
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12N2501/155
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed are methods and compositions for inducing proliferation in cardiomyocyte cells or for high-throughput assays.
Claims
1. A method of promoting cardiomyocyte cell proliferation comprising: providing a demethylase to the cardiomyocyte, wherein the demethylase has enzymatic activity that removes a methyl group from Region 2 of the Bmp10 mRNA.
2. The method of claim 1, wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO: 4 and SEQ ID NO: 5.
3. (canceled)
4. (canceled)
5. The method of claim 1, wherein the demethylase removes a methyl group from site M2+M3 of Region 2.
6. The method of claim 5, wherein site M2+M3 comprises the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
7. The method of claim 6, wherein site M2 comprises SEQ ID NO 11.
8. The method of claim 1, wherein i) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 3 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10; or ii) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 4 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10; or iii) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 5 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10.
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. A method of inducing proliferation in a cardiomyocyte cell comprising: providing an m.sup.6A methylation inhibitor to the cardiomyocyte cell; and/or providing an engineered protein comprising a demethylase to the cell, wherein the demethylase enhances the Brg1 demethylation activity, wherein the engineered protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2, wherein the Brg1 demethylation activity is enhanced relative to the base line level of Brg1.
14. The method of claim 13, wherein said method comprises providing 3-DZA to the cardiomyocyte cell.
15. The method of claim 13, wherein the engineered protein comprises an amino acid sequence having 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO 4, and SEQ ID NO: 5.
16. The method of claim 13, wherein said method comprises providing 3-DZA and a demethylase to the cardiomyocyte cell, wherein the demethylase comprises an amino acid sequence having 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO 4, and SEQ ID NO 5.
17. (canceled)
18. A method of enhancing proliferation of cardiomyocytes in accordance with claim 13, said method, comprising: contacting the cardiomyocytes with an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition includes a demethylase, said demethylase having enzymatic activity that removes a methyl group from Region 2 of Bmp10 mRNA, and a pharmaceutically acceptable carrier.
19. The method of claim 18, wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 4.
20. The method of claim 18, wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5.
21. The method of claim 18, wherein the demethylase is designed to remove a methyl group from site M2+M3 of Region 2.
22. The method of claim 18, wherein site M2+M3 comprises the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
23. The method of claim 22, wherein site M2+M3 consists essentially of SEQ ID NO 11.
24. The method of claim 18, wherein i) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 3 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10; or ii) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 4 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10; or iii) the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 5 and Region 2 comprises a nucleic acid sequence having 95% sequence identity to SEQ ID NO 10.
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. A culture medium, comprising: a buffer, and a pharmaceutical composition that includes a demethylase and a pharmaceutically acceptable carrier, wherein the amount of demethylase present in the culture medium is effective to remove a methyl group from Region 2 of Bmp10 mRNA when in contact with a cardiomyocyte.
30. (canceled)
31. (canceled)
32. The culture media of claim 29 wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO: 4 and SEQ ID NO: 5.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Definitions
[0052] In describing and claiming the invention, the following terminology will be used in accordance with the definitions set forth below.
[0053] As used herein, the term “Brg1” refers to Brahma-related gene-1, a protein generally understood to be involved in numerous chromatin-modifying enzymatic complexes, and in this disclosure was discovered to have a separate mechanism of action as a demethylase in cardiomyocytes as well as other cell types.
[0054] As used herein, the term “D1D2” refers to Brg1's helicase domain comprising D1 coupled to Insertion coupled to D2 as illustrated in
[0055] As used herein, the term “R/R+H/D” refers to specific amino acid sequences found within the Insertion and D2 regions of the Brg1 helicase domain, as shown in
[0056] As used herein, the term “Bmp10” mRNA refers to bone morphogenetic protein-10 mRNA.
[0057] As used herein, the term “Region 2” refers to a section of Bmp10 mRNA where D1D2 interacts and removes the m.sup.6A from Bmp10 mRNA, and is illustrated in
CCAUGAAGAGGUCGUCAUGGCUGA[A]CUGCGGUUGU[A]C[A]CGCUGGUGCAGAG AGAUCGCAUGAUGUAUGAUGGCGUGG[A]CCGUAAAAUUACCAUUUUUGAGGU[A]CUAGAGAGUGCAG[A]CGGUAGCGAGGAGGAGAGGAGCA; wherein the underlined nucleic acids identify sites M1, M4, M5, and M6, respectively, and the double underlined nucleic acids identify site M2+M3. The [A] represents the position of a possible methylated adenosine.
[0058] As used herein, the term “site M2+M3” refers to represents amino acids GU[A]C[A]C, wherein the [A] represents a site for a methylated adenosine, within Region 2 of the Bmp10 mRNA.
[0059] As used herein, the term “Site M2” refers to GU[A] within Region 2.
[0060] As used herein, the term “lead compound” refers to a compound of commercial or research interest by an institution or organization.
[0061] As used herein, the term “engineered” includes the term recombinant, and refers to protein or mRNA not naturally found in the body because it includes some kind of modification including, but not limited to, a truncation of the wildtype amino acid or nucleic acid sequence, a mutation, or the addition of a probe. In some embodiments, engineered also encompasses a sequence, mRNA, or protein that is substantially purified.
Embodiments
[0062] This disclosure provides methods and compositions based on a newly discovered mechanism of Brg1. Brg1, a protein, is understood to play a role in chromatin remodeling and organization. Here, the methods and compositions take advantage of a newly discovered non-chromatin remodeling mechanism of Brg1. This disclosure provides methods and compositions based on the newly discovered demethylase activity of Brg1.
[0063] In one embodiment, an engineered protein is provided. The engineered protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2. In one embodiment, the engineered protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 1. The engineered protein may be used to induce or enhance cardiomyocyte cell proliferation for various uses including cell therapies or for the development of high-throughput screening assays.
[0064] In one embodiment, the engineered protein comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein further comprises a probe. The probe may be an antibody, a protein, a bead, a fluorophore, a radioisotope, or a combination thereof. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence having at least 95% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence of SEQ ID NO 1. In one embodiment, the engineered protein comprises a biotin coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1. In one embodiment, an engineered protein comprises a biotin coupled to SEQ ID NO 1. In one embodiment, an engineered protein consists essentially of a biotin coupled to SEQ ID NO 1. In one embodiment, an engineered protein comprises an amino acid sequence of SEQ ID NO 27 coupled to SEQ ID NO 1.
[0065] In one embodiment, the engineered protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, an engineered protein comprises a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, an engineered protein consists essentially of a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence of SEQ ID NO 2. In one embodiment, the engineered protein comprises a biotin coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, an engineered protein comprises a biotin coupled to SEQ ID NO 2. In one embodiment, an engineered protein consists essentially of a biotin coupled to SEQ ID NO 2. In one embodiment, an engineered protein comprises an amino acid sequence of SEQ ID NO 27 coupled to SEQ ID NO 2.
[0066] In one embodiment, the engineered protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, an engineered protein comprises a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, an engineered protein consists essentially of a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence of SEQ ID NO 3. In one embodiment, the engineered protein comprises a biotin coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, an engineered protein comprises a biotin coupled to SEQ ID NO 3. In one embodiment, an engineered protein consists essentially of a biotin coupled to SEQ ID NO 3. In one embodiment, an engineered protein comprises an amino acid sequence of SEQ ID NO 27 coupled to SEQ ID NO 3.
[0067] In one embodiment, the engineered protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 4. In one embodiment, an engineered protein comprises a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 4. In one embodiment, an engineered protein consists essentially of a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 4. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence of SEQ ID NO 4. In one embodiment, the engineered protein comprises a biotin coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 4. In one embodiment, an engineered protein comprises a biotin coupled to SEQ ID NO 4. In one embodiment, an engineered protein consists essentially of a biotin coupled to SEQ ID NO 4. In one embodiment, an engineered protein comprises an amino acid sequence of SEQ ID NO 27 coupled to SEQ ID NO 4.
[0068] In one embodiment, the engineered protein comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 5. In one embodiment, an engineered protein comprises a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 5. In one embodiment, an engineered protein consists essentially of a probe and an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 5. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence of SEQ ID NO 5. In one embodiment, the engineered protein comprises a biotin coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 5. In one embodiment, an engineered protein comprises a biotin coupled to SEQ ID NO 5. In one embodiment, an engineered protein consists essentially of a biotin coupled to SEQ ID NO 5. In one embodiment, an engineered protein comprises an amino acid sequence of SEQ ID NO 27 coupled to SEQ ID NO 5.
[0069] In one embodiment, a high-throughput method of testing lead compounds is provided. In one embodiment, the method comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method further includes the step of analyzing the effect of the lead compound on the engineered protein's ability to remove the methyl group from the mRNA.
[0070] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. In one embodiment, the engineered protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, the engineered protein further comprises a probe coupled to the engineered protein. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2. In one embodiment, the engineered protein consists essentially of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 2.
[0071] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. In one embodiment, the engineered protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein further comprises a probe coupled to the engineered protein. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein consists essentially of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In another embodiment, the engineered protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein further comprises a probe. In another embodiment, the engineered protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 3. In one embodiment, the engineered protein further comprises a probe.
[0072] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. In one embodiment, the engineered protein comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein further comprises a probe coupled to the engineered protein. In one embodiment, the engineered protein consists essentially of a probe coupled to an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1. In one embodiment, the engineered protein consists essentially of an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 1.
[0073] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. In one embodiment, the methylated mRNA comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 8. In one embodiment, the methylated mRNA further comprises a probe, wherein the probe may be an antibody, protein, bead, fluorophore, radioisotope, or a combination thereof. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 8. In one embodiment, the methylated mRNA consists essentially of a probe coupled to a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 8. In one embodiment, the methylated mRNA comprises a nucleic acid sequence of SEQ ID NO 8. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence of SEQ ID NO 8.
[0074] In one embodiment, a methylated mRNA comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 9. In one embodiment, the methylated mRNA further comprises a probe, wherein the probe may be an antibody, protein, bead, fluorophore, radioisotope, or a combination thereof. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 9. In one embodiment, the methylated mRNA consists essentially of a probe coupled to a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 9. In one embodiment, the methylated mRNA comprises a nucleic acid sequence of SEQ ID NO 9. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence of SEQ ID NO 9.
[0075] In one embodiment, a methylated mRNA comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 10. In one embodiment, the methylated mRNA further comprises a probe, wherein the probe may be an antibody, protein, bead, fluorophore, radioisotope, or a combination thereof. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 10. In one embodiment, the methylated mRNA consists essentially of a probe coupled to a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 10. In one embodiment, the methylated mRNA comprises a nucleic acid sequence of SEQ ID NO 10. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence of SEQ ID NO 10.
[0076] In one embodiment, a methylated mRNA comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 11. In one embodiment, the methylated mRNA further comprises a probe, wherein the probe may be an antibody, protein, bead, fluorophore, radioisotope, or a combination thereof. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 11. In one embodiment, the methylated mRNA consists essentially of a probe coupled to a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 11. In one embodiment, the methylated mRNA comprises a nucleic acid sequence of SEQ ID NO 11. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence of SEQ ID NO 11.
[0077] In one embodiment, a methylated mRNA comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 12. In one embodiment, the methylated mRNA further comprises a probe, wherein the probe may be an antibody, protein, bead, fluorophore, radioisotope, or a combination thereof. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 12. In one embodiment, the methylated mRNA consists essentially of a probe coupled to a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO 12. In one embodiment, the methylated mRNA comprises a nucleic acid sequence of SEQ ID NO 12. In one embodiment, the methylated mRNA consists essentially of a nucleic acid sequence of SEQ ID NO 12. In one embodiment, a methylated mRNA comprises a biotin coupled to SEQ ID NO 12. In one embodiment, a methylated mRNA consists essentially of a biotin coupled to SEQ ID NO 12.
[0078] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method comprises providing a engineered protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2; providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10, and a methylated adenosine; providing a lead compound; and analyzing the effect of the lead compound on the engineered protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0079] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method comprises providing an engineered protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3; providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10 and a methylated adenosine; providing a lead compound; and analyzing the effect of the lead compound on the engineered protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0080] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method comprises providing an engineered protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2; providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NOs 12 which includes a methylated adenosine; providing a lead compound; and analyzing the effect of the lead compound on the engineered protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0081] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method comprises providing an engineered protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3; providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 12 and a methylated adenosine; providing a lead compound; and analyzing the effect of the lead compound on the engineered protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0082] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein, a methylated mRNA, and a lead compound. The method comprises providing an engineered protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 1; providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 12; providing a lead compound; and analyzing the effect of the lead compound on the engineered protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0083] In one embodiment, a high-throughput method of testing lead compounds comprises providing an engineered protein having at least 95% sequence identity selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, and SEQ ID NO 7; providing a methylated mRNA comprising a nucleic acid sequence having at least 95% sequence identity selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, and SEQ ID NO 12; providing a lead compound, and analyzing the effect of the lead compound on the engineered protein's ability to remove the methyl group from the methylated mRNA. In some embodiments, the engineered protein, the methylated mRNA, the lead compound, or a combination thereof include a probe.
[0084] In one embodiment, the step of analyzing includes methods known to those of skill in the art. In one embodiment, the step of analyzing includes microscopy or electrophoretic mobility shift assay.
[0085] In one embodiment, the present disclosure is directed to a method of inducing proliferation in a cardiomyocyte cell. The method comprises providing a demethylase to the cardiomyocyte cell, wherein the demethylase is designed to remove a methyl group from Region 2 of a Bmp10 mRNA. In one embodiment, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2. In one embodiment, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3. In one embodiment, the demethylase comprises an amino acid sequence having at least 95% identity to SEQ ID NO 4. In one embodiment, the demethylase comprises an amino acid sequence having at least 95% identity to SEQ ID NO 5.
[0086] In one embodiment, the demethylase is designed to remove a methyl group from site M2+M3 of Region 2. In one embodiment, the site M2+M3 comprises the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10. In one embodiment, the demethylase is designed to remove a methyl group from site M2, wherein site M2 comprises SEQ ID NO: 11. In some embodiments, the demethylase is an engineered protein comprising a probe and having at least 95% sequence identity to SEQ ID NO 2, or SEQ ID NO 3.
[0087] In one embodiment, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3 and is designed to remove a methyl group from Region 2 of Bmp10 mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10. In some embodiments, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2 or SEQ ID NO 3 and is designed to remove a methyl group from adenosine of Bmp10 mRNA.
[0088] In one embodiment, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 4 and is designed to remove an m.sup.6A from Region 2, wherein Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0089] In one embodiment, the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5 and is designed to remove an m.sup.6A from Region 2, wherein Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0090] In one embodiment, a method for inducing proliferation in a cardiomyocyte cell is provided. The method comprises providing an m.sup.6A methylation inhibitor to the cardiomyocyte cell in an amount effective to remove a methyl group from Region 2 of Bmp10 mRNA. In one embodiment, the m.sup.6A methylation inhibitor comprising 3-DZA. In one embodiment, the m.sup.6A methylation inhibitor consists essentially of 3-DZA.
[0091] In one embodiment a method of enhancing proliferation of cardiomyocytes is provided. The method comprises contacting the cardiomyocytes with an effective amount of a pharmaceutical composition to remove a methyl group from Region 2 of Bmp10 mRNA, wherein the pharmaceutical composition includes a demethylase and a pharmaceutically acceptable carrier. In some embodiments, the demethylase is an engineered protein comprising a sequence having at least 95% sequence identity selected from a group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5. In some embodiments, the methyl group is bound to an adenosine of Region 2.
[0092] In one embodiment, a method of culturing cardiomyocyte cells is provided. The method comprises introducing a pharmaceutical composition into a cardiomyocyte culture medium, wherein the pharmaceutical composition includes a demethylase and pharmaceutically acceptable carrier. In some embodiments, the demethylase is an engineered protein comprising a sequence having at least 95% sequence identity selected from a group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5.
[0093] In one embodiment a culture medium is provided the culture medium comprises a buffer, and a pharmaceutical composition that includes a demethylase and a pharmaceutically acceptable carrier, wherein the amount of demethylase present in the culture medium is effective to remove a methyl group from Region 2 of Bmp10 mRNA when in contact with a cardiomyocyte. In some embodiment, the demethylase is an engineered protein comprising a sequence having at least 95% sequence identity selected from a group consisting of SEQ ID SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5. In some embodiments, the demethylase further comprises a means of transporting across the memebrane of a cardiomyocyte. Means of transport are known to those skilled in the art and include but are not limited to, peptides, liposome, nanoparticles, and viruses. In some embodiments, the peptide is able to transport the demethylase from the culture medium into the cytosol and nucleus of the cardiomyocyte.
[0094] In one embodiment, a method of producing a culture medium is provided. The method comprises: mixing a buffer with a demethylase, wherein the amount of demethylase present in the buffer is effective to remove a methyl group from Region 2 of Bmp10 mRNA when the demethylase is in contact with a cardiomyocyte. In some embodiment, the demethylase is an engineered protein comprising a sequence having at least 95% sequence identity selected from a group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5.
[0095] In accordance with embodiment 1, a method of promoting cardiomyocyte cell proliferation is provided. The method comprises providing a demethylase to the cardiomyocyte, wherein the demethylase is designed to remove a methyl group from Region 2 of the Bmp10 mRNA.
[0096] In accordance with embodiment 2, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to Brg1 helicase domain SEQ ID NO 3.
[0097] In accordance with embodiment 3, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having 95% identity to SEQ ID NO 4.
[0098] In accordance with embodiment 4, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having at least 95% identity to SEQ ID NO 5.
[0099] In accordance with embodiment 5, the method of embodiment 1 is provided wherein the demethylase is designed to remove a methyl group from site M2+M3 of Region 2.
[0100] In accordance with embodiment 6, the method of embodiment 5 is provided wherein site M2+M3 comprises the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0101] In accordance with embodiment 7 the method of embodiment 6 is provided wherein site M2 comprises of SEQ ID NO 11.
[0102] In accordance with embodiment 8, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0103] In accordance with embodiment 9, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 4 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0104] In accordance with embodiment 10, the method of embodiment 1 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0105] In accordance with embodiment 11, a method of inducing proliferation in a cardiomyocyte cell is provided. The method comprises providing an m.sup.6A methylation inhibitor to the cardiomyocyte cell.
[0106] In accordance with embodiment 12, the method of embodiment 11 is provided wherein the m.sup.6A methylation inhibitor comprising 3-DZA.
[0107] In accordance with embodiment 13, a method of inducing proliferation in a cardiomyocyte cell is provided. The method comprises providing a recombinant protein comprising a demethylase to the cell, wherein the demethylase enhances the Brg1 demethylation activity, wherein the recombinant protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2,wherein the Brg1 demethylation activity is enhanced relative to the base line level of Brg1.
[0108] In accordance with embodiment 14, the method of embodiment 13 is provided wherein the recombinant protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3.
[0109] In accordance with embodiment 15, the method of embodiment 13 is provided wherein the Brg1 helicase domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 4.
[0110] In accordance with embodiment 16, the method of embodiment 13 is provided wherein the Brg1 helicase domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5.
[0111] In accordance with embodiment 17, a method of testing lead compounds in a high-throughput assay is provided. The method comprises providing a recombinant protein comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2; [0112] providing a recombinant mRNA comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10 and a methylated adenosine; [0113] providing a lead compound; and [0114] analyzing the effect of the lead compound on the recombinant protein's ability to demethylate the methylated adenosine of the recombinant mRNA.
[0115] In accordance with embodiment 18, a method of enhancing proliferation of cardiomyocytes is provided. The method comprises contacting the cardiomyocytes with an effective amount of a pharmaceutical composition to remove a methyl group from Region 2 of Bmp10 mRNA, wherein the pharmaceutical composition includes a demethylase and a pharmaceutically acceptable carrier.
[0116] In accordance with embodiment 19, the method of embodiment 18 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 4.
[0117] In accordance with embodiment 20, the method of embodiment 18 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5.
[0118] In accordance with embodiment 21, the method of embodiment 18 is provided wherein the demethylase is designed to remove a methyl group from site M2+M3 of Region 2.
[0119] In accordance with embodiment 22, the method of claim 18 is provided wherein site M2+M3 comprises the nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0120] In accordance with embodiment 23, the method of embodiment 22 is provided wherein site M2+M3 consists essentially of SEQ ID NO 11.
[0121] In accordance with embodiment 24, the method of embodiment 18 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 3 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0122] In accordance with embodiment 25, the method of embodiment 18 is provided wherein the demethylase comprises an amino acid sequence having 95% sequence identity to SEQ ID NO 4 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0123] In accordance with embodiment 26, the method of embodiment 18 is provided wherein the demethylase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO 5 and Region 2 comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO 10.
[0124] In accordance with embodiment 27, a method of culturing cardiomyocyte cells is provided. The method comprises introducing a pharmaceutical composition into a cardiomyocyte culture medium, wherein the pharmaceutical composition includes a demethylase and pharmaceutically acceptable carrier.
[0125] In accordance with embodiment 28, the method of embodiment 27 is provided wherein, the pharmaceutical composition is introduced in an amount to effectively remove a methyl group from Region 2 of Bmp10 mRNA.
[0126] In accordance with embodiment 29, a culture medium is provided. The culture medium comprises: [0127] a buffer, and [0128] a pharmaceutical composition that includes a demethylase and a pharmaceutically acceptable carrier, wherein the amount of demethylase present in the culture medium is effective to remove a methyl group from Region 2 of Bmp10 mRNA when in contact with a cardiomyocyte.
[0129] In accordance with embodiment 30, a method of producing a culture medium is provided. The method comprises: mixing a buffer with a demethylase, wherein the amount of demethylase present in the buffer is effective to remove a methyl group from Region 2 of Bmp10 mRNA when the demethylase is in contact with a cardiomyocyte.
[0130] In accordance with embodiment 31, an engineered protein is provided. The embodiment comprises an amino acid sequence having an amino acid sequence having at least 95% sequence identity to SEQ ID NO 2.
[0131] All amino acid sequences are intended to include their respective nucleic acid sequences, and all nucleic acid sequences, when appropriate, are intended to include their respective amino acid sequences. All nucleic acid sequences are intended to include their complementary nucleic acid sequences.
[0132] The following Examples are provided to expand and further support the above disclosed embodiments. The Examples are not meant to be limiting. Further, any reference or patent application mentioned in this specification are each hereby incorporated in their entirety.
EXAMPLE 1: MICE
[0133] CD1 male and female mice were purchased from Charles River (Strain Code: 022). Sm22αCre.sup.1-3 and Brg1.sup.fl/fl mice were generated similar to the protocol described in Sumi-Ichinose, C., Ichinose, H., Metzger, D. & Chambon, P. SNF2beta-BRG1 is essential for the viability of F9 murine embryonal carcinoma cells. Mol Cell Biol 17, 5976-5986 (1997). The date of observing of a vaginal plug was set as embryonic day E0.5 by convention.
EXAMPLE 2: CELL CULTURE, SIRNA KNOCKDOWN, AND PLASMID TRANSFECTION
[0134] 293T, H1299, and SW13 cell lines were purchased from ATCC. Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (PBS) and 1% 100× Pen/Strep (Gibco). Mouse embryonic ventricular cardiomyocytes were cultured using acceptable protocols. Briefly, ventricles of E18.5 CD1 mouse hearts were excised and digested with type II collagenase 4 or 5 times, for about 15 minutes each. Cells were then collected and resuspended in DMEM with 10% FBS. Cells were plated for about 1 hour to allow the attachment and the removal of non-myocyte cells. Cardiomyocytes were then plated at a density of about 2×10.sup.5/ml and cultured for further experiments. Gene knockdown experiments were conducted using Brg1 siRNA (Santa Cruz, sc-29827), YTHDF2 siRNA (ThermoFisher Scientific, s102905), or negative control siRNA (QIAGEN, 1027281). Transfections were achieved using Lipofectamine RNAiMAX (Invitrogen) for siRNA, Lipofectamine 2000 for single plasmid transfection, or Lipofectamine LTX Plus (Invitrogen) for co-transfection of two or more types of plasmids assay, according to manufacturer's protocols.
EXAMPLE 3: M.SUP.6.A DOT BLOT ASSAY
[0135] RNA samples were quantified using NanoDrop™ 2000c Spectrophotometers (Thermo Fisher Scientific), and equal amounts were spotted onto positively charged nylon membranes (Sigma-Aldrich, 11209299001). The membranes were then UV crosslinked, blocked, and exposed to rabbit anti-m.sup.6A antibody (Synaptic System Cat. No. 202003) at 4° C. overnight. Membranes were then washed in 0.1% PBST, followed by incubation in secondary antibody (HRP-conjugated mouse anti-rabbit IgG, diluted 1:20000 in block) for about 1 hour (hr) at about Room Temperature (RT). Membranes were again washed in 0.1% PBST and developed with SuperSignal ECL Western blot detection kits (Pierce, 34095) using LI-COR odyssey image system.
EXAMPLE 4: TWO-DIMENSION THIN LAYER CHROMATOGRAPHY (2D-TLC) ASSAY
[0136] 2D-TLC was performed. 100 to 200 ng of polyA+ RNAs purified with PolyATtract™ mRNA Isolation Systems IV (Promega, Z5310) were digested using 2000 units of RNAse T1 (ThermoFisher Scientific, FEREN0541) in a final volume of 25 μl in 1× PNK (70 mM Tris-HCl, 10 mM MgCl.sub.2, 5 mM DTT, pH 7.6) (NEB, B0201S) buffer incubated at about 37° C. for about 1 hour. The RNAs were labeled with 10 units of PNK and 1 μl [-.sup.32P] ATP (6000 Ci/mmol, Perkin-Elmer). The reactions were cleaned with a G25 column, and RNAs precipitated with ethanol, re-suspended in 5 μl of 50 mM sodium acetate (pH 5.5), and digested with 1 unit of nuclease P1 (Sigma-Aldrich, N8630-1VL). 1 μl was loaded on a cellulose TLC glass plate (EMD chemicals, 5716-7). The first dimension was resolved in isobutyric acid: 0.5 M NH.sub.4OH (5:3, v/v), and the second dimension resolved in isopropanol:HCl:water. The plates were exposed on a phosphor screen and scanned on a Bio-Rad imaging system.
EXAMPLE 5: M.SUP.6.A-RNA-IP
[0137] m.sup.6A RNA immunoprecipitation was conducted as described with some modifications. Total RNA was isolated using TRIzol reagent. Polyadenylated RNAs (polyA+ RNAs) were enriched from total RNAs using one round of PolyATtract® mRNA Isolation Systems (Promega) followed by further removal of contaminated rRNA using RiboMinus Eukaryote Kit v2 (Ambion). mRNA samples were chemically fragmented into ˜100-nucleotide-long fragments by 5 minutes of incubation at 94° C. in NEBNext® Magnesium RNA Fragmentation buffer (Cat. E6150S). Fragmentation reactions were terminated with RNA Fragmentation Stop Solution, followed by RNA purification using Clean & Concentrator kit (Zymo Research Cat. No. R1017). 20 ng of fragmented mRNAs were saved as input, whereas 200 ng of fragmented mRNAs were incubated with 2 μg of anti-m.sup.6A antibody (Synaptic System Cat. No. 202003) in 1× IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% NP-40) on a rotating wheel overnight at 4° C. The m.sup.6A-RNA mixture was then incubated with 20 μl Protein G (Bio-Rad Cat. No.161-4023) for 2 hours at 4° C. on a rotating wheel. After washing three times (15 minutes each) with 1× IP buffer, bound mRNAs were eluted using 100 μl elution buffer containing 6.7 mM N6-Methyladenosine 5′-monophosphate sodium salt (Sigma-Aldrich, M2780) in 1× IP buffer, followed by mRNA recovery using RNA Clean & Concentrator kit (Zymo Research Cat. No. R1017). Recovered mRNAs were used for reverse transcription reaction, real-time PCR, and cDNA library construction for high-throughput sequencing.
EXAMPLE 6: REAL-TIME QUANTITATIVE PCR (RT-QPCR)
[0138] RT-qPCR was performed to assess the relative abundance of mRNAs. The RT-PCR primers were designed to span exon-exon junctions to eliminate amplifications of genomic DNA and unspliced mRNAs. Reverse transcription was performed by PrimeScript™ RT reagent Kit (Clontech, RR037A). Real-time PCR reactions were performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, 4367659) with an Applied Biosystems realplex, and the primer sets were tested to be quantitative. Transcription factor IIb (TFIIb) was used as normalizer. Threshold cycles and melting curve measurements were performed with ABI software. P-values were calculated by the Student-t test. Error bars indicate standard error of mean.
List of primers used in the Examples.
TABLE-US-00001 Bmp10 R1-F: (SEQ ID NO 13) CTTCACGGAGCAAGATGGTA; Bmp10 R1-R: (SEQ ID NO 14) TGTACTCTGGTGGATCCACT; Bmp10 R2-F: (SEQ ID NO 15) CCATGAAGAGGTCGTCAT; Bmp10 R2-R: (SEQ ID NO 16) TGCTCCTCTCCTCCTCGCT; Bmp10 R3-F: (SEQ ID NO 17) CTGATGACCAAAGCAATGAC; Bmp10 R3-R: (SEQ ID NO 18) AGAGCCTCTTCATCGGGCCCA; Bmp10 R4-F: (SEQ ID NO 19) ATGATTCGTCCGCTCGGATCA; Bmp10 R4-R: (SEQ ID NO 20) CGGCACTCATAGGCTTCATA; Bmp10 q-F: (SEQ ID NO 21) CTCAAGACGCTGAACTTGTCG; Bmp10 q-R: (SEQ ID NO 22) GAGAGGATATTTCCGGAGCCC;
m.SUP.6.A-IP-Seq
[0139] Sequencing was carried out on Ion Proton System (ThermoFisher Scientific) according to manufacturer's instructions, using Ion Total RNA-Seq Kit v2 (ThermoFisher Scientific) for library construction and deep sequencing. Sequencing depth is about 10 million reads per sample.
EXAMPLE 7: DETECTION OF M.SUP.6.A SITES AND M.SUP.6.A MOTIF ANALYSIS
[0140] UCSC mm10 canonical Known Genes are used to map reads. WinScore was computed using acceptable methods, and windows with WinScore >=2 (four-fold change in IP vs Control) were merged as putative peaks. Two replicates were combined to get Ctrl m.sup.6A peaks and Brg1 knockout (KO) m.sup.6A Peaks (IP vs Input). In addition, Brg1 KO specific peaks were identified using WinScore with Brg1 KO vs Ctrl, and Ctrl specific peaks were identified using WinSore with Ctrl vs. Brg1 KO. To identify motif search method, we took the top 2000 WT peaks, used negative peaks (WinScore <1) as background, and used homer program to search for motifs with 5, 7, or 9 bases.
EXAMPLE 8: RNA IMMUNOPRECIPITATION
[0141] RNA immunoprecipitation (RNA-IP, RIP) was conducted. Briefly, E10.5 hearts or SW13 cells were crosslinked and lysed with lysis buffer (10 mM HEPES pH 7.5, 85 mM KCl, 0.5% NP-40, 1 mM dithiothreitol (DTT), 1× protease inhibitor) for tissues or lysis buffer (10 mM Tris-HCl pH 8.1, 10 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, 1 mM DTT, 1× protease inhibitor) for cells. Nuclei were isolated and sonicated using Bioruptor (Diagenode) (30 s on, 30 s off, power setting H, 5 min, performed twice) in nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 150 mM NaCl, 0.1% NP-40, 1 mM DTT, protease inhibitor, ribonuclease inhibitor). The nuclear extract was collected then incubated with anti-Ythdf2 antibody (EMD Millipore ABE542, 1:100) and normal IgG control at 4° C. overnight together with Manga ChIP Protein G Magnetic Beads (Millipore). The beads were washed by wash buffer I (20 mM Tris-Hcl pH 8.1, 150 mM NaCl, 1% Triton X-100 and 0.1% SDS) three times, and wash buffer II (20 mM Tris-Hcl pH8.1, 500 mM NaCl, 1% Triton X-100 and 0.1% SDS) three times. Beads were then resuspended in 150 ml 150 mM RIPA (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) with 5 ml Proteinase K and incubated for 1 h at 65° C. We added 1 ml of TRIzol to the sample, and RNA was extracted using the Quick-RNA Mini Kit with the on-column DNase I digest (Zymo Research). RT and qPCR were then conducted with the purified RNA.
EXAMPLE 9: WESTERN BLOT ANALYSIS
[0142] Cells were collected and washed once with ice-cold phosphate-buffered saline (PBS) and pellets were resuspended in RIPA lysis buffer (0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA). After sonication and centrifugation, the supernatants were collected and boiled. The blots were reacted with antibodies for YTHDF2 (EMD Millipore ABE542, 1:1,000 WB); Brg1 (Santa Cruz sc-17796, 1:1,000 WB); FTO (Abcam ab92821, 1:1,000 WB); METTL3 (Abnova H00056339-B01P, 1:1,000 WB); METTL14 (Sigma-Aldrich HPA038002, 1:1,000 WB), ALKBH5 (Sigma-Aldrich HPA007196, 1:1,000 WB), and GAPDH (Santa Cruz sc-25778, 1: 2000) followed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG or HRP-conjugated anti-rabbit IgG (Jackson). Chemiluminescence was detected with with SuperSignal ECL Western blot detection kits (Pierce, 34095) using LI-COR odyssey image system.
EXAMPLE 10: M.SUP.6.A INDIVIDUAL-NUCLEOTIDE-RESOLUTION CROSS-LINKING AND IMMUNOPRECIPITATION (MICLIP)
[0143] miCLIP was performed. Briefly, total RNAs extracted from embryonic hearts were fragmented using fragmentation reagent (Life Technologies) to a size between 90 and 200 nt. After stopping the reaction, the fragmented RNA pool was transferred to IP buffer (50 mM Tris pH 7.4, 100 mM NaCl, 0.05% NP40) and incubated with 5-10 ug anti-m.sup.6A antibody from Abcam (ab151230) for 2 h at 4° C. rotating head over tail. After 2 h, the mixture of RNA and antibody was transferred into a 3-cm cell culture dish and crosslinked twice with 0.15 J cm.sup.−2 UV light (254 nm) in a Stratalinker (Agilent). RNAs immunoprecipitated by m.sup.6A antibody were isolated and reverse-transcribed, and a 79bp fragment from Bmp10 R2 region and covering M2 sites was PCR-amplified and cloned into TOPO vector for sequencing. RNAs were pooled from 10 to 12 E10.5 hearts or from 6 to 8 E12.5 hearts for one single miCLIP experiment. Crosslink-induced mutations at position 0, +1 and +2 relative to the m.sup.6A introduced during reverse transcription were analyzed to determine positions of m.sup.6A sites. Bmp10 Region2 79nt primer F: ATGAAGAGGTCGTCATGGCT (SEQ ID NO 23); R: CGGTCCACGCCATCATACAT (SEQ ID NO 24). 79nt is part of Bmp10 Region 2(140nt) and covers M2 sites.
EXAMPLE 11: MAZF DIGESTION AND QPCR ASSAY
[0144] MazF-qPCR was performed. Briefly, mRNAs isolated from embryonic hearts were incubated with or without MazF (Clontech, 2415A) enzyme digestion in the reaction buffer at 37° C. for about 30 min. After digestion, mRNAs were purified by RNA Clean & Concentrator-5 kit (ZYMO Research) and reverse-transcribed into cDNAs for qPCR to quantitate methylation of the first adenosine of the ACA motif.
EXAMPLE 12: ATPASE ASSAY
[0145] ATPase activity was determined using an ATPase Colorimetric Assay Kit (Catalogue no. 601-0120, Novus Biologicals). Briefly, 100 μl of 40 nM (4 pmol) purified protein was mixed with 100 μl of the reaction buffer (the final concentration is 20 nm purified protein, 50 mM Tris, 2.5 mM MgCl2, and 0.5 mM ATP), incubating for about 30 minutes at Room Temperature (RT). The reaction is then stopped by adding 50 ul of Gold mix. After 2 minutes, the stabilizer is added and mixed thoroughly. The ATPase activity was calculated by measurement of liberated inorganic phosphate (Pi) at a wavelength in the range 590-660 nm and the standard curve.
EXAMPLE 13: MRNA STABILITY ASSAY
[0146] Mouse E18.5 cardiomyocytes with Control, Brg1, or YTHDF2 siRNA knockdown were treated with actinomycin D (1 μg/ml) (Sigma, A9415) from 0-4 hr. RNA extraction and RT-qPCR were performed as described above.
EXAMPLE 14: 4-THIOURACIL (4tU) PULSE AND URACIL CHASE ASSAY
[0147] The 4tU was performed. Briefly, E17-E18 embryonic cardiomyocytes were isolated and cultured overnight. RNAi transfection was then performed, and cells were cultured for 48 hrs. 4-Thiouracil (4tU) (100 μM, Sigma, 440736) was added to the fresh media and cultured for 12 hrs. The culture media was then replaced by new media with uracil (12 mM, Sigma, U1128) and cultured until cell collection at times indicated. Total RNAs were then isolated with TRIzol (ThermoFisher Scientific) and biotinylation of 4tU-labeled RNA was performed using (N-(6-(Biotinamido)hexyl)-3′-(2′-pyridyldithio)-propionamide (EZ-Link Biotin-HPDP, ThermoFisher Scientific). Biotin-labeled 4tU incorporated RNA was then pulled down by Dynabeads® MyOne™ Streptavidin C1 (ThermoFisher Scientific) and eluted for reverse transcription (SuperScript™ IV VILO™, ThermoFisher Scientific). Bmp10 was quantitated by qPCR (StepOnePlus Real-Time PCR System, ThermoFisher Scientific).
EXAMPLE 15: 3-DEAZAADENOSINE TREATMENT
[0148] 3-Deazaadenosine (3-DZA, D8296, Sigma-Aldrich) was injected intraperitoneally of about 2 mg/kg/day from embryonic day 7.5 to 10.5.
EXAMPLE 16: HISTOLOGY AND BRDU STAINING
[0149] Histology of embryonic hearts was performed as described. Embryos were fixed overnight in 4% PFA in PBS. They were then dehydrated through an ethanol series, treated with xylenes, and embedded in paraffin wax overnight with several changes. Embryos were oriented for transverse sections and cut in 7 μm sections using a Leica microtome. Following re-hydration, the sections were consecutively stained with Hematoxylin & Eosin (Sigma-Aldrich, St. Louis, Mo.), dehydrated, and mounted in Permount (Sigma) prior to imaging. The thickness of compact myocardium was measured at 20 points along the circumference of the left ventricle, and the results were presented by normalizing with the compact myocardium thickness in the Ctrl and Vehicle group. Pregnant mice were injected with BrdU (Sigma, 100 μg/g, intraperitoneal injection) for 6 hours prior to embryo isolation at E10.5. Incorporated BrdU was stained in the tissue-section of the heart according to the manufacturer's protocol (Invitrogen, BrdU Staining Kit 93-3943). BrdU incorporation was quantitated by the percentage of BrdU-positive cells in the myocardium. p-values were calculated by the Student-t test. Error bars indicate one standard deviation.
EXAMPLE 17: CARDIAC TROPONIN T AND PHOSPHO-HISTONE H3 CO-STAINING
[0150] E10.5 hearts was collected and embedded in paraffin. After hydration and antigen retrieval, E10.5 heart was blocked with PBS containing 5% NGS, and stained with Cardiac Troponin T (Abcam, Cat. No. ab8295, 1: 200) and phospho-Histone H3 (Cell Signaling Technology, Cat. No. 9701, 1:100) antibodies in blocking solution, followed by secondary antibodies conjugated to Alexa488 and Alexa594 (Invitrogen). E10.5 hearts were photographed by confocal microscope (Leica TCS SP8). Phospho-Histone H3 positive Cardiomyocytes were quantitated by normalizing to cardiomyocytes in the myocardium. p-values were calculated by the Student-t test.
EXAMPLE 18: M.SUP.6.A IMMUNOSTAINING
[0151] Cells transfected with different Brg1 constructs were fixed with 2.5% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 30 minutes, permeabilized with 0.5% Triton X-100 for 1 hour and treated with 2N HCl for 1 hour. Cells were then blocked with PBS containing 5% NGS, and stained with Brg1 (Santa Cruz sc-374197X, 1:500) and m.sup.6A (Synaptic System Cat. No. 202003, 1:50) antibodies in blocking solution, followed by secondary antibodies conjugated to Alexa488 and Alexa594 (Invitrogen). Cells were photographed by confocal microscope (Leica TCS SP8). The fluorescence intensity of Brg1-transfected cells was analyzed by Image J software.
EXAMPLE 19: PROTEIN EXPRESSION AND PURIFICATION
[0152] Expression and purification of MBP fusion proteins were performed. Mouse Brg1 DExx-box domain (D1) (Amino acids 774-913 of Brg1), Helicase-C domain (D2) (Amino acids 1086-1204 of Brg1), the entire helicase region (D1D2) (774-1204) as well as the D1D2 domains of snf2 (790-1212) were amplified by PCR and cloned into pMAL vector. Construct mutations were achieved by GeneArt® Site-Directed Mutagenesis kit (ThermoFisher Scientific, A13282). MBP fusion proteins were induced by IPTG and purified by Amylose resin (E8021S, NEB).
EXAMPLE 20: BIOCHEMICAL ASSAY OF M.SUP.6.A DEMETHYLATION ACTIVITY IN VITRO
[0153] The demethylation activity assay was performed. In brief, the assay was performed in standard 100 μl of reaction mixture containing RNA with m.sup.6A, purified protein, 2.83 mM of (NH.sub.4).sub.2Fe(SO.sub.4).sub.2.Math.6H.sub.2O, 300 μM of α-KG, 20 mM of L-ascorbic acid, 50 μg ml.sup.−1 of BSA, and 50 mM of HEPES buffer (pH 7.0). The reaction was incubated at room temperature, quenched by addition of 5 mM EDTA and heating for 5 min at 95° C., followed by m.sup.6A dot blot analysis.
EXAMPLE 21: NUCLEOSOME ASSEMBLY ASSAY
[0154] Nucleosomes were assembled using EpiMark Nucleosome Assembly Kit (E5350S, NEB) following the manufacturer's instruction with minor modifications. In brief, recombinant human core histone octamers (2:1 mix of histone H2A/H2B dimer and histone H3.1/H4 tetramer) were mixed with purified double-stranded DNA of Myh6 core promoter (202 bp, −428 to −227) amplified by primers of CAGATAGCCAGGGTTGAAAG and TGGGCAGATAGAGGAGAGAC at the molecular ratio of 1:1. The salt concentration was gradually lowered by dilution to allow the formation of nucleosomes. After nucleosome assembly, recombinant proteins (1.25 pmol of MBP, Brg1-D1D2 and D1D2 mutants) were mixed with 1.25 pmol nucleosomes in the presence of ATP (2 mM). The mixtures were placed on ice for 15 min and then analyzed with 1% agarose gel.
EXAMPLE 22: LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY (LC-MS/MS)
[0155] Cell mRNA for LC-MS/MS: 200 ng of mRNA was digested by nuclease P1 (2 U) in 25 μl of buffer containing 10 mM of NH.sub.4OAc (pH=5.3) at 42° C. for 2 h, followed by the addition of NH.sub.4HCO.sub.3 (1 M, 3 μl, freshly made) and alkaline phosphatase (0.5 U). After an additional incubation at 37° C. for 2 h, sample was diluted to 50 μl and filtered (0.22 μm pore size, 4 mm diameter, Millipore), and 5 μl of the solution was injected into LC-MS/MS.
[0156] RNA probe for LC-MS/MS: After in vitro biochemistry reaction, all samples were inactivated by adding 1 μl EDTA (0.5 M) and heated at 95° C. for 5 min, following by adding 0.5 μl proteinase K (20 mg/ml) into each reaction at 56° C. for at least 15 min to completely digest the precipitated protein. The mixture was treated with 72° C. for 15 min to inactivate proteinase K. The reacted nucleic acid substrates (1 μg) were enzymatically digested into single nucleoside by mixture with 1 U nuclease P1 (Sigma) and 1 U alkaline phosphatase (NEB). Nucleosides were separated by reverse phase ultra-performance liquid chromatography (LC) on a C18 column with on-line mass spectrometry detection using Finnigan LTQ-Orbitrap hybrid tandem mass spectrometer (Thermo Fisher Scientific) in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside to base ion mass transitions of 282 to 150 (m.sup.6A), and 268 to 136 (A). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. The ratio of m.sup.6A to A was calculated based on the calibrated concentrations.
EXAMPLE 23: RNA ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) AND KD CALCULATION
[0157] Biotin-labelled RNA control probe: [0158] Biotin—5′UCGUCAUGGCUGAACUGCGGUUGUACACGCUGGUGCAGAGAGAUCGCAU-3′ (SEQ ID NO 25); and Biotin-labelled RNA probe with m.sup.6A: [0159] Biotin—5′UCGUCAUGGCUGAACUGCGGUUGU(m.sup.6A)CACGCUGGUGCAGAGAGAUCGCAU-3′ (SEQ ID NO 26); were synthesized by Gene Link (Orlando, Fla.). EMSA was performed by using the LightShift™ Chemiluminescent RNA EMSA Kit (Cat. 20158, Thermo Scientific). The labelled probe was incubated with appropriate amounts of recombinant proteins in 10 μl in the 1× binding buffer (10 mM HEPES-KOH, pH 7.3, 10 mM NaCl, 1 mM MgCl2, 1 mM DTT) with 5 μg tRNA carrier at room temperature for 30 min. The reactions were then loaded onto 1% 0.5× TBE agarose gel and transferred to Bright Star Plus positive charged membrane. The biotin-labelled probes were detected and quantified subsequently using Odyssey Infrared Imaging System. The shifted signals were quantified and plotted against amount of the MBP-D1D2 protein ((SEQ ID NO 27) coupled to (SEQ ID NO 2)) with GraphPad Prism (GraphPad). The software facilitates the fitting of nonlinear regression model and calculation of Kd values based on the fitting curve. The errors and r.sup.2 values were also generated from the fitting curve.
EXAMPLE 24: BRG1 R973Q INDUCIBLE POINT MUTATION MOUSE LINE
[0160] The Brg1 R973Q inducible point mutation knock-in mouse line was generated by ES cell gene targeting (Data not shown). The targeting vector was constructed by recombination. The 5′ distal LoxP site was inserted 192 bp upstream of Brg1 exon20 in forward direction. The FRT-flanked Neo cassette was placed 214 bp downstream of exon20, which was followed by the engineered inversion cassette containing duplicated exon20 with the specific point mutation (CGG>CAG, R973Q) and their flanking genomic sequences for correct splicing. The inversion cassette was flanked by mutant Lox71/Lox66 sites. The long homology arm was extending about 6.2 kb to the 5′ distal LoxP site, and the short homology arm was about 2 kb 3′ to the inversion cassette. The targeting vector was then linearized and electroporated into mouse embryonic stem (ES) cells. Neomycin-resistant clones were tested for correct gene targeting by Southern Blot. Following verification of correct targeting and karyotypes, two positive ES clones were expanded and injected into blastocysts for mouse generation. The obtained chimeric mouse lines were crossed to C57BL/6 lines for germline transmission. Chimeric mice that were positive for the targeted ES cells were germline-transferred to the F1 generation and bred on a C57BL/6 background. Heterozygous Brg1.sup.iR973Q/+ was crossed with Sm22αCre driver line to induce Brg1 R973Q point mutation in cardiomyocytes. Upon Cre expression, Lox71 and Lox66 recombined to invert their flanked sequence. As a result, a wild type LoxP site and a new mutant site Lox72 would be created, and the inverted sequences containing R973 within exon20 would be configured in the sense direction. Then the new LoxP site would recombine with 5′ distal LoxP, which disabled wild type gene expression and activated expression of the mutant version, respectively. Such site-specific Brg1 R973Q point mutation was detected by PCR and verified by sequencing (Data not shown).
EXAMPLE 25: YEAST GENETICS AND RNA M.SUP.6.A ANALYSIS
[0161] URA gene was cloned from YIp5 [pRB12] (37061, ATCC) plasmid, and inserted into DNA templates with R988Q or H1188A/D1190A point mutations after the stop codon of SNF2 (Data not shown). The yeast wild type and IME4 knockout (ime4Δ) strains purchased from Transomic Technologies (BY4741, Cat No: TKY0002) were transformed with linearized double strand DNA templates using PEG and LiAc. The transformation mixture was directly spread onto SD-URA selection plates. Colonies on the plates were picked, and their genomic DNA extracted for genotyping. Genotyping was performed by restriction enzyme (SmlI and HindIII) digestion followed by DNA sequencing. Total RNAs was purified from the cells using acid-phenol extraction. Enrichment of polyadenylated RNAs (polyA+ RNA) from total RNAs was performed using one round of PolyATtract® mRNA Isolation Systems (Promega).
EXAMPLE 26: STATISTICAL ANALYSIS
[0162] Statistical significances and enzyme kinetics constants were calculated using GraphPad Prism 5 and 7 (GraphPad Software, La Jolla, Calif.). A value of P<0.05 was considered statistically significant. The software of GraphPad Prism facilitates the fitting of non-linear regression model and calculation of kinetics constants. The errors and r-square (r.sup.2) were generated from the fitting curve.
EXAMPLE 27
[0163] Previous studies revealed that the SWI/SNF-like chromatin remodeler Brg1 maintains Bmp10 mRNA levels in embryonic hearts to promote cardiomyocyte proliferation, and the regulation of Bmp10 by Brg1 doesn't require promoter activation by Brg1, given that Brg1 doesn't bind Bmp10 promoter (Data not shown). We further tested the transcriptional activity of Brg1 on Bmp10 promoter by reporter assays. We cloned Bmp10 promoter (−5307 to +1134 bp) into pREP4 episomal reporter that allows promoter chromatinization in mammalian cells. We then transfected Brg1-expressing plasmids with the reporter into SW13 cells. In these cells Brg1 showed no activity in Bmp10 promoter, while Brg1 caused 1.7-fold increase of Myh7 promoter and 40% reduction of Myh6 promoter activity (Data not shown). Reporter studies, combined with in vivo ChIP results, indicate that Brg1 doesn't activate Bmp10 promoter, in contrast to Brg1's transcriptional activities on Myh promoters. Given that Brg1 can directly bind RNA with high affinity through its helicase domain (also known as ATPase domain), we examined the helicase domain for clues of its RNA function. Brg1 helicase domain contains two RecA-like subdomains DExxC (D1) and HELICc (D2) connected by an insertion peptide (
EXAMPLE 28
[0164] To assess whether Brg1 was an m.sup.6A demethylase, we began by examining m.sup.6A marks of polyadenylated RNAs in mouse embryonic hearts with or without Brg1 in cardiomyocytes. To delete Brg1 in fetal cardiomyocytes, we used Sm22α promoter to drive the expression of Cre recombinase in fetal cardiomyocytes to recombine Brg1 floxed alleles (Brg1.sup.fl/fl) in Sm22αCre;Brg1.sup.fl/fl mice, allowing the disruption of myocardial Brg1 by embryonic day 8.5-9.5 (E8.5-9.5). To quantitate m.sup.6A methylation, we used dot blot analysis with m.sup.6A-specific antibody and found that m.sup.6A marks of total polyadenylated RNAs of E10.5 mouse hearts increased by 46% when Brg1 was absent in cardiomyocytes (Sm22αCre;Brg1.sup.fl/fl), relative to the littermate control hearts (Sm22αCre;Brg1.sup.fl/+, Brg1.sup.fl/+ or Brg1.sup.fl/fl) (See
EXAMPLE 29
[0165] Given that Brg1 is essential for maintaining Bmp10 mRNA levels to promote fetal cardiomyocyte proliferation, we asked whether the m.sup.6A status of Bmp10 mRNA was altered in embryonic hearts lacking Brg1 in cardiomyocytes. To test that, we used m.sup.6A-RNA immunoprecipitation coupled with RT-qPCR (m.sup.6A-RNA-IP) to precipitate m.sup.6A from E10.5 hearts and quantitate Bmp10 mRNA. In this assay, mRNA was fragmented for IP, and specific qPCR primers were used to target four different regions (R1-R4) in Bmp10 mRNA to assess m.sup.6A enrichment (See
EXAMPLE 30
[0166] To determine the in vivo Bmp10 site of m.sup.6A demethylation by Brg1, we profiled m.sup.6A methylation of polyadenylated RNAs in embryonic hearts, using m.sup.6A-RNA-IP and RNA sequencing. Polyadenylated RNAs were prepared from E10.5 hearts and fragmented to ˜100 nucleotides in length, followed by m.sup.6A-RNA-IP and RNA sequencing. Such m.sup.6A mRNA profiling—consistent with the dot blot and 2D-TLC analyses (See
EXAMPLE 31
[0167] Modifications of m.sup.6A that control RNA stability are enriched around stop codons, in 3′UTR, or within long exons. The R2 location in the longer exon of Bmp10 fit the profile of m.sup.6A modifications. We then focused on R2 to determine the primary adenosine site of Bmp10 m.sup.6A methylation. Based on similarities to the consensus m.sup.6A sequence (Data not shown), we selected six potential m.sup.6A sites (M1-M6) from R2 for mutagenesis (A-to-G point mutation) to disable m.sup.6A modification (See
EXAMPLE 32
[0168] Collectively, the identification of a specific Bmp10 m.sup.6A site (GUACAC) (SEQ ID NO 10) demethylated by Brg1 was accomplished by multiple methods, including sequence walking, m.sup.6A-RNA-IP, point mutagenesis, miCLIP, and MazF studies. Furthermore, we tested whether Brg1 was required for the demethylation of m.sup.6A.sub.m at the penultimate base of RNAs. 2D-TLC analyses showed no changes of the ratio between m.sup.6A.sub.m and A.sub.m either by BRG1 knockdown (84% efficiency) in 293T cells or E10.5 Brg1-null hearts (Data not shown). These observations suggest a primary function of Brg1 in m.sup.6A but not m.sup.6A.sub.m demethylation.
EXAMPLE 33
[0169] We then asked whether m.sup.6A demethylation of Bmp10 by Brg1 was relevant to Bmp10 stability in vivo. Given that m.sup.6A is selectively bound by YTH domain family 2 protein (YTHDF2) to trigger mRNA degradation, we examined the binding of endogenous Ythdf2 proteins to Bmp10 mRNA in mouse embryonic hearts with or without Brg1. RNA IP using anti-Ythdf2 antibody showed that the binding of Ythdf2 to Bmp10 mRNA was increased by 3.8-fold in E10.5 Brg1-null hearts (Sm22αCre;Brg1.sup.fl/fl)(See
EXAMPLE 34
[0170] We further confirmed the interaction between Brg1 and YTHDF2 in controlling Bmp10 mRNA stability in cardiomyocytes. We first cultured cardiomyocytes isolated from E18.5 mouse hearts and measured Bmp10 m.sup.6A methylation and stability. Brg1 knockdown by siRNA in cardiomyocytes increased Bmp10 m.sup.6A signal at R2 (but not other R regions) by 50%, and this was associated with 70% Bmp10 mRNA reduction and 45% Bmp10 protein reduction, as well as a switch of Myh isoform expression (Myh7-to-Myh6 switch) (Data not shown). These results from cultured cardiomyocytes recapitulated the in vivo effects of Brg1 in embryonic hearts on Bmp10 and Myh expression and on Bmp10 m.sup.6A methylation (See
EXAMPLE 35
[0171] Given the ability of Brg1 to erase m.sup.6A marks from Bmp10 (See
EXAMPLE 36
[0172] To further pursue this new molecular action of Brg1, we tested whether Brg1 had a general role in RNA m.sup.6A demethylation in other cells beyond cardiomyocytes. We used siRNA to knock down BRG1 in human 293T cells that do not express Bmp10 and obtained 84% efficiency of BRG1 disruption without affecting the expression of m.sup.6A reader (YTHDF2), methyltransferases (METTL3 and 14), or demethylases (FTO, ALKBH5) (Data not shown). Quantitation of m.sup.6A of mRNAs showed that BRG1 knockdown caused 64% increase of m.sup.6A (dot blot) and 58% increase of m.sup.6A-to-A ratio (2D-TLC) on polyadenylated RNAs (Data not shown). The increase of m.sup.6A was further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which showed a 48% increase of m.sup.6A/A signal intensity ratio when BRG1 was knocked down by 65% (Data not shown). Therefore, without changing the expression of known m.sup.6A reader, writer, or eraser, Brg1 is necessary for RNA m.sup.6A demethylation in 293T cells. Therefore, Brg1 is capable of erasing m.sup.6A marks from RNAs in multiple cell types.
EXAMPLE 37
[0173] Given that Brg1 helicase domain carried putative motifs of an m.sup.6A demethylase (See
EXAMPLE 38
[0174] Next we tested whether Brg1 was in itself an m.sup.6A demethylase. We used recombinant proteins and synthetic RNA probes to examine the biochemical activity of Brg1 as an RNA m.sup.6A demethylase. We purified maltose-binding-protein (MBP)-tagged Brg1 helicase domain proteins (D1D2, amino acid 774-1202) (Data not shown) and synthesized an m.sup.6A-containing Bmp10 mRNA probe (5′-CGGUUGUA*CACGCUG-3′, A*: m.sup.6A) SEQ ID NO 12 with m.sup.6A at the M2 adenosine site (See
EXAMPLE 39
[0175] We then asked whether the R/R and H/D motifs of Brg1 helicase were required for Brg1's m.sup.6A demethylation activity. We mutated R973 of Brg1 R/R motif (RLHKVLR*PFLLRR, R*: R973) (modified SEQ ID NO 4) to Q973 (
EXAMPLE 40
[0176] To evaluate the in vivo necessity of these motifs, we generated similar R/R and H/D mutations in the context of full-length Brg1 and tested their m.sup.6A activities in BRG1 knockdown 293T cells and BRG1/BRM-null SW13 cells. Wildtype and mutant Brg1 constructs (Brg1, Brg1-R973Q, or Brg1-H1181A/D1183A) were respectively transfected to both types of cells to reconstitute Brg1 proteins (Data not shown). Dot blot m.sup.6A quantitation showed that Brg1-R973Q and Brg1-H1181A/D1183A mutant proteins lost their ability to demethylate m.sup.6A in cells (Data not shown). The loss of m.sup.6A demethylation by Brg1 mutant proteins was further confirmed by cellular co-immunostaining of m.sup.6A and Brg1 proteins in SW13 cells transfected with Brg1 mutant constructs. Cells that incorporated Brg1-R973Q or Brg1-H1181A/D1183A proteins maintained their m.sup.6A marks comparable to control cells (Data not shown), corroborating the results of biochemical studies (Data not shown). Next, we tested whether ATP hydrolysis by Brg1 was essential for m.sup.6A demethylation. Cells reconstituted with Brg1-K798R that had ATPase disabled (Data not shown) showed ˜50% reduction of m.sup.6A signal, comparable to that achieved by wildtype Brg1 (Data not shown). Conversely, R973Q and H1181A/D1183A proteins, though incapable of m.sup.6A demethylation, maintained their biochemical ability to hydrolyze ATP (Data not shown), suggesting a separation of ATPase and m.sup.6A demethylation function of Brg1. Furthermore, to test whether R/R or H/D mutations affected chromatin remodeling, we used histone octamers to chromatinize in vitro the Myh6 promoter DNA (−428 to −227 upstream of Myh6 TSS), which Brg1 is capable of binding, remodeling, and transcriptionally repressing. We found that nucleosomes assembled on the Myh6 promoter were remodeled by Brg1 R973Q and H1181A/D1183A proteins, as evidenced by the removal of histones from Myh6 DNA, releasing the free DNA (Data not shown). Therefore, R973 and H1181/D1183 are not essential for chromatin remodeling. Collectively, these biochemical and cell-based studies identify the catalytic motifs and establish Brg1 as an RNA m.sup.6A demethylase, independent of its ATPase and chromatin remodeling activity.
EXAMPLE 41
[0177] R973 in R/R motif played a dominant role in m.sup.6A demethylation, and remarkably, heterozygous R973 missense mutations (R973Q and R973W) were frequently found in human cancers. Such R973 mutations, however, had no known effects on Brg1's chromatin engagement or ATP hydrolysis, consistent with our biochemical studies (Data not shown). Given its implications in disease biology, we tested whether R973 was essential for Brg1's m.sup.6A demethylase activity in vivo by generating a murine germline R973Q (CGG-to-CAG) mutation. To our surprise, despite multiple attempts, we were unable to obtain any heterozygous line viable to birth, suggesting embryonic lethality of R973Q heterozygosity. To overcome that, we designed a genetic knock-in scheme to enable inducible, tissue-specific R973Q mutation (Data not shown). By embryonic stem cell gene targeting, we introduced into Brg1 locus an inversion cassette, flanked by LoxP and Lox71/Lox66 sites and comprising duplicated exon20 of Brg1—a wild type exon20 and an inverted one with R973Q (Data not shown). In the absence of Cre recombinase, the cassette allowed wildtype exon20 to express; conversely, with Cre activity the cassette was inverted and switched the expression to R973Q-exon20. We then used Sm22αCre to induce cardiomyocyte-specific Brg1-R973Q mutation (Data not shown). Even with such tissue-specific R973Q mutation, we encountered heterozygous lethality between E14.5 to birth in embryos (Data not shown). In E10.5-12.5 R973Q heterozygous hearts (Sm22αCre;Brg1.sup.iR973Q/+), dot blot analysis showed that m.sup.6A-modified mRNAs increased by 115% (Data not shown), and miCLIP analyses of E12.5 R973Q hearts revealed that Bmp10 mutations occurred at 0 (A-to-C/T) and +2 (A-to-C) positions relative to the M2 adenosine with significantly higher frequencies than the littermate control hearts (3.6±0.88% vs 1.3±1.1%)(Data not shown). These results indicate that R973 is essential for Brg1 to demethylate the M2 adenosine of Bmp10 in embryonic hearts. In those R973Q heterozygous hearts, Brg1 protein levels were comparable to controls, while Bmp10 mRNA and protein levels decreased by 30% (Data not shown). The changes of Bmp10 m.sup.6A and mRNA were associated with a 45% decrease of myocardial wall thickness at E13.5 (Data not shown). The in vivo genetic studies thus demonstrate that R973 is essential for Brg1-mediated m.sup.6A demethylation necessary for heart muscle development.
EXAMPLE 42
[0178] Given that Brg1 represents the vertebrate SWI/SNF family of ATP-dependent chromatin-remodeling factors, we asked whether the m.sup.6A-demethylating activity of Brg1 was conserved in its yeast orthologue Snf2 of Saccharomyces cerevisiae. The yeast contains methyltransferase complex (MIS), composed of Ime4 (METTL3 orthologue), Mum2 (WTAP orthologue), and Slz1. Mutations of the MIS proteins abrogate m.sup.6A methylation of mRNAs and delay meiotic entry. Despite the biological importance of m.sup.6A methylation, enzymes that demethylate m.sup.6A in yeasts have not been identified.
EXAMPLE 43
[0179] We found that the yeast chromatin remodeler Snf2 also contained R/R and H/D motifs identical to those of mouse Brg1 (See
EXAMPLE 44
[0180] To test whether Snf2 counteracted Ime4 in RNA m.sup.6A regulation, we generated yeast haploid strains with SNF2 and IME4 double mutations—ime4Δsnf2R988Q and ime4Δsnf2H1188A, D1190A. Remarkably, the snf2R/R or H/D point mutation fully rescued the reduction of global RNA m.sup.6A signal in ime4Δ cells (Data not shown). Such opposing regulation of m.sup.6A by Ime4 and Snf2 at the global level strongly supports the role of Snf2 as an m.sup.6A demethylase. However, unlike SNF2 mutants, the ime4Δ haploid cells produced colonies with comparable size to that of the parent wildtype strain, indicating that Ime4 isn't essential for regulating specific RNA(s) required for haploid cell growth. Consistent with this non-essentiality for cell growth, ime4Δ couldn't rescue the growth defects of SNF2 mutants (snf2R988Q and snf2H1188A, D1190A)(Data not shown). The results suggest that these two m.sup.6A regulators, despite having common RNA targets globally, show distinct, non-overlapping subsets of downstream RNAs that await further characterization.
[0181] These studies describe a new class of RNA m.sup.6A demethylase evolutionarily conserved from yeast to mammals. The Brg1-mediated RNA m.sup.6A regulation not only depicts a new mechanistic terrain for heart development, but also glimpses a wider vista of the molecular action of SWI/SNF proteins beyond their traditional role as chromatin regulators (See
EXAMPLE 45
[0182] RNA m.sup.6A modification adds to the diversity of Brg1 function in chromatin remodeling, transcription control, and RNA regulation (Data not shown). To execute those functions, the Brg1 helicase domain mediates ATP hydrolysis, chromatin engagement, and RNA binding and m.sup.6A demethylation. Brg1 has at least two different modes of direct interaction with RNA molecules. Binding to the lncRNA Mhrt allows Brg1 to modulate its chromatin binding and gene targeting, whereas binding to m.sup.6A-modified Bmp10 or other RNAs enables Brg1 to maintain RNA stability after transcription. Besides stabilizing RNAs through exonic/3′-UTR m.sup.6A demethylation, Brg1 may act on 5′-UTR m.sup.6A to regulate cap-independent RNA translation, given the enhanced m.sup.6A signals at 5′-UTR in Brg1-null hearts. Brg1 helicase is structurally distinct from FTO and ALKBH5, and further studies will be needed to determine how Brg1's m.sup.6A activity is structurally supported and how Brg1 helicase physically switches between different functional modes to execute a fine control of gene expression at different levels to cope with pathophysiological demands
[0183] Brg1 is thought to act as an ATPase subunit of the multi-protein BAF chromatin-remodeling complex, and its function outside BAF is essentially unknown. As a tumor suppressor, Brg1 often has its R973 in the ATPase/helicase domain mutated to Q or W in human cancers. Those mutations, unlike other cancer-related mutations in the helicase domain, have no effects on ATP hydrolysis or nucleosome-remodeling activity of Brg1 (current study). Accordingly, the inability of R973Q mutation to demethylate m.sup.6A reveals a new m.sup.6A-based mechanism of Brg1 in tumor suppression. Brg1 may m.sup.6A-demethylate a subset of tumor suppressor genes to stabilize their mRNAs and inhibit cancer progression. Consistent with this model, the m.sup.6A methyltransferase METTL3 was shown to repress a tumor suppressor gene SOCS2 through an m.sup.6A-dependent mechanism in hepatocellular carcinoma to promote cancer progression. Identification of m.sup.6A targets of Brg1 in cancer cells may be useful to fully evaluate the m.sup.6A mechanism of Brg1 in cancer biology.
EXAMPLE 46
[0184] The m.sup.6A action of SWI/SNF chromatin remodelers delivers the first example of yeast RNA m.sup.6A demethylase (Swi2/Snf2), providing a major missing component in the eukaryotic m.sup.6A regulatory circuit. In yeast, m.sup.6A-modified RNAs are thought to accumulate only during meiosis and absent in haploid cells. By multiple independent chemical methods, we demonstrated the presence of m.sup.6A-modified mRNAs in haploid cells and their antithetical m.sup.6A regulations by the Ime4 methyltransferase and Snf2 demethylase, extending the m.sup.6A biology beyond meiosis. The findings introduce an extra layer of gene regulation by SWI/SNF proteins besides their chromatin-based gene transcription control. Future studies are essential to decipher how the SWI/SNF complex may couple m.sup.6A regulation to its chromatin and transcription function and how the four helicase-defined families of ATP-dependent chromatin remodelers differ in this new aspect of their biology. Given these unexpected molecular actions of SWI/SNF proteins, gene changes caused by chromatin remodelers will need to be re-interpreted and understood in the context of chromatin regulation and post-transcriptional m.sup.6A modification.
TABLE-US-00002 SEQUENCE LISTING SEQ ID NO 1: Brg1-Mouse MSTPDPPLGGTPRPGPSPGPGPSPGAMLGPSPGPSPGSAHSMMGPSPGPPSAGHPMPTQ GPGGYPQDNMHQMHKPMESMHEKGMPDDPRYNQMKGMGMRSGAHTGMAPPPSPM DQHSQGYPSPLGGSEHASSPVPASGPSSGPQMSSGPGGAPLDGSDPQALGQQNRGPTPF GPGPGPGPGPGPGPGPAPPNYSRPHGMGGPNMPPPGPSGVPPGMPGQPPGGPPKPWPE GPMANAAAPTSTPQKLIPPQPTGRPSPAPPAVPPAASPVMPPQTQSPGQPAQPAPLVPLH QKQSRITPIQKPRGLDPVEILQEREYRLQARIAHRIQELENLPGSLAGDLRTKATIELKAL RLLNFQRQLRQEVVVCMRRDTALETALNAKAYKRSKRQSLREARITEKLEKQQKIEQE RKRRQKHQEYLNSILQHAKDFREYHRSVTGKLQKLTKAVATYHANTEREQKKENERI EKERMRRLMAEDEEGYRKLIDQKKDKRLAYLLQQTDEYVANLTELVRQHKAAQVAK EKKKKKKKKKAENAEGQTPAIGPDGEPLDETSQMSDLPVKVIHVESGKILTGTDAPKA GQLEAWLEMNPGYEVAPRSDSEESGSEEEEEEEEEEQPQPAQPPTLPVEEKKKIPDPDS DDVSEVDARHIIENAKQDVDDEYGVSQALARGLOSYYAVAHAVTERVDKQSALMVN GVLKQYQIKGLEWLVSLYNNNLNGILADEMGLGKTIQTIALITYLMEHKRINGPFLIIVP LSTLSNWAYEFDKWAPSVVKVSYKGSPAARRAFVPQLRSGKFNVLLTTYEYIIKDKHIL AKIRWKYMIVDEGHRMKNHHCKLTQVLNTHYVAPRRLLLTGTPLQNKLPELWALLNF LLPTIFKSCSTFEQWFNAPFAMTGEKVDLNEEETILIIRRLHKVLRPFLLRRLKKEVEAQ LPEKVEYVIKCDMSALQRVLYRHMQAKGVLLTDGSEKDKKGKGGTKTLMNTIMQLR KICNHPYMFQHIEESFSEHLGFTGGIVQGLDLYRASGKFELLDRILPKLRATNHKVLLFC QMTSLMTIMEDYFAYRGFKYLRLDGTTKAEDRGMLLKTFNEPGSEYFIFLLSTRAGGL GLNLQSADTVIIFDSDWNPHQDLQAQDRAHRIGQQNEVRVLRLCTVNSVEEKILAAAK YKLNVDQKVIQAGMFDQKSSSHERRAFLQAILEHEEQDEEEDEVPDDETVNQMIARHE EEFDLFMRMDLDRRREEARNPKRKPRLMEEDELPSWIIKDDAEVERLTCEEEEEKMFG RGSRHRKEVDYSDSLTEKQWLKTLKAIEEGTLEEIEEEVRQKKSSRKRKRDSEAGSSTP TTSTRSRDKDEESKKQKKRGRPPAEKLSPNPPNLTKKMKKIVDAVIKYKDSSSGRQLSE VFIQLPSRKELPEYYELIRKPVDFKKIKERIRNHKYRSLNDLEKDVMLLCQNAQTFNLE GSLIYEDSIVLQSVFTSVRQKIEKEDDSEGEESEEEEEGEEEGSESESRSVKVKIKLGRKE KAQDRLKGGRRRPSRGSRAKPVVSDDDSEEEQEEDRSGSGSEED SEQ ID NO 2: D1D2 (mouse) amino acid NGILADEMGLGKTIQTIALITYLMEHKRINGPFLIIVPLSTLSNWAYEFDKWAPSVVKVS YKGSPAARRAFVPQLRSGKFNVLLTTYEYIIKDKHILAKIRWKYMIVDEGHRMKNHHC KLTQVLNTHYVAPRRLLLTGTPLQNKLPELWALLNFLLPTIFKSCSTFEQWFNAPFAMT GEKVDLNEEETILIIRRLHKVLRPFLLRRLKKEVEAQLPEKVEYVIKCDMSALQRVLYR HMQAKGVLLTDGSEKDKKGKGGTKTLMNTIMQLRKICNHPYMFQHIEESFSEHLGFT GGIVQGLDLYRASGKFELLDRILPKLRATNHKVLLFCQMTSLMTIMEDYFAYRGFKYL RLDGTTKAEDRGMLLKTFNEPGSEYFIFLLSTRAGGLGLNLQSADTVIIFDSDWNPHQD LQAQDRAHRIGQQNEVRVLRL SEQ ID NO 3: R/R + H/D Motif amino acid IRRLHKVLRPFLLRRLKKEVEAQLPEKVEYVIKCDMSALQRVLYRHMQAKGVLLTDGS EKDKKGKGGTKTLMNTIMQLRKICNHPYMFQHIEESFSEHLGFTGGIVQGLDLYRASG KFELLDRILPKLRATNHKVLLFCQMTSLMTIMEDYFAYRGFKYLRLDGTTKAEDRGML LKTFNEPGSEYFIFLLSTRAGGLGLNLQSADTVIIFDSDWNPHQDLQAQDRAHRI SEQ ID NO 4: R/R Motif : (mouse) amino acid IRRLHKVLRPFLLRRLK SEQ ID NO 5: H/D Motif: (mouse) amino acid DWNPHQDLQAQDRAHRI SEQ ID NO 6: D1 (MOUSE) amino acid NGILADEMGLGKTIQTIALITYLMEHKRINGPFLIIVPLSTLSNWAYEFDKWAPSVVKVS YKGSPAARRAFVPQLRSGKFNVLLTTYEYIIKDKHILAKIRWKYMIVDEGHRMKNHHC KLTQVLNTHYVAPRRLLLTGTPL SEQ ID NO 7: D2 (MOUSE) amino acid NHKVLLFCQMTSLMTIMEDYFAYRGFKYLRLDGTTKAEDRGMLLKTFNEPGSEYFIFL LSTRAGGLGLNLQSADTVIIFDSDWNPHQDLQAQDRAHRIGQQNEVRVLRL SEQ ID NO 8: Bmp10 DNA (mouse) CTAGGTTGGCCTGGGAGCTGAGCAGAGAGTCATGGGGTCTCTGGTTCTGCCGCTGA GCGCCGTCTTCTGCCTGGTGGCTCACTCGGCTTCTGGCAGCCCCATTATGGGCCTTG AGCAGTCGCCCCTGGAAGAAGACATGCCCTTCTTTGATGATATCTTCACGGAGCAA GATGGTATTGACTTCAACACACTGCTGCAGAGCATGAAGAACGAGTTTCTCAAGAC GCTGAACTTGTCGGACATTCCTGTGCAGGACACGGGCAGAGTGGATCCACCAGAGT ACATGCTGGAGCTCTACAACAAATTCGCCACAGACCGGACCTCCATGCCGTCTGCT AACATCATCCGGAGCTTCAAGAACGAAGATCTGTTTTCTCAACCAGTCACTTTTAAT GGGCTCCGGAAATATCCTCTCCTCTTCAATGTGTCTATCCCTCACCATGAAGAGGTC GTCATGGCTGAACTGCGGTTGTACACGCTGGTGCAGAGAGATCGCATGATGTATGA TGGCGTGGACCGTAAAATTACCATTTTTGAGGTACTAGAGAGTGCAGACGGTAGCG AGGAGGAGAGGAGCATGCTGGTCTTAGTATCAACAGAGATCTACGGAACCAACAG TGAGTGGGAGACATTTGACGTCACAGATGCCACCAGACGTTGGCAAAAGTCAGGC CCATCAACCCATCAGCTGGAGATCCACATAGAAAGCAGACAAAACCAAGCTGAGG ACACCGGAAGGGGACAACTGGAAATAGATATGAGTGCCCAAAATAAGCATGACCC TTTGCTGGTTGTGTTTTCTGATGACCAAAGCAATGACAAGGAGCAGAAAGAAGAAC TGAACGAATTGATCACCCATGAGCAGGATCTGGACCTGGACTCAGATGCTTTCTTC AGTGGGCCCGATGAAGAGGCTCTGCTGCAGATGAGGTCGAACATGATTGATGATTC GTCCGCTCGGATCAGGAGGAACGCCAAGGGGAACTACTGTAAGAAGACCCCACTA TACATCGACTTCAAGGAGATTGGGTGGGACTCCTGGATCATCGCTCCTCCTGGGTA TGAAGCCTATGAGTGCCGGGGTGTGTGTAACTACCCTCTGGCGGAGCACCTCACAC CTACAAAACACGCAATTATTCAGGCCTTGGTCCACCTCAAGAATTCCCAGAAAGCT TCCAAAGCCTGCTGTGTGCCCACGAAGCTGGATCCCATCTCCATCCTCTATTTAGAT AAAGGTGTCGTCACCTACAAGTTTAAATATGAAGGGATGGCTGTGTCTGAGTGTGG CTGTAGATAGGAGAGGAGAGGCGTCCCATG SEQ ID NO 9: Region 2 of mRNA CCAUGAAGAGGUCGUCAUGGCUGAACUGCGGUUGUACACGCUGGUGCAGAGAGA UCGCAUGAUGUAUGAUGGCGUGGACCGUAAAAUUACCAUUUUUGAGGUACUAG AGAGUGCAGACGGUAGCGAGGAGGAGAGGAGCA SEQ ID NO 10: Site M2 + M3 of Bmp10 mRNA GUACAC SEQ ID NO 11: Site M2 of mRNA GUA SEQ ID NO 12: Generic recombinant mRNA sequence 5′-AUUGUCAA*CAGCAGC-3′, A*: m.sup.6A List of primers used in the study. SEQ ID NO: 13 Bmp10 R1-F: CTTCACGGAGCAAGATGGTA; SEQ ID NO: 14 Bmp10 R1-R: TGTACTCTGGTGGATCCACT SEQ ID NO: 15 Bmp10 R2-F: CCATGAAGAGGTCGTCAT; SEQ ID NO 16 Bmp10 R2-R: TGCTCCTCTCCTCCTCGCT SEQ ID NO 17 Bmp10 R3-F: CTGATGACCAAAGCAATGAC; SEQ ID NO 18 Bmp10 R3-R: AGAGCCTCTTCATCGGGCCCA SEQ ID NO 19 Bmp10 R4-F: ATGATTCGTCCGCTCGGATCA; SEQ ID NO 20: Bmp10 R4-R: CGGCACTCATAGGCTTCATA SEQ ID NO 21 Bmp10 q-F: CTCAAGACGCTGAACTTGTCG; SEQ ID NO 22 Bmp10 q-R: GAGAGGATATTTCCGGAGCCC SEQ ID NO 23 Bmp10 Region2 79 nt primer F: ATGAAGAGGTCGTCATGGCT; SEQ ID NO 24: Bmp10 Region2 79 nt primer R: CGGTCCACGCCATCATACAT. SEQ ID NO 25: Biotin labelled RNA Control probe: Biotin- 5′UCGUCAUGGCUGAACUGCGGUUGUACACGCUGGUGCAGAGAGAUCGCAU-3′ SEQ ID NO 26: Biotin-labelled RNA probe with m.sup.6A: Biotin- 5′UCGUCAUGGCUGAACUGCGGUUGU(m.sup.6A)CACGCUGGUGCAGAGAGAUCGCAU-3′ SEQ ID NO 27: MBP (amino acids) MSYYHHHHHHDYDIPTTENLYFQGAMGIRNSKAYVD