Reporter of genomic methylation and uses thereof

Abstract

In some aspects, described herein is a DNA methylation reporter. In some aspects, the DNA methylation reporter comprises a promoter whose activity can be affected by exogenous methylation changes without being independently regulated by the DNA methylation machinery, operably linked to a DNA sequence that encodes a reporter molecule. In some embodiments the DNA methylation reporter comprises (i) a promoter derived from a mammalian imprinted gene promoter; and (ii) a sequence that encodes a reporter molecule that is detectable in individual mammalian cells, wherein the promoter is operably linked to the sequence that encodes the reporter molecule. Also described are nucleic acids that comprise the DNA methylation reporter, cells that have the DNA methylation reporter integrated into their genome, and non-human mammals comprising cells that have the DNA methylation reporter integrated into their genome. Also described are methods of measuring DNA methylation of a region of interest located in proximity to the DNA methylation reporter in the genome of a cell by detecting the reporter molecule.

1 . A nucleic acid comprising: (i) a mammalian imprinted gene promoter; and (ii) a sequence that encodes a reporter molecule that is detectable in individual mammalian cells, wherein the promoter is operably linked to the sequence that encodes the reporter molecule. 
     
     
         2 . The nucleic acid of  claim 1 , wherein the mammalian imprinted gene promoter comprises at least a portion of a parent-of-origin differentially methylated region (DMR). 
     
     
         3 . The nucleic acid of  claim 1 , further comprising a first homology arm located 5′ from the promoter and a second homology arm located 3′ from the sequence that encodes a reporter molecule, wherein the homology arms comprise sequences that are homologous to sequences that flank a target location in a mammalian genome. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The nucleic acid of  claim 3 , wherein the target location is in proximity to an enhancer, superenhancer, promoter, gene body, CpG island, or low CpG region. 
     
     
         7 .- 10 . (canceled) 
     
     
         11 . The nucleic acid of  claim 1 , wherein the imprinted gene promoter is from the Snrpn gene. 
     
     
         12 . The nucleic acid of  claim 1 , wherein the sequence of the promoter comprises SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         13 . The nucleic acid of  claim 1  wherein the reporter molecule comprises a fluorescent protein or a luciferase. 
     
     
         14 .- 23 . (canceled) 
     
     
         24 . A cell comprising the nucleic acid or vector of  claim 3 . 
     
     
         25 . (canceled) 
     
     
         26 . A cell comprising a nucleic acid comprising (i) a mammalian imprinted gene promoter; and (ii) a sequence that encodes a reporter molecule, wherein the promoter is operably linked to the sequence that encodes the reporter molecule, and wherein the nucleic acid is integrated into the genome of the cell. 
     
     
         27 .- 29 . (canceled) 
     
     
         30 . The cell of  claim 26 , wherein the imprinted gene promoter is from the Snrpn gene. 
     
     
         31 . (canceled) 
     
     
         32 . The cell of  claim 26  wherein the reporter molecule is detectable in individual cells. 
     
     
         33 . The cell of  claim 26  wherein the reporter molecule comprises a fluorescent protein or a luciferase. 
     
     
         34 .- 47 . (canceled) 
     
     
         48 . The cell of  claim 26 , wherein the cell is a mammalian cell, and wherein the genomic DNA of the cell comprises at least one region with aberrant DNA methylation. 
     
     
         49 .- 84 . (canceled) 
     
     
         85 . A method of detecting the methylation state of a DNA region of interest in the genome of a cell comprising:
 (a) providing one or more cells of  claim 26 , wherein the nucleic acid is integrated in proximity to a region of interest in the genome of the cell; and   (b) measuring expression of the reporter molecule by the one or more cells, wherein the level of expression of the reporter molecule is indicative of the level of methylation of the region of interest, thereby detecting the methylation state of the region of interest.   
     
     
         86 . The method of  claim 85 , wherein expression of the reporter molecule is indicative of hypomethylation of the DNA region of interest and lack of expression of the reporter molecule is indicative of hypermethylation of the DNA region of interest. 
     
     
         87 .- 101 . (canceled) 
     
     
         102 . A method of monitoring the methylation state of a region of interest in a cell over a period of time comprising steps of:
 (a) providing one or more cells of  claim 26 , wherein the nucleic acid is integrated in proximity to a region of interest in the genome of the cell; and   (b) measuring expression of the reporter molecule by the one or more cells at two or more time points, wherein the level of expression of the reporter molecule is indicative of the level of methylation of the region of interest, thereby monitoring the methylation state of the region of interest over a period of time.   
     
     
         103 .- 113 . (canceled) 
     
     
         114 . The method of  claim 102 , wherein the method comprises: exposing the cell to an agent or condition of interest; measuring expression of the reporter molecule at two or more time points; comparing the level of expression of the reporter molecule between two or more of the time points, wherein a difference in the level of the reporter molecule between at least two of the time points indicates that the agent or condition affects methylation of the region of interest. 
     
     
         115 .- 121 . (canceled) 
     
     
         122 . A method of evaluating the effect of an agent on the methylation state of a DNA region of interest in a cell comprising steps of: contacting one or more cells of  claim 26  with a test agent; measuring expression of the reporter molecule; and comparing the level of expression of the reporter molecule with a control value, wherein a difference between the measured value and the control value indicates that the test agent modulates the methylation state of the region of interest. 
     
     
         123 .- 124 . (canceled) 
     
     
         125 . The method of  claim 122 , wherein the method comprises detecting an increase in the level of expression of the reporter molecule as compared to the control value, thereby determining that the agent decreases methylation of the region of interest. 
     
     
         126 . The method of  claim 122 , wherein the method comprises detecting a decrease in the level of expression of the reporter molecule as compared to the control value, thereby determining that the agent increases DNA methylation of the region of interest. 
     
     
         127 .- 137 . (canceled)