Phosphorylation of histones and uses thereof

Abstract

Phosphorylation of histones was observed at certain tyrosine residues which have not been associated with epigenetic modification. These sites include H2B Tyr37, H4 Tyr88 and Tyr51 and H3 Tyr99. Kinases responsible for the phosphorylation as well as downstream genes regulated by such phosphorylation were also identified. Antibodies that are specific to such phosphorylated histones have been generated, which are useful for detecting the phosphorylation and related events. With such findings, the present disclosure provides compositions and methods for disease diagnosis, prognosis and therapy selection, in particular for cancer, obesity and diabetes.

1 . A method for detecting the activity of a WEE 1 protein in a sample, comprising:
 contacting the sample with a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 (KRSRKESYSVYVYKVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 62, wherein the Y(Tyr)8 residue is unphosphorylated, under conditions allowing the WEE1 protein to phosphorylate the Tyr8 residue; and detecting phosphorylation at the Tyr8 residue,   wherein detected phosphorylation indicates WEE1 protein activity in the sample.   
     
     
         2 . The method of  claim 1 , wherein the phosphorylation is detected with a probe that specifically recognizes the polypeptide with the phosphorylated Tyr8 residue. 
     
     
         3 . The method of  claim 2 , wherein the probe comprises an antibody. 
     
     
         4 . The method of  claim 3 , wherein the antibody is a phosphospecific antibody having specificity to the polypeptide with the phosphorylated Tyr8 residue. 
     
     
         5 . The method of  claim 1 , wherein the sample comprises a cancer cell or a cell isolated from a subject suspected of having cancer. 
     
     
         6 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 63 (KRSRKESpYSVYVYKVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 63, wherein the Y(Tyr)8 residue is phosphorylated. 
     
     
         7 . An isolated antibody that specifically recognizes SEQ ID NO: 63 (KRSRKESpYSVYVYKVL), wherein the Y(Tyr)8 residue is phosphorylated. 
     
     
         8 . An isolated antibody that specifically recognizes a histone H2B protein comprising a phosphorylated Tyr37 residue. 
     
     
         9 . A method for identifying a cell with an activated WEE1 protein, comprising detecting phosphorylation or a histone H2B protein at the Tyr37 residue in the cell, wherein phosphorylation of the Tyr37 residue indicates presence of an activated WEE1 protein in the cell. 
     
     
         10 . A method for identifying a subject as having cancer or risk of developing cancer, comprising:
 detecting one or more of:
 (a) phosphorylation of a histone H2B protein at the Tyr37 residue in a cell isolated from a subject; 
 (b) expression of one or more genes in the Hist1 gene cluster of cell; or 
 (c) expression of one or more microRNA selected from the group consisting of Mir26b, Mir149, Mir346, Mir350, Mir491, Mir599, Mir670, Mir708, Mir760, Mir879, Mir1192, Mir1965, Mir3058 and Mir5098; or 
 (d) expression of one or more genes selected from the group consisting of IDH2, DNMT3B, JARID2, EYA3, JMJD2c, JMJD1C, CREBBP, SOX4 and SOX18, and 
   identifying that the subject has cancer or is at risk of developing cancer if:
 (e) the phosphorylation is detected in the cell; 
 (f) the expression of the genes in the Hist1 gene cluster is decreased as compared to a suitable control sample; 
 (g) the expression of the microRNA is different as compared to a suitable control sample; or 
 (h) the expression of the genes of (d) is different as compared to a suitable control sample. 
   
     
     
         11 . A method for selecting a cancer patient for a therapy comprising a WEE1 kinase inhibitor, comprising:
 detecting one or more of:
 (a) phosphorylation of a histone H2B protein at the Tyr37 residue in a cell isolated from a cancer patient; 
 (b) expression of one or more genes in the Hist1 gene cluster of cell; or 
 (c) expression of one or more microRNA selected from the group consisting of Mir26b, Mir149, Mir346, Mir350, Mir491, Mir599, Mir670, Mir708, Mir760, Mir879, Mir1192, Mir1965, Mir3058 and Mir5098; or 
 (d) expression of one or more genes selected from the group consisting of IDH2, DNMT3B, JARID2, EYA3, JMJD2c, JMJD1C, CREBBP, SOX4 and SOX18, and 
   selecting the cancer patient for the therapy if:
 (e) the phosphorylation is detected in the cell; 
 (f) the expression of the genes in the Hist1 gene cluster is decreased as compared to a suitable control sample; 
 (g) the expression of the microRNA is different as compared to a suitable control sample; or 
 (h) the expression of the genes of (d) is different as compared to a suitable control sample. 
   
     
     
         12 . The method of  claim 11 , further comprising administering the therapy to the cancer patient. 
     
     
         13 . The method of  claim 11 , wherein the cancer patient suffers from brain cancer, glioblastomas, breast cancer, melanoma, lung cancer, prostate cancer, ovarian cancer, pancreatic cancer, lymphoma, or leukemia. 
     
     
         14 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 65 (TVTAMDVVpYALKRQGRT) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 65, wherein the Y(Tyr)9 residue is phosphorylated. 
     
     
         15 . An isolated antibody that specifically recognizes SEQ ID NO: 65 (TVTAMDVVpYALKRQGRT), wherein the Y(Tyr)9 residue is phosphorylated. 
     
     
         16 . An isolated antibody that specifically recognizes a histone H4 protein comprising a phosphorylated Tyr88 residue. 
     
     
         17 . A method for detecting the activity of a WEE1 protein or an Ack1 protein in a sample, comprising:
 contacting the sample with a polypeptide comprising an amino acid sequence of SEQ ID NO: 64 (TVTAMDVVYALKRQGRT) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 64, wherein the Y(Tyr)9 residue is unphosphorylated, under conditions allowing the WEE1 protein or the Ack1 protein to phosphorylate the Tyr9 residue; and then   detecting phosphorylation at the Tyr9 residue,   wherein detected phosphorylation indicates that WEE1 protein activity and/or Ack1 protein activity is likely present in the sample.   
     
     
         18 . A method for identifying a cell with an activated WEE1 protein or an activated Ack1 protein, comprising detecting phosphorylation of a histone H4 protein at the Tyr88 residue in the cell, wherein phosphorylation of the Tyr88 residue indicates likely presence of an activated WEE1 protein and/or an activated Ack1 protein in the cell. 
     
     
         19 . A method for identifying a subject as having cancer or at risk of developing cancer, comprising detecting phosphorylation of a histone H4 protein at the Tyr88 residue in a cell isolated from the subject, and identifying that the subject has cancer or is at risk of developing cancer if phosphorylation of the Tyr88 residue is detected. 
     
     
         20 . A method for selecting a cancer patient for a therapy comprising a WEE1 kinase inhibitor, an Ack1 inhibitor and/or an EGFR inhibitor, comprising detecting phosphorylation of a histone H4 protein at the Tyr88 residue in a cell isolated from the subject, and selecting the cancer patient for the therapy if phosphorylation of the Tyr88 residue is detected. 
     
     
         21 . The method of  claim 20 , further comprising administering the therapy to cancer patient. 
     
     
         22 . The method of  claim 20 , wherein the cancer patient suffers from brain cancer, breast cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer. 
     
     
         23 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 67 (KRISGLIpYEETRGVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 67, wherein the Y(Tyr)8 residue is phosphorylated and wherein the polypeptide does not include a histone H4 protein. 
     
     
         24 . An isolated antibody that specifically recognizes SEQ ID NO: 67 (KRISGLIpYEETRGVL), wherein the Y(Tyr)8 residue is phosphorylated. 
     
     
         25 . An antibody that specifically recognizes a histone H4 protein comprising a phosphorylated Tyr51 residue. 
     
     
         26 . A method for detecting the activity of an Ack1 protein, an EGFR protein or an insulin receptor in a sample, comprising:
 contacting the sample with a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 (KRISGLIYEETRGVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 66, wherein the Y(Tyr)8 residue is unphosphorylated, under conditions allowing the Ack1 protein, the EGFR protein or the insulin receptor to activates phosphorylation of the Tyr8 residue; and then   detecting phosphorylation at the Tyr8 residue,   wherein detected phosphorylation indicates that Ack1 protein activity, EGFR protein activity or insulin receptor activity is likely present in the sample.   
     
     
         27 . A method for identifying a cell with an activated Ack1 protein, EGFR protein or insulin receptor, comprising detecting phosphorylation of a histone H4 protein at the Tyr51 residue in the cell, wherein phosphorylation of the Tyr51 residue indicates likely presence of an activated Ack1 protein, EGFR protein and/or insulin receptor protein in the cell. 
     
     
         28 . A method for identifying a subject as having cancer or at risk of developing cancer, comprising detecting phosphorylation of a histone H4 protein at the Tyr51 residue in a cell isolated from the subject, and identifying that the subject has cancer or is at risk of developing cancer if phosphorylation of the Tyr51 residue is detected. 
     
     
         29 . A method for selecting a cancer patient for a therapy comprising an Ack1 inhibitor, an EGFR inhibitor and/or an insulin receptor inhibitor, comprising detecting phosphorylation of a histone H4 protein at the Tyr51 residue in a cell isolated from the subject, and selecting the cancer patient for the therapy if phosphorylation of the Tyr51 residue is detected. 
     
     
         30 . The method of  claim 29 , further comprising administering the therapy to the cancer patient. 
     
     
         31 . The method of  claim 29 , wherein the cancer patient suffers from brain cancer, breast cancer, melanoma or lung cancer. 
     
     
         32 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 69 (ALQEACEApYLVGLFED) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 69, wherein the Y(Tyr)9 residue is phosphorylated and wherein the polypeptide does not include a histone H3 protein. 
     
     
         33 . An isolated antibody that specifically recognizes SEQ ID NO: 69 (ALQEACEApYLVGLFED), wherein the Y(Tyr)9 residue is phosphorylated. 
     
     
         34 . An isolated antibody that specifically recognizes a histone H3 protein comprising a phosphorylated Tyr99 residue. 
     
     
         35 . A method for detecting the activity of an Ack1 protein, an EGFR protein, an FGFR protein or an insulin receptor in a sample, comprising:
 contacting the sample with a polypeptide comprising an amino acid sequence of SEQ ID NO: 68 (ALQEACEAYLVGLFED) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 68, wherein the Y(Tyr)9 residue is unphosphorylated, under conditions allowing the Ack1 protein, the LGFR protein, the FGFR protein or the insulin receptor to activate phosphorylation of the Tyr8 residue; and then   detecting phosphorylation at the Tyr8 residue,   wherein detected phosphorylation indicates that Ack1 protein activity, EGFR protein activity, FGFR activity or insulin receptor activity is likely present in the sample.   
     
     
         36 . A method for identifying a cell with an activated Ack1 protein, EGFR protein, FGFR protein or insulin receptor, comprising detecting phosphorylation of a histone H3 protein at the Tyr99 residue in the cell, wherein phosphorylation of the Tyr99 residue indicates likely presence of an activated Ack1 protein, EGFR protein, FGFR protein and/or insulin receptor protein in the cell. 
     
     
         37 . A method for identifying a subject as having cancer or at risk of developing cancer, comprising detecting phosphorylation of a histone H3 protein at the Tyr99 residue in a cell isolated from the subject, and identifying that the subject has cancer or is at risk of developing cancer if phosphorylation of the Tyr99 residue is detected. 
     
     
         38 . A method for selecting a cancer patient for a therapy comprising an Ack1 inhibitor, an EGFR inhibitor, an FGFR inhibitor and/or an insulin receptor inhibitor, comprising detecting phosphorylation of a histone H3 protein at the Tyr99 residue in a cell isolated from the subject, and selecting the cancer patient for the therapy if phosphorylation of the Tyr99 residue is detected. 
     
     
         39 . The method of  claim 38 , further comprising administering the therapy to the cancer patient. 
     
     
         40 . The method of  claim 38 , wherein the cancer patient suffers from prostate cancer, breast cancer, pancreatic cancer, melanoma or lung cancer. 
     
     
         41 . A method for reducing the body weight or treating obesity or diabetes in a subject, comprising administering to the subject an Ack1 inhibitor or an agent that decrease the expression or activity of Ack1. 
     
     
         42 . The method of  claim 41 , wherein the Ack1 inhibitor is a small molecule inhibitor or siRNA. 
     
     
         43 . The method of  claim 42 , wherein the Ack1 inhibitor comprises AIM-100 or an isolated Ack1 antibody. 
     
     
         44 . A kit comprising (a) one or more proteins selected from:
 (i) a polypeptide comprising an amino acid sequence of SEQ ID NO: 62 (KRSRKESYSVYVYKVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 62, wherein the Y(Tyr)8 residue is unphosphorylated;   (ii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 64 (TVTAMDVVYALKRQGRT) or an amino acid sequence having at least about 95% sequence identity to SEQ Ill NO: 64, wherein the Y(Tyr)9 residue is unphosphorylated;   (iii) a polypeptide comprising an amino acid sequence of SEQ ID NO: 66 (KRISGLIYEETRGVL) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 66, wherein the Y(Tyr)8 residue is unphosphorylated; or   (iv) a polypeptide comprising an amino acid sequence of SEQ ID NO: 68 (ALQEACEAYLVGLFED) or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 68, wherein the Y(Tyr)9 residue is unphosphorylated, and (b) one or more antibodies that specifically recognize the one or more proteins of (a).   
     
     
         45 . The kit of  claim 44 , wherein the one or more proteins are affixed on a scaffold.