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 Tyr 51 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 WEE1 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 and a carrier. 
     
     
         7 . An isolated antibody that specifically recognizes SEQ ID NO: 63 (KRSRKESpYSVYVYKVL), wherein the Y(Tyr)8 residue is phosphorylated, with the proviso that the antibody is not a polyclonal antibody or an isolated naturally occurring antibody. 
     
     
         8 . An isolated antibody that specifically recognizes a histone H2B protein comprising a phosphorylated Tyr37 residue, with the proviso that the antibody is not a polyclonal antibody or an isolated naturally occurring antibody. 
     
     
         9 . A method for identifying a cell with an activated WEE1 protein, comprising detecting phosphorylation of 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 at risk of developing cancer and further progressing to metastatic disease, 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  or  12 , 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 and a carrier. 
     
     
         15 . An isolated antibody that specifically recognizes SEQ ID NO: 65 (TVTAMDVVpYALKRQGRT), wherein the Y(Tyr)9 residue is phosphorylated, with the proviso that the antibody is not a polyclonal antibody or an isolated naturally occurring antibody. 
     
     
         16 . An isolated antibody that specifically recognizes a histone H4 protein comprising a phosphorylated Tyr88 residue, with the proviso that the antibody is not a polyclonal antibody or an isolated naturally occurring antibody. 
     
     
         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 - 50 . (canceled)