Ubiquitin E3 ligase

#The present invention concerns a ubiquitin E3 ligase and methods of identifying modulators thereof.

#Covalent modification of histones play important roles in regulating chromatin dynamics and transcription (Jenuwein et al., (2001) Science 293:1074-80; Zhang et al., (2001) Genes Dev 15:2343-60). One example of histone modification is ubiquitination, which mainly occurs on histones H2A and H2B (Zhang, (2003) Genes Dev 17:2733-40). Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination (Robzyk et al., (2000) Science 287:501-4; Hwang et al., (2003) Mol Cell 11: 261-6; Wood et al., (2003) Mol Cell 11:267-74) and a “cross-talk” between H2B ubiquitination and histone methylation (Sun et al., (2002) Nature 418:104-8; Dover et al., (2002) J. Biol. Chem. 277:28368-71), the responsible enzymes and the functions of H2A ubiquitination are unknown.

#Ligation of ubiquitin involves a series of enzymatic reactions. First, ubiquitin itself is activated with ATP by the ubiquitin-activating enzyme (E1), then transferred to a ubiquitin carrier protein (E2), and finally it is transferred to ubiquitin ligase (E3). E3 enzyme catalyzes the covalent modification of lysine residues of the target protein with activated ubiquitin (Chau et al. (1989) Science 243:1576-1583). Recently, an additional conjugation factor, named E4, has been shown to be important in the ubiquitination process in conjunction with E1, E2, and E3 (Koegl et al. (1999) Cell 96(5):635-44).

#FIGS. 1A-1C show the identification of an H2A ubiquitin ligase activity in HeLa cells. FIG. 1A, Ubiquitin ligase assay using HeLa nuclear proteins fractionated on DE52 and P11 columns. Numbers on top of the panels indicate the salt concentration (M) for step elution. NE and NP represent nuclear extracts and nuclear pellet, respectively. Molecular weight markers are indicated at the left of the panel. Left and right panels use histone octamer and oligonucleosome substrates, respectively. FIG. 1B, The ligase activity depends on the presence of ATP, E1, E2, ubiquitin, nucleosomal histones (NUC.), and proteins present in the 0.5 M P11 nuclear pellet fraction. Molecular weight markers are indicated at the left of the panel. FIG. 1C, Mass spectrometry analysis of the ubiquitinated protein revealed the presence of histone H2A and ubiquitin. A peptide that contains amino acids from both ubiquitin and H2A identifies lysine 119 as the ubiquitination site.

#FIGS. 2A-2E show the purification and identification of the H2A ubiquitination ligase complex. FIG. 2A, Schematic representation of the steps used to purify the H2A ubiquitination ligase complex. Numbers represent the salt concentrations (mM) at which the E3 ligase activity elutes from the columns. FIG. 2B, The H2A ubiquitin ligase activity assay (top panel) and western blot analysis (bottom two panels) of the protein fractions derived from the gel-filtration S300 column. The elution profile of the protein markers is indicated on top of the panel. Antibodies used for the western blot are indicated. FIG. 2C, Silver stained SDS-polyacrylamide gel (top panel), H2A ubiquitin ligase activity assay (second panel) and western blot analysis (bottom two panels) of the fractions derived from the MONOQ™ column. The candidate proteins that co-fractionated with the E3 ligase activity are indicated by *. The positions of the protein size markers on SDS-polyacrylamide gel electrophoresis (PAGE) are indicated to the left of the panel. FIG. 2D, Silver stained SDS-polyacrylamide gels containing samples derived from input (In), flow-through (Ft), and Bound (B) proteins immunoaffinity purified with rabbit IgG and Ring1 antibodies. The identities of the proteins co-immunoprecipitating with Ring1 are indicated. Peptides derived from each protein band are also indicated. Numbers represent the amino acid number of the respective proteins. NS indicates nonspecific-binding proteins that are also present in the control IP (lane 3). FIG. 2E, The same samples presented in FIG. 2D were analyzed by H2A ubiquitin ligase activity assay (top panel) and western blot analysis (bottom two panels). Antibodies used are indicated.

#FIGS. 3A-3E show that Ring2 is the catalytic subunit and is required for H2A ubiquitination in vivo. FIG. 3A, Diagram of the human Ring1, Ring2, their Drosophila homolog dRing, and human Bim1. A sequence alignment of the ring finger domain of these proteins is shown. Amino acids that are completely conserved are boxed. The two Ring2 mutants used in FIG. 3B are indicated by *. Numbers represent the amino acid number. FIG. 3B, Ubiquitin ligase activity (bottom panel) of recombinant proteins (top panel, lanes 2-6, 8 and 9) and native hPRC1L (lane 7). FIG. 3C, Estimation of the Ring2 knock-down level in the KD1 and KD2 cell lines relative to the mock knock-down cells transfected with empty vector. Tubulin was used as a loading control. Antibodies used are indicated on the left of the panel. FIG. 3D, Sequence alignment between ubiquitinated human H2A (hH2A) and Drosophila H2A (dH2A). Amino acids that are completely conserved are boxed. Ubiquitinated Lys119 is indicated. FIG. 3E, Ring2 knock-down results in morphological change and cell growth inhibition. Top panels show morphological changes of control and knock-down HeLa cells. Bottom panel shows the growth curve of control and knock-down HeLa cells. Viable cells were counted by trypan blue staining at different times after initial seeding of 3×104 cells.

#The present invention will now be described in more detail with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

#Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

#As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.