Purification of histone ubiquitin ligases from HeLa cells

Abstract

Posttranslational histone modifications play an important role in regulating chromatin based nuclear processes including transcription. Of these modifications, histone ubiquitination is among the least understood. Histone ubiquitination predominately targets histones H2A and H2B. While ubiquitination of H2B is evolutionarily conserved from budding yeast to mammals, ubiquitination of H2A has not been detected in budding yeast, worms, or plants. Until recently, studies of histone ubiquitination lagged far behind the study of other histone modifications, largely because antibodies specific for ubiquitinated histones are difficult to generate. Despite this obstacle, the identification of the enzymatic machineries involved in histone ubiquitination, together with the successful use of a combination of genetic and immunoblot approaches to detect ubiquitinated histones, have helped to reveal important regulatory roles for this modification in transcriptional initiation and elongation, cell cycle progression, and DNA damage response. With the aid of the recently developed ubiquitinated histone-specific antibodies, an intriguing link between histone ubiquitination and cancer development has been established. While the enzymes involved in H2B ubiquitination were identified first in budding yeast and subsequently in higher organisms based on gene homology, the identification of the enzymatic machineries involved in H2A ubiquitination largely depended on a biochemical purification approach. The unbiased search for ubiquitin ligases targeting histones also led to the identification of a H3 and H4 ubiquitin ligase. Here we detail a protocol for the biochemical approach to identify histone ubiquitin ligase(s) from HeLa cells. Similar approaches have been successfully used to identify histone methyltransferases, histone demethylases, chromatin remodeling factors, and general transcription factors. So long as an in vitro enzymatic assay can be established, the approach we describe can be easily adapted to identify other histone and non-histone modifying enzymes.

Figure 1

Schematic depiction of histone ubiquitination. Ubiquitin is first activated by an E1 activating enzyme. Ubiquitin is then transferred to an E2 ubiquitin conjugating enzyme. HECT family E3 ligases receive ubiquitin from an E2 and directly conjugate ubiquitin to their target molecules. RING and U-box domain containing E3 ligases mediate the interaction between E2 ubiquitin conjugating enzymes and substrates and oversee the conjugation of ubiquitin to target molecules.

Figure 2

Purification of oligonucleosomes and mononucleosomes from HeLa S3 cells. a. MNase test digestion of 500 μl nuclei suspension for increasing lengths of time. b. DNA extraction of from sucrose gradient fractions. Mononucleosomes are enriched in fraction containing a majority of DNA approximately 150bp in length (fractions 10–12). Oligonucleosomes are enriched in fractions 18–20. c. TCA precipitation of different sucrose gradient fractions. Early fractions contain high molecular weight contaminants.

Figure 3

Identification of an H2A ubiquitin ligase activity in HeLa cells. a, 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. Left and right panels use histone octamer and oligonucleosome substrates, respectively. b, The ligase activity depends on the presence of ATP, E1, E2, ubiquitin, nucleosomal histones, and proteins present in the 0.5M P11 nuclear pellet fraction.

Figure 4

Purification and identification of the H2A ubiquitination ligase complex. a, 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. b, Silver staining of a polyacrylamide–SDS 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–PAGE are indicated to the left of the panel.

Figure 5

Purification of a previously unidentified histone ubiquitin E3 ligase complex. a. Schematic representation of the steps used to purify the histone ubiquitin E3 ligase complex. Numbers represent the salt concentrations (mM) at which the E3 ligase activity elutes from the columns. b. Histone ubiquitin ligase assay of protein fractions derived from a Mono Q column. In addition to the ubiquitin ligase activity specific for histone H2A (hPRC1L), a previously unidentified ubiquitin ligase activity for all the core histones was observed in the flowthrough (Ft). c. Silver staining of a polyacrylamide-SDS gel (top) and histone ubiquitin ligase activity (bottom) of fractions derived from a Mono S column. The protein bands that cofractionated with the histone ubiquitin E3 ligase activity are indicated by an asterisk (*). The protein size marker is indicated on the left side of the panel.