Ubiquityl-Histone H2B (Lys120)

5546:

ChIP Agarose , ChIP Magnetic , Western Blotting

Ubiquityl-Histone H2B (Lys120) (D11) XP^® Rabbit mAb detects endogenous levels of histone H2B protein only when ubiquitylated on Lys120. The antibody does not cross-react with other ubiquitylated proteins or free ubiquitin.

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the carboxy terminus of the human histone H2B protein in which Lys120 is mono-ubiquitylated.

Western Blotting

Western blot analysis of extracts from various cell lines using Ubiquityl-Histone H2B (Lys120) (D11) XP^® Rabbit mAb.

Chromatin IP

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 10⁶ HeLa cells and either 10 μl of Ubiquityl-Histone H2B (Lys120) (D11) XP^® Rabbit mAb or 2 μl of Normal Rabbit IgG #2729 using SimpleChIP^® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using SimpleChIP^® Human γ-Actin Promoter Primers #5037, SimpleChIP^® Human γ-Actin Intron 3 Primers #5047, SimpleChIP^® Human GAPDH Promoter Primers #4471, and SimpleChIP^® Human GAPDH Intron 2 Primers #4478. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.

The nucleosome, made up of four core histone proteins (H2A, H2B, H3 and H4), is the primary building block of chromatin. Originally thought to function as a static scaffold for DNA packaging, histones have now been shown to be dynamic proteins, undergoing multiple types of post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitylation (1). Ubiquitin is a conserved 76 amino acid peptide unit that can be covalently linked to many cellular proteins by the ubiquitylation process. Three components are involved in this protein-ubiquitin conjugation process. Ubiquitin is first activated by forming a thiolester complex with the activation component E1; the activated ubiquitin is subsequently transferred to the ubiquitin-carrier protein E2, then from E2 to ubiquitin ligase E3 for final delivery to the epsilon-NH2 of the target protein lysine residue (2). Histone H2B is mono-ubiquitylated on lysine 120 during transcriptional activation by the RAD6 E2 protein in conjunction with the BRE1A/BRE1B E3 ligase (also known as RNF20/RNF40) (3). The RAD6/BRE1 complex is recruited to gene promoters during activation by the PAF complex, an RNA polymerase II-associated protein complex that regulates transcriptional elongation (3-5). Mono-ubiquitylated histone H2B lysine 120 is associated with the transcribed region of active genes (3,6). Mono-ubiquitylation of histone H2B stimulates transcriptional elongation by facilitating FACT-dependent chromatin remodeling (7,8). In addition, it is essential for subsequent methylation of histone H3 lysines 4 and 79, two additional histone modifications that regulate transcriptional initiation and elongation (9). Interestingly, de-ubiquitylation of histone H2B lysine 120 by USP22, a subunit of the human SAGA histone acetyltransferase complex, is a required step in transcriptional activation (10). Thus, it appears that the ubiquitylation state of histone H2B is dynamic during transcription and may serve as an intermediate step in transcriptional activation.

1. Peterson, C.L. and Laniel, M.A. (2004) Curr Biol 14, R546-51.
2. Liu, F. and Walters, K.J. (2010) Trends Biochem Sci 35, 352-60.
3. Kim, J. et al. (2009) Cell 137, 459-71.
4. Wood, A. et al. (2003) J Biol Chem 278, 34739-42.
5. Xiao, T. et al. (2005) Mol Cell Biol 25, 637-51.
6. Minsky, N. et al. (2008) Nat Cell Biol 10, 483-8.
7. Pavri, R. et al. (2006) Cell 125, 703-17.
8. Fleming, A.B. et al. (2008) Mol Cell 31, 57-66.
9. Shilatifard, A. (2006) Annu Rev Biochem 75, 243-69.
10. Wyce, A. et al. (2004) Novartis Found Symp 259, 63-73; discussion 73-7, 163-9.

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