Assays for Acetylation and Other Acylations of Lysine Residues.

Lysine acetylation refers to addition of an acetyl moiety to the epsilon-amino group of a lysine residue and is important for regulating protein functions in various organisms from bacteria to humans. This is a reversible and precisely controlled covalent modification that either serves as an on/off switch or participates in a codified manner with other post-translational modifications to regulate different cellular and developmental processes in normal and pathological states. This unit describes methods for in vitro and in vivo determination of lysine acetylation. Such methods can be easily extended for analysis of other acylations (such as propionylation, butyrylation, crotonylation, and succinylation) that are also present in histones and many other proteins. © 2017 by John Wiley & Sons, Inc.

MOZ and MORF histone acetyltransferases interact with the Runt-domain transcription factor Runx2.

The monocytic leukemia zinc finger protein MOZ and its homologue MORF have been implicated in leukemogenesis. Both MOZ and MORF are histone acetyltransferases with weak transcriptional repression domains and strong transcriptional activation domains, suggesting that they may function as transcriptional coregulators. Here we describe that MOZ and MORF both interact with Runx2 (or Cbfa1), a Runt-domain transcription factor that is known to play important roles in T cell lymphomagenesis and bone development. Through its C-terminal SM (serine- and methionine-rich) domain, MORF binds to Runx2 in vitro and in vivo. Consistent with this, the SM domain of MORF also binds to Runx1 (or AML1), a Runx2 homologue that is frequently altered by leukemia-associated chromosomal translocations. While MORF does not acetylate Runx2, its SM domain potentiates Runx2-dependent transcriptional activation. Moreover, endogenous MORF is required for transcriptional activation by Runx2. Intriguingly, Runx2 negatively regulates the transcriptional activation potential of the SM domain. Like that of MORF, the SM domain of MOZ physically and functionally interacts with Runx2. These results thus identify Runx2 as an interaction partner of MOZ and MORF and suggest that both acetyltransferases are involved in regulating transcriptional activation mediated by Runx2 and its homologues.

Reconstitution of active and stoichiometric multisubunit lysine acetyltransferase complexes in insect cells.

Protein lysine acetyltransferases (KATs) catalyze acetylation of the ε-amino group on a specific lysine residue, and this posttranslational modification is important for regulating the function and activities of thousands of proteins in diverse organisms from bacteria to humans. Interestingly, many known KATs exist in multisubunit complexes and complex formation is important for their proper structure, function, and regulation. Thus, it is necessary to reconstitute enzymatically active complexes for studying the relationship between subunits and determining structures of the complexes. Due to inherent limitations of bacterial and mammalian expression systems, baculovirus-mediated protein expression in insect cells has proven useful for assembling such multisubunit complexes. Related to this, we have adopted such an approach for reconstituting active tetrameric complexes of monocytic leukemia zinc (MOZ, finger protein, recently renamed MYST3 or KAT6A) and MOZ-related factor (MORF, also known as MYST4 or KAT6B), two KATs directly linked to development of leukemia and self-renewal of stem cells. Herein, we use these complexes as examples to describe the related procedures. Similar methods have been used for reconstituting active complexes of histone deacetylases, lysine demethylases, and ubiquitin ligases, so this simple approach can be adapted for molecular dissection of various multisubunit complexes.

Analysis of protein lysine acetylation in vitro and in vivo.

Protein lysine acetylation, referring to acetylation of the epsilon-amino group of a lysine residue, has recently emerged as an important post-translational modification for regulating protein functions in various organisms. Like phosphorylation, lysine acetylation is a rapidly reversible and precisely controlled covalent modification that serves as a simple on/off switch or participates in a codified manner with other post-translational modifications to regulate protein functions in different cellular and developmental processes. This unit describes and discusses methods used for in vitro and in vivo determination of lysine acetylation.

Molecular architecture of quartet MOZ/MORF histone acetyltransferase complexes.

The monocytic leukemia zinc finger protein MOZ and the related factor MORF form tetrameric complexes with ING5 (inhibitor of growth 5), EAF6 (Esa1-associated factor 6 ortholog), and the bromodomain-PHD finger protein BRPF1, -2, or -3. To gain new insights into the structure, function, and regulation of these complexes, we reconstituted them and performed various molecular analyses. We found that BRPF proteins bridge the association of MOZ and MORF with ING5 and EAF6. An N-terminal region of BRPF1 interacts with the acetyltransferases; the enhancer of polycomb (EPc) homology domain in the middle part binds to ING5 and EAF6. The association of BRPF1 with EAF6 is weak, but ING5 increases the affinity. These three proteins form a trimeric core that is conserved from Drosophila melanogaster to humans, although authentic orthologs of MOZ and MORF are absent in invertebrates. Deletion mapping studies revealed that the acetyltransferase domain of MOZ/MORF is sufficient for BRPF1 interaction. At the functional level, complex formation with BRPF1 and ING5 drastically stimulates the activity of the acetyltransferase domain in acetylation of nucleosomal histone H3 and free histones H3 and H4. An unstructured 18-residue region at the C-terminal end of the catalytic domain is required for BRPF1 interaction and may function as an "activation lid." Furthermore, BRPF1 enhances the transcriptional potential of MOZ and a leukemic MOZ-TIF2 fusion protein. These findings thus indicate that BRPF proteins play a key role in assembling and activating MOZ/MORF acetyltransferase complexes.

Expression, purification, and analysis of MOZ and MORF histone acetyltransferases.

Histone acetylation is one major mechanism by which chromatin structure and function are regulated. Besides histones, many nonhistone proteins are also acetylated in vivo. Aberrant acetylation has been linked to the development of various human diseases. Through acetylating histone and nonhistone proteins, histone acetyltransferases (HATs) play fundamental roles in regulating chromatin remodeling, transcription, and other nuclear processes. Known HATs belong to several groups, including the GCN5/PCAF, p300/CBP, and MYST families. ESA1, SAS3, MOF, TIP60, HBO1, MOZ, and MORF are the MYST family members with demonstrated HAT activity. The MOZ and MORF genes are rearranged by chromosome abnormalities associated with several types of leukemia, so these two HATs have been implicated in leukemogenesis. Compared with p300, CBP, and PCAF, much less is known about MOZ and MORF. To elucidate the function and regulation of these two interesting HATs, we have conducted their initial characterization. Here we describe the expression, purification, and activity analysis of MOZ and MORF. For comparison, we also include the procedure for expression and purification of PCAF. These methods are useful not only for functional characterization of MOZ, MORF, PCAF, and other HATs, but also for preparation of HAT proteins to screen compound libraries and obtain inhibitors with potential therapeutic value.

Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms.

LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.