Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex

Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8.     

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.     

Unique Role of the WD-40 Repeat Protein 5 (WDR5) Subunit within the Mixed Lineage Leukemia 3 (MLL3) Histone Methyltransferase Complex

The MLL3 (mixed lineage leukemia 3) protein is a member of the human SET1 family of histone H3 lysine 4 methyltransferases and contains the conserved WDR5 interaction (Win) motif and the catalytic suppressor of variegation, enhancer of zeste, trithorax (SET) domain. The human SET1 family includes MLL1–4 and SETd1A/B, which all interact with a conserved subcomplex containing WDR5, RbBP5, Ash2L, and DPY-30 (WRAD) to form the minimal core complex required for full methyltransferase activity. However, recent evidence suggests that the WDR5 subunit may not be utilized in an identical manner within all SET1 family core complexes. Although the roles of WDR5 within the MLL1 core complex have been extensively studied, not much is known about the roles of WDR5 in other SET1 family core complexes. In this investigation, we set out to characterize the roles of the WDR5 subunit in the MLL3 core complex. We found that unlike MLL1, the MLL3 SET domain assembles with the RbBP5/Ash2L heterodimer independently of the Win motif-WDR5 interaction. Furthermore, we observed that WDR5 inhibits the monomethylation activity of the MLL3 core complex, which is dependent on the Win motif. We also found evidence suggesting that the WRAD subcomplex catalyzes weak H3K4 monomethylation within the context of the MLL3 core complex. Furthermore, solution structures of the MLL3 core complex assembled with and without WDR5 by small angle x-ray scattering show similar overall topologies. Together, this work demonstrates a unique role for WDR5 in modulating the enzymatic activity of the MLL3 core complex.     

A Novel 3-Methylhistidine Modification of Yeast Ribosomal Protein Rpl3 Is Dependent upon the YIL110W Methyltransferase

We have shown that Rpl3, a protein of the large ribosomal subunit from baker''s yeast (Saccharomyces cerevisiae), is stoichiometrically monomethylated at position 243, producing a 3-methylhistidine residue. This conclusion is supported by top-down and bottom-up mass spectrometry of Rpl3, as well as by biochemical analysis of Rpl3 radiolabeled in vivo with S-adenosyl-l-[methyl-³H]methionine. The results show that a +14-Da modification occurs within the GTKKLPRKTHRGLRKVAC sequence of Rpl3. Using high-resolution cation-exchange chromatography and thin layer chromatography, we demonstrate that neither lysine nor arginine residues are methylated and that a 3-methylhistidine residue is present. Analysis of 37 deletion strains of known and putative methyltransferases revealed that only the deletion of the YIL110W gene, encoding a seven β-strand methyltransferase, results in the loss of the +14-Da modification of Rpl3. We suggest that YIL110W encodes a protein histidine methyltransferase responsible for the modification of Rpl3 and potentially other yeast proteins, and now designate it Hpm1 (Histidine protein methyltransferase 1). Deletion of the YIL110W/HPM1 gene results in numerous phenotypes including some that may result from abnormal interactions between Rpl3 and the 25 S ribosomal RNA. This is the first report of a methylated histidine residue in yeast cells, and the first example of a gene required for protein histidine methylation in nature.     

Structural and biochemical studies of human lysine methyltransferase Smyd3 reveal the important functional roles of its post-SET and TPR domains and the regulation of its activity by DNA binding

The SET- and MYND-domain containing (Smyd) proteins constitute a special subfamily of the SET-containing lysine methyltransferases. Here we present the structure of full-length human Smyd3 in complex with S-adenosyl-l-homocysteine at 2.8 Å resolution. Smyd3 affords the first example that other region(s) besides the SET domain and its flanking regions participate in the formation of the active site. Structural analysis shows that the previously uncharacterized C-terminal domain of Smyd3 contains a tetratrico-peptide repeat (TPR) domain which together with the SET and post-SET domains forms a deep, narrow substrate binding pocket. Our data demonstrate the important roles of both TPR and post-SET domains in the histone lysine methyltransferase (HKMT) activity of Smyd3, and show that the hydroxyl group of Tyr239 is critical for the enzymatic activity. The characteristic MYND domain is located nearby to the substrate binding pocket and exhibits a largely positively charged surface. Further biochemical assays show that DNA binding of Smyd3 can stimulate its HKMT activity and the process may be mediated via the MYND domain through direct DNA binding.     

Native Variants of the MRB1 Complex Exhibit Specialized Functions in Kinetoplastid RNA Editing

Adaptation and survival of Trypanosoma brucei requires editing of mitochondrial mRNA by uridylate (U) insertion and deletion. Hundreds of small guide RNAs (gRNAs) direct the mRNA editing at over 3,000 sites. RNA editing is controlled during the life cycle but the regulation of substrate and stage specificity remains unknown. Editing progresses in the 3’ to 5’ direction along the pre-mRNA in blocks, each targeted by a unique gRNA. A critical editing factor is the mitochondrial RNA binding complex 1 (MRB1) that binds gRNA and transiently interacts with the catalytic RNA editing core complex (RECC). MRB1 is a large and dynamic complex that appears to be comprised of distinct but related subcomplexes (termed here MRBs). MRBs seem to share a ‘core’ complex of proteins but differ in the composition of the ‘variable’ proteins. Since some proteins associate transiently the MRBs remain imprecisely defined. MRB1 controls editing by unknown mechanisms, and the functional relevance of the different MRBs is unclear. We previously identified two distinct MRBs, and showed that they carry mRNAs that undergo editing. We proposed that editing takes place in the MRBs because MRBs stably associate with mRNA and gRNA but only transiently interact with RECC, which is RNA free. Here, we identify the first specialized functions in MRBs: 1) 3010-MRB is a major scaffold for RNA editing, and 2) REH2-MRB contains a critical trans-acting RNA helicase (REH2) that affects multiple steps of editing function in 3010-MRB. These trans effects of the REH2 include loading of unedited mRNA and editing in the first block and in subsequent blocks as editing progresses. REH2 binds its own MRB via RNA, and conserved domains in REH2 were critical for REH2 to associate with the RNA and protein components of its MRB. Importantly, REH2 associates with a ~30 kDa RNA-binding protein in a novel ~15S subcomplex in RNA-depleted mitochondria. We use these new results to update our model of MRB function and organization.     

Mammalian Target of Rapamycin (mTOR) Activation Increases Axonal Growth Capacity of Injured Peripheral Nerves

Unlike neurons in the central nervous system (CNS), injured neurons in the peripheral nervous system (PNS) can regenerate their axons and reinnervate their targets. However, functional recovery in the PNS often remains suboptimal, especially in cases of severe damage. The lack of regenerative ability of CNS neurons has been linked to down-regulation of the mTOR (mammalian target of rapamycin) pathway. We report here that PNS dorsal root ganglial neurons (DRGs) activate mTOR following damage and that this activity enhances axonal growth capacity. Furthermore, genetic up-regulation of mTOR activity by deletion of tuberous sclerosis complex 2 (TSC2) in DRGs is sufficient to enhance axonal growth capacity in vitro and in vivo. We further show that mTOR activity is linked to the expression of GAP-43, a crucial component of axonal outgrowth. However, although TSC2 deletion in DRGs facilitates axonal regrowth, it leads to defects in target innervation. Thus, whereas manipulation of mTOR activity could provide new strategies to stimulate nerve regeneration in the PNS, fine control of mTOR activity is required for proper target innervation.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.     

The motility of a human parasite, Toxoplasma gondii, is regulated by a novel lysine methyltransferase

Protozoa in the phylum Apicomplexa are a large group of obligate intracellular parasites. Toxoplasma gondii and other apicomplexan parasites, such as Plasmodium falciparum, cause diseases by reiterating their lytic cycle, comprising host cell invasion, parasite replication, and parasite egress. The successful completion of the lytic cycle requires that the parasite senses changes in its environment and switches between the non-motile (for intracellular replication) and motile (for invasion and egress) states appropriately. Although the signaling pathway that regulates the motile state switch is critical to the pathogenesis of the diseases caused by these parasites, it is not well understood. Here we report a previously unknown mechanism of regulating the motility activation in Toxoplasma, mediated by a protein lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). AKMT depletion greatly inhibits activation of motility, compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Interestingly, AKMT redistributes from the apical complex to the parasite body rapidly in the presence of egress-stimulating signals that increase [Ca²⁺] in the parasite cytoplasm, suggesting that AKMT regulation of parasite motility might be accomplished by the precise temporal control of its localization in response to environmental changes.     

A Conserved Interaction between the SDI Domain of Bre2 and the Dpy-30 Domain of Sdc1 Is Required for Histone Methylation and Gene Expression

In Saccharomyces cerevisiae, lysine 4 on histone H3 (H3K4) is methylated by the Set1 complex (Set1C or COMPASS). Besides the catalytic Set1 subunit, several proteins that form the Set1C (Swd1, Swd2, Swd3, Spp1, Bre2, and Sdc1) are also needed to mediate proper H3K4 methylation. Until this study, it has been unclear how individual Set1C members interact and how this interaction may impact histone methylation and gene expression. In this study, Bre2 and Sdc1 are shown to directly interact, and it is shown that the association of this heteromeric complex is needed for proper H3K4 methylation and gene expression to occur. Interestingly, mutational and biochemical analysis identified the C terminus of Bre2 as a critical protein-protein interaction domain that binds to the Dpy-30 domain of Sdc1. Using the human homologs of Bre2 and Sdc1, ASH2L and DPY-30, respectively, we demonstrate that the C terminus of ASH2L also interacts with the Dpy-30 domain of DPY-30, suggesting that this protein-protein interaction is maintained from yeast to humans. Because of the functionally conserved nature of the C terminus of Bre2 and ASH2L, this region was named the SDI (Sdc1 Dpy-30 interaction) domain. Finally, we show that the SDI-Dpy-30 domain interaction is physiologically important for the function of Set1 in vivo, because specific disruption of this interaction prevents Bre2 and Sdc1 association with Set1, resulting in H3K4 methylation defects and decreases in gene expression. Overall, these and other mechanistic studies on how H3K4 methyltransferase complexes function will likely provide insights into how human MLL and SET1-like complexes or overexpression of ASH2L leads to oncogenesis.