Christine E. Schaner Tooley

NRMT2 is an N-terminal monomethylase that primes for its homologue NRMT1.

NRMT (N-terminal regulator of chromatin condensation 1 methyltransferase) was the first eukaryotic methyltransferase identified to specifically methylate the free α-amino group of proteins. Since the discovery of this N-terminal methyltransferase, many new substrates have been identified and the modification itself has been shown to regulate DNA-protein interactions. Sequence analysis predicts one close human homologue of NRMT, METTL11B (methyltransferase-like protein 11B, now renamed NRMT2). We show in the present paper for the first time that NRMT2 also has N-terminal methylation activity and recognizes the same N-terminal consensus sequences as NRMT (now NRMT1). Both enzymes have similar tissue expression and cellular localization patterns. However, enzyme assays and MS experiments indicate that they differ in their specific catalytic functions. Although NRMT1 is a distributive methyltransferase that can mono-, di- and tri-methylate its substrates, NRMT2 is primarily a monomethylase. Concurrent expression of NRMT1 and NRMT2 accelerates the production of trimethylation, and we propose that NRMT2 activates NRMT1 by priming its substrates for trimethylation.

NRMT1 knockout mice exhibit phenotypes associated with impaired DNA repair and premature aging.

Though defective genome maintenance and DNA repair have long been known to promote phenotypes of premature aging, the role protein methylation plays in these processes is only now emerging. We have recently identified the first N-terminal methyltransferase, NRMT1, which regulates protein-DNA interactions and is necessary for both accurate mitotic division and nucleotide excision repair. To demonstrate if complete loss of NRMT1 subsequently resulted in developmental or aging phenotypes, we constructed the first NRMT1 knockout (Nrmt1(-/-)) mouse. The majority of these mice die shortly after birth. However, the ones that survive, exhibit decreased body size, female-specific infertility, kyphosis, decreased mitochondrial function, and early-onset liver degeneration; phenotypes characteristic of other mouse models deficient in DNA repair. The livers from Nrmt1(-/-) mice produce less reactive oxygen species (ROS) than wild type controls, and Nrmt1(-/-) mouse embryonic fibroblasts show a decreased capacity for handling oxidative damage. This indicates that decreased mitochondrial function may benefit Nrmt1(-/-) mice and protect them from excess internal ROS and subsequent DNA damage. These studies position the NRMT1 knockout mouse as a useful new system for studying the effects of genomic instability and defective DNA damage repair on organismal and tissue-specific aging.

New roles for old modifications: Emerging roles of N‐terminal post‐translational modifications in development and disease

The importance of internal post‐translational modification (PTM) in protein signaling and function has long been known and appreciated. However, the significance of the same PTMs on the alpha amino group of N‐terminal amino acids has been comparatively understudied. Historically considered static regulators of protein stability, additional functional roles for N‐terminal PTMs are now beginning to be elucidated. New findings show that N‐terminal methylation, along with N‐terminal acetylation, is an important regulatory modification with significant roles in development and disease progression. There are also emerging studies on the enzymology and functional roles of N‐terminal ubiquitylation and N‐terminal propionylation. Here, will discuss the recent advances in the functional studies of N‐terminal PTMs, recount the new N‐terminal PTMs being identified, and briefly examine the possibility of dynamic N‐terminal PTM exchange.

Select human cancer mutants of NRMT1 alter its catalytic activity and decrease N-terminal trimethylation

A subset of B‐cell lymphoma patients have dominant mutations in the histone H3 lysine 27 (H3K27) methyltransferase EZH2, which change it from a monomethylase to a trimethylase. These mutations occur in aromatic resides surrounding the active site and increase growth and alter transcription. We study the N‐terminal trimethylase NRMT1 and the N‐terminal monomethylase NRMT2. They are 50% identical, but differ in key aromatic residues in their active site. Given how these residues affect EZH2 activity, we tested whether they are responsible for the distinct catalytic activities of NRMT1/2. Additionally, NRMT1 acts as a tumor suppressor in breast cancer cells. Its loss promotes oncogenic phenotypes but sensitizes cells to DNA damage. Mutations of NRMT1 naturally occur in human cancers, and we tested a select group for altered activity. While directed mutation of the aromatic residues had minimal catalytic effect, NRMT1 mutants N209I (endometrial cancer) and P211S (lung cancer) displayed decreased trimethylase and increased monomethylase/dimethylase activity. Both mutations are located in the peptide‐binding channel and indicate a second structural region impacting enzyme specificity. The NRMT1 mutants demonstrated a slower rate of trimethylation and a requirement for higher substrate concentration. Expression of the mutants in wild type NRMT backgrounds showed no change in N‐terminal methylation levels or growth rates, demonstrating they are not acting as dominant negatives. Expression of the mutants in cells lacking endogenous NRMT1 resulted in minimal accumulation of N‐terminal trimethylation, indicating homozygosity could help drive oncogenesis or serve as a marker for sensitivity to DNA damaging chemotherapeutics or γ‐irradiation.

BMP-9 dependent pathways required for the chondrogenic differentiation of pluripotent stem cells

Current cartilage repair therapies focus on the delivery of chondrocytes differentiated from mesenchymal stem cells, and thus understanding the factors that promote chondrogenesis may lead to improved therapies. Several bone morphogenetic proteins (BMPs) have been implicated in chondrogenic differentiation and/or chondrocyte function. Although the signaling pathways downstream of BMPs have been studied in other systems, their role in chondrogenesis is less well characterized. Here, we investigated the effects of BMP-9 in chondroprogenitor cells. Compared to BMP-2 and BMP-6, we showed that BMP-9 was significantly more potent in inducing chondrogenic differentiation in mouse C3H10T1/2 and ATDC5 cells. Moreover, we demonstrated that BMP-9 induces the phosphorylation of SMAD1/5 in a dose and time dependent manner. Confocal immunofluorescence microscopy further demonstrated an accumulation of phosphorylated SMAD1/5 in the nuclei of BMP-9 treated cells. Consistent with activation of the SMAD signaling pathway, we also observed an up-regulation of Id1 and PAI-I expression. Importantly, we demonstrated that the simultaneous knockdown of SMAD1 and SMAD5 was able to inhibit chondrogenesis. Additionally, we also observed activation of p38 by BMP-9, and pharmacological inhibition of this pathway blocked chondrogenesis. In contrast, inhibition of p44/42 ERK had no effect. Finally, we tested the ability of Noggin to block the actions of BMP-9. While Noggin potently inhibited the ability of BMP-2 to mediate differentiation, it had no significant effect on BMP-9. Our findings provide a clearer understanding of the cellular pathways utilized by BMP-9 for chondrogenesis that may help improve current therapies for regenerative cartilage repair.

The N-terminal methyltransferase homologs NRMT1 and NRMT2 exhibit novel regulation of activity through heterotrimer formation: Interactions Between NRMT1 and NRMT2

Protein, DNA, and RNA methyltransferases have an ever‐expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered by complex formation with close homologs. We work with the N‐terminal methyltransferase homologs NRMT1 and NRMT2. NRMT1 is a ubiquitously expressed distributive trimethylase. NRMT2 is a monomethylase expressed at low levels in a tissue‐specific manner. They are both nuclear methyltransferases with overlapping consensus sequences but have distinct enzymatic activities and tissue expression patterns. Co‐expression with NRMT2 increases the trimethylation rate of NRMT1, and here we aim to understand how this occurs. We use analytical ultracentrifugation to show that while NRMT1 primarily exists as a dimer and NRMT2 as a monomer, when co‐expressed they form a heterotrimer. We use co‐immunoprecipitation and molecular modeling to demonstrate in vivo binding and map areas of interaction. While overexpression of NRMT2 increases the half‐life of NRMT1, the converse is not true, indicating that NRMT2 may be increasing NRMT1 activity by stabilizing the enzyme. Accordingly, the catalytic activity of NRMT2 is not needed to increase NRMT1 activity or increase its affinity for less preferred substrates. Monomethylation can also not rescue phenotypes seen with loss of trimethylation. Taken together, these data support a model where NRMT2 expression activates NRMT1 activity, not through priming, but by increasing its stability and substrate affinity.

Abstract 5386: Investigating the role of N-terminal protein methylation in colon cancer progression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC We have recently identified the first mammalian N-terminal methyltransferase, NRMT. NRMT is a highly conserved, ubiquitously expressed nuclear enzyme that is predicted to methylate more than 300 target proteins, including the proven targets Retinoblastoma Protein (RB), DDB2, and the oncoprotein SET. N-terminal methylation has been shown to regulate protein-DNA interactions and has also been implicated in regulating protein stability. Multiple microarray analyses have shown that NRMT is overexpressed in human colon cancer, and our preliminary data indicate the more aggressive the cancer, the higher the NRMT expression. However, there are currently no commercially available inhibitors specific for this enzyme and very little is known about the downstream signaling consequences of NRMT misregulation. We have used in silico screening of the ZINC small molecule library to identify potential NRMT-specific inhibitors. Screening of the top 100 candidates produced 10 compounds that could inhibit NRMT methyltransferase activity in vitro. The top compound of these 10 can also inhibit NRMT methyltransferase activity in cell culture and specifically stops the growth of colon cancer cell lines that have high NRMT expression. We are currently working to optimize this small molecule inhibitor for stability and solubility, understand its downstream signaling consequences, and test its efficacy in a murine model system. We are especially interested in the potential of NRMT inhibitors as a treatment for patients with mutant kRAS. These patients are often insensitive to anti-EGFR therapy, but oncogenic RAS seems to require functional RB. Therefore, impairment of NRMT activity could also impair oncogenic RAS signaling through destabilization of RB. Data from this study will enhance our understanding of how colon cancer develops and could lead to novel treatments for the disease. Citation Format: John G. Tooley, John O. Trent, Christine E. Schaner Tooley. Investigating the role of N-terminal protein methylation in colon cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2013-5386