Methylation at arginine 17 of histone H3 is linked to gene activation

The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1-mediated R17 methylation on histone H3 takes place in vivo during this active state.     

O-GlcNAc Transferase Regulates Mitotic Chromatin Dynamics

Mitosis must faithfully divide the genome such that each progeny inherits the same genetic material. DNA condensation is crucial in ensuring that chromosomes are correctly attached to the mitotic spindle for segregation, preventing DNA breaks or constrictions from the contractile ring. Histones form an octameric complex of basic proteins important in regulating DNA organization and accessibility. Histone post-translational modifications are altered during mitosis, although the roles of these post-translational modifications remain poorly characterized. Here, we report that N-acetylglucosamine (O-GlcNAc) transferase (OGT), the enzyme catalyzing the addition of O-GlcNAc moieties to nuclear and cytoplasmic proteins at serine and threonine residues, regulates some aspects of mitotic chromatin dynamics. OGT protein amounts decrease during M phase. Modest overexpression of OGT alters mitotic histone post-translational modifications at Lys-9, Ser-10, Arg-17, and Lys-27 of histone H3. Overexpression of OGT also prevents mitotic phosphorylation of coactivator-associated arginine methyltransferase 1 (CARM1) and prevents its correct cellular localization during mitosis. Moreover, OGT overexpression results in an increase in abnormal chromosomal bridge formation. Together, these results show that regulating the amount of OGT during mitosis is important in ensuring correct chromosomal segregation during mitosis.     

The linker histone in Saccharomyces cerevisiae interacts with actin-related protein 4 and both regulate chromatin structure and cellular morphology

Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear proteins that influence it. The intricate interaction among all these structural and functional nuclear proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (actin-related protein 4) which is a key subunit of three chromatin modifying complexes – INO80, SWR1 and NuA4. A single – point mutation in the actin – fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization.     

Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology

Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in Escherichia coli forms insoluble inclusion bodies that are difficult to denature and refold. The presented results demonstrate the use of a novel HaloTag? technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10-50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1's methyltransferase activity.     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.