Meso scale discovery-based assays for the detection of aggregated huntingtin.

Huntington's disease (HD) is a monogenic neurodegenerative disorder caused by an expansion of the CAG trinucleotide repeat domain in the huntingtin (HTT) gene, leading to an expanded poly-glutamine (polyQ) stretch in the HTT protein. This mutant HTT (mHTT) protein is highly prone to intracellular aggregation, causing significant damage and cellular loss in the striatal, cortical, and other regions of the brain. Therefore, modulation of mHTT levels in these brain regions in order to reduce intracellular mHTT and aggregate levels represents a direct approach in the development of HD therapeutics. To this end, assays that can be used to detect changes in HTT levels in biological samples are invaluable tools to assess target engagement and guide dose selection in clinical trials. The Meso Scale Discovery (MSD) ELISA-based assay platform is a robust and sensitive method previously employed for the quantification of HTT. However, the currently available MSD assays for HTT are primarily detecting the monomeric soluble form of the protein, but not aggregated species. In this study, we describe the development of novel MSD assays preferentially detecting mHTT in an aggregated form. Recombinant monomeric HTT(1-97)-Q46, which forms aggregates in a time-dependent manner, was used to characterize the ability of each established assay to distinguish between HTT monomers and HTT in a higher assembly state. Further validation of these assays was performed using brain lysates from R6/2, zQ175 knock-in, and BACHD mouse models, to replicate a previously well-characterized age-dependent increase in brain aggregate signals, as well as a significant reduction of aggregate levels in the striatum following mHTT knockdown with a CAG-directed allele-specific zinc-finger repressor protein (ZFP). Lastly, size exclusion chromatography was used to separate and characterize HTT species from brain tissue lysates to demonstrate specificity of the assays for the fractions containing aggregated HTT. In summary, we demonstrate that the newly developed assays preferentially detect aggregated HTT with improved performance in comparison to previous assay technologies. These assays complement the existing MSD platform assays specific for soluble HTT monomers, allowing for a more comprehensive analysis of disease-relevant HTT species in preclinical models of HD.

Cell cycle regulation by the PRMT6 arginine methyltransferase through repression of cyclin-dependent kinase inhibitors.

PRMT6 belongs to the family of Protein Arginine Methyltransferase (PRMT) enzymes that catalyze the methylation of guanidino nitrogens of arginine residues. PRMT6 has been shown to modify the tail of histone H3, but the in vivo function of PRMT6 is largely unknown. Here, we show that PRMT6 regulates cell cycle progression. Knockdown of PRMT6 expression in the human osteosarcoma cell line U2OS results in an accumulation of cells at the G2 checkpoint. Loss of PRMT6 coincides with upregulation of p21 and p27, two members of the CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors. Gene expression and promoter analysis show that p21 and p27 are direct targets of PRMT6, which involves methylation of arginine-2 of histone H3. Our findings imply arginine methylation of histones by PRMT6 in cell cycle regulation.

MMP13 mediates cell cycle progression in melanocytes and melanoma cells: in vitro studies of migration and proliferation.

BACKGROUND: Melanoma cells are usually characterized by a strong proliferative potential and efficient invasive migration. Among the multiple molecular changes that are recorded during progression of this disease, aberrant activation of receptor tyrosine kinases (RTK) is often observed. Activation of matrix metalloproteases goes along with RTK activation and usually enhances RTK-driven migration. The purpose of this study was to examine RTK-driven three-dimensional migration of melanocytes and the pro-tumorigenic role of matrix metalloproteases for melanocytes and melanoma cells. RESULTS: Using experimental melanocyte dedifferentiation as a model for early melanomagenesis we show that an activated EGF receptor variant potentiates migration through three-dimensional fibrillar collagen. EGFR stimulation also resulted in a strong induction of matrix metalloproteases in a MAPK-dependent manner. However, neither MAPK nor MMP activity were required for migration, as the cells migrated in an entirely amoeboid mode. Instead, MMPs fulfilled a function in cell cycle regulation, as their inhibition resulted in strong growth inhibition of melanocytes. The same effect was observed in the human melanoma cell line A375 after stimulation with FCS. Using sh- and siRNA techniques, we could show that MMP13 is the protease responsible for this effect. Along with decreased proliferation, knockdown of MMP13 strongly enhanced pigmentation of melanocytes. CONCLUSIONS: Our data show for the first time that growth stimuli are mediated via MMP13 in melanocytes and melanoma, suggesting an autocrine MMP13-driven loop. Given that MMP13-specific inhibitors are already developed, these results support the evaluation of these inhibitors in the treatment of melanoma.

lin9 is required for mitosis and cell survival during early zebrafish development.

LIN9 has been described as a regulator of G(1)/S and G(2)/M progression of the cell cycle in invertebrates and human cell lines. To elucidate the in vivo function of LIN9 during vertebrate development, we took advantage of the teleost zebrafish (Danio rerio). By means of antisense morpholinos we show here that Lin9-depleted embryonic cells accumulate in mitosis. Flow cytometry and confocal microscopy data demonstrate that the delay in mitotic progression is followed by apoptosis, which strongly manifests in the developing central nervous system. In accordance with these findings, we identified a cohort of Lin9-regulated genes required for different mitotic processes, including mitotic entry, metaphase/anaphase transition, and cytokinesis. Our data establish LIN9 as an essential regulator of mitosis in vertebrate development.

The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation.

Protein arginine methyltransferases (PRMT) have been implicated in the regulation of transcription. They are recruited to promoters via interaction with transcription factors and exert their coactivator function by methylating arginine residues in histones and other chromatin proteins. Here, we employ an unbiased approach to identify novel target genes, which are under the control of two members of the enzyme family, PRMT1 and CARM1/PRMT4 (coactivator associated arginine methyltransferase 1). By using cDNA microarray analysis, we find that the siRNA-mediated single knockdown of neither CARM1 nor PRMT1 causes significant changes in gene expression. In contrast, double knockdown of both enzymes results in the deregulated expression of a large group of genes, among them the CITED2 gene. Cytokine-stimulated expression analysis indicates that transcriptional activation of CITED2 depends on STAT5 and the coactivation of both PRMTs. ChIP analysis identifies the CITED2 gene as a direct target gene of STAT5, CARM1 and PRMT1. In reporter gene assays, we show that STAT5-mediated transcription is cooperatively enhanced by CARM1 and PRMT1. Interaction assays reveal a cytokine-induced association of STAT5 and the two PRMTs. Our data demonstrate a widespread cooperation of CARM1 and PRMT1 in gene activation as well as repression and that STAT5-dependent transcription of the CITED2 gene is a novel pathway coactivated by the two methyltransferases.

SET-mediated promoter hypoacetylation is a prerequisite for coactivation of the estrogen-responsive pS2 gene by PRMT1.

Induction of transcription requires an ordered recruitment of coregulators and specific combinations of histone modifications at the promoter. Occurrence of histone H4 arginine (Arg) 3 methylation by protein arginine methyltransferase 1 (PRMT1) represents an early promoter event in ER (estrogen receptor)-regulated gene activation. However, its in vivo significance in ER signaling and the prerequisites for PRMT1 recruitment to promoters have not been established yet. We show here that endogenous PRMT1 is a crucial and non-redundant coactivator of ER-mediated pS2 gene induction in MCF7 cells. By investigating promoter requirements for PRMT1 recruitment we find that the patient SE translocation (SET) protein, which was reported to protect histone tails from acetylation, associates with the uninduced pS2 gene promoter and dissociates early upon estrogen treatment. Knockdown of SET or trichostatin A (TSA) treatment causes premature acetylation of H4 and abrogation of H4 Arg3 methylation at the pS2 gene promoter resulting in diminished transcriptional induction. Thus, SET prevents promoter acetylation and is a prerequisite for the initial acetylation-sensitive steps of pS2 gene activation, namely PRMT1 function. Similar to pS2 we identify lactoferrin as a PRMT1-dependent and TSA-sensitive ER target gene. In contrast, we find that the C3 gene, another ER target, is activated in a PRMT1-independent manner and that SET is involved in C3 gene repression. These findings establish the existence of PRMT1-dependent and -independent ER target genes and show that proteins guarding promoter hypoacetylation, like SET, execute a key function in the coactivation process by PRMT1.