Nuclear actin structure regulates chromatin accessibility.

Polymerized ß-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR.

Pathophysiology associated with Huntington's disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles.

Plant-based expression system has many potential advantages to produce biopharmaceuticals, but plants cannot be directly used to express human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit plant-based expression system for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco in order to generate capacities for ß1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human ß1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) was co-expressed to produce rhuEPO bearing ß1,4-galactose-extended N-glycan chains as well as compare their ß1,4-galactosylation efficiencies. Five mammalian genes encoding enzymes/transporter for sialic acid biosynthesis, transport and transfer were co-expressed to build sialylation capacity in plants. The human MGAT3 was co-expressed to produce N-glycan chains with bisecting GlcNAc. Our results demonstrated that the above transgenes were incorporated into tobacco genome and transcribed. ST/GalT was found to be more efficient than GalT for ß1,4-galactosylation. Furthermore, co-expressing MGAT3 generated N-glycans likely bearing bisected GlcNAc. However, our current efforts did not result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of ß1,4-galactose-extended N-glycan chains for tissue protective purposes.