Investigation of RNAi-dependent heterochromatin establishment in Schizosaccharomyces pombe

In S. pombe, heterochromatin assembly is RNAi-dependent. Short-interfering RNAs (siRNAs) are loaded into the RNA-induced transcriptional silencing (RITS) complex and guide RITS to silence nascent RNA sequences. RITS also recruits the Clr4 methyltransferase complex (CLRC) complex to initiate H3K9 methylation at chromatin. Centromeric heterochromatin assembly in S. pombe is separated into two distinct steps: establishment and maintenance. Currently we know fairly well how heterochromatin is maintained, but it is not well understood about how nascent siRNAs and H3K9me are generated and whether there are additional factors for de novo heterochromatin assembly. In this project, I aim to identify new potential establishment factors and investigate the underlying mechanisms of heterochromatin establishment. I constructed a cross-based establishment assay by deleting CLRC or RNAi components and reintroducing them by genetic cross. I found that wild-type progeny of a rik1Δ × ago1Δ cross showed inefficient heterochromatin establishment as assessed via silencing of an ade6⁺reporter inserted at centromeric outer repeat, reflected by the proportion of red (silent) versus white (expressing) colonies. Deletion of genes encoding factors known to be required for heterochromatin establishment, tri1⁺ or mkt1⁺, reduced establishment efficiency, suggesting the rik1Δ × ago1Δ assay is applicable as a screen for potential establishment factors. In parallel, I found that the efficiency of heterochromatin establishment can be improved through decreasing histone H3 acetylation by deleting the histone acetyltransferase gcn5⁺, or overexpressing histone deacetylase Sir2 and/or Clr3, as well as through increasing ubiquitination levels by deletion of ubp14⁺,, a putative deubiquitinase. By using the rik1Δ × ago1Δ assay, I identified two potential candidates, SPAC18G6.13 and cpn1⁺, both of which appeared to be required for heterochromatin establishment. For SPAC18G6.13, I conducted limited analysis: deletion of SPAC18G6.13 showed silencing defects at centromeres and subtelomeres, but no defects at heterochromatin islands. However, deletion of cpn1⁺ had a specific defect on heterochromatin establishment, independent of maintenance. Therefore, I concentrated more on the role of Cpn1, the ortholog of Human CAPRIN1, in heterochromatin assembly. The effects of cpn1⁺ on heterochromatin establishment were confirmed through Clr4 reintroduction and minichromosome assay. Moreover, the arginine-glycine-glycine (RGG)/RG motif of Cpn1 is important for its function in heterochromatin establishment. In addition, deletion of cpn1⁺ showed some similarities with mkt1Δ: both genes are not required for RNA-independent heterochromatin assembly, but are required for the maintenance at specific HOODs. By using IP-mass spectrometry, two proteins were identified to have interaction with Cpn1: Nxt3 and Ubp3, which have been reported to localize to stress granules. Subsequently, Cpn1 was also observed to localize to stress granules under various stress conditions. Additionally, Cpn1, as well as its RGG/RG motif, is required for stress granule formation under heat shock. In general, there are several findings that support a link between heterochromatin assembly and stress granule formation: stress granule formation was promoted when heterochromatin was perturbed under native conditions; the RNAi components, Ago1 and Dcr1 were observed to localize to stress granules; stress granule formation was suppressed when heterochromatin integrity was disrupted in response to thermal stress. Moreover, the pericentromeric transcripts were found to be localized to stress granules under glucose starvation. More pericentromeric transcripts were found to accumulate to centromeres in the absence of Cpn1 when heterochromatin integrity was disrupted in clr4Δ. Therefore, these finding supports a model where Cpn1 promotes heterochromatin assembly by preventing the over-accumulation of pericentromeric transcripts to centromeres, which are degraded in the stress granules formed when transcription at centromeres is high (either in an establishment scenario, or when heterochromatin integrity is disrupted)

Initiation of scutellum-derived callus is regulated by an embryo-like developmental pathway in rice

Abstract In rice (Oryza sativa) tissue culture, callus can be induced from the scutellum in embryo or from the vasculature of non-embryonic organs such as leaves, nodes, or roots. Here we show that the auxin signaling pathway triggers cell division in the epidermis of the scutellum to form an embryo-like structure, which leads to callus formation. Our transcriptome data show that embryo-, stem cell-, and auxin-related genes are upregulated during scutellum-derived callus initiation. Among those genes, the embryo-specific gene OsLEC1 is activated by auxin and involved in scutellum-derived callus initiation. However, OsLEC1 is not required for vasculature-derived callus initiation from roots. In addition, OsIAA11 and OsCRL1, which are involved in root development, are required for vasculature-derived callus formation but not for scutellum-derived callus formation. Overall, our data indicate that scutellum-derived callus initiation is regulated by an embryo-like development program, and this is different from vasculature-derived callus initiation which borrows a root development program

Investigation of Controllable Nanoscale Heat-Denatured Bovine Serum Albumin Films on Graphene

Two-dimensional graphene devices are widely used for biomolecule detection. Nevertheless, the surface modification of graphene is critical to achieve the high sensitivity and specificity required for biological detection. Herein, native bovine serum albumin (BSA) in inorganic solution is denatured on the graphene surface by heating, leading to the formation of nanoscale BSA protein films adsorbed on the graphene substrate via π-stacking interactions. This technique yields a controllable, scalable, uniform, and high-coverage method for graphene biosensors. Further, the application of such nanoscale heat-denatured BSA films on graphene as a universal graphene biosensor platform is explored. The thickness of heat-denatured BSA films increased with heating time and BSA concentration but decreased with solvent concentration as confirmed by atomic force microscopy. The noncovalent interaction between denatured BSA films and graphene was investigated by Raman spectroscopy. BSA can act as a p-type and n-type dopant by modulating pH-dependent net charges on the layered BSA–graphene surface, as assessed by current–voltage measurements. Chemical groups of denatured BSA films, including amino and carboxyl groups, were verified by X-ray photoelectron microscopy, attenuated total reflectance-Fourier transform infrared spectra, and fluorescent labeling. The tailoring of the BSA–graphene surfaces through chemical modification, controlled thickness, and doping type via noncovalent interactions provides a controllable, multifunctional biosensor platform for molecular diagnosis without the possibility of nonspecific adsorption on graphene