Structural mechanism of HP1α-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1α and nucleosomes. We have elucidated the cryo-EM structure of an HP1α dimer bound to an H2A.Z nucleosome, revealing that the HP1α dimer interfaces with nucleosomes at two distinct sites. The primary binding site is located at the N-terminus of histone H3, specifically at the trimethylated K9 (K9me3) region, while a novel secondary binding site is situated near histone H2B, close to nucleosome superhelical location 4 (SHL4). Our biochemical data further demonstrates that HP1α binding influences the dynamics of DNA on the nucleosome. It promotes DNA unwrapping near the nucleosome entry and exit sites while concurrently restricting DNA accessibility in the vicinity of SHL4. This study offers a model that explains how HP1α functions in heterochromatin maintenance and gene silencing, particularly in the context of H3K9me-dependent mechanisms. Additionally, it sheds light on the H3K9me-independent role of HP1 in responding to DNA damage.

Histone variants and chromatin structure, update of advances.

Histone proteins are highly conserved among all eukaryotes. They have two important functions in the cell: to package the genomic DNA and to regulate gene accessibility. Fundamental to these functions is the ability of histone proteins to interact with DNA and to form the nucleoprotein complex called chromatin. One of the mechanisms the cells use to regulate chromatin and gene expression is through replacing canonical histones with their variants at specific loci to achieve functional consequence. Recent cryo-electron microscope (cryo-EM) studies of chromatin containing histone variants reveal new details that shed light on how variant-specific features influence the structures and functions of chromatin. In this article, we review the current state of knowledge on histone variants biochemistry and discuss the implication of these new structural information on histone variant biology and their functions in transcription.

The Arabidopsis transcription factor NAI1 activates the NAI2 promoter by binding to the G-box motifs.

Brassicaceae plants, including Arabidopsis thaliana, develop endoplasmic reticulum (ER)-derived structures called ER bodies, which are involved in chemical defense against herbivores. NAI1 is a basic helix-loop-helix (bHLH) type transcription factor that regulates two downstream genes, NAI2 and BGLU23, that are responsible for the ER body formation and function. Here, we examined the transcription factor function of NAI1, and found that NAI1 binds to the promoter region of NAI2 and activates the NAI2 promoter. The recombinant NAI1 protein recognizes the canonical and non-canonical G-box motifs in the NAI2 promoter. Furthermore, we examined the DNA binding activity of NAI1 toward several E-box motifs in the NAI2 and BGLU23 promoters and found that NAI1 binds to a DNA fragment that includes an E-box motif from the BGLU23 promoter. Subcellular localization of NAI1 was evident in the nucleus, which is consistent with its transcription factor function. Transient expression experiments in Nicotiana benthamiana leaves showed that GFP-NAI1 protein activated the NAI2 promoter by binding to the two G-boxes of the promoter. Disruption of the G-boxes abolished the NAI1-dependent activation of the NAI2 promoter. These results indicate that NAI1 has a DNA binding activity in a motif-dependent manner and suggest that NAI1 regulates NAI2 and BGLU23 gene expressions through binding to these DNA motifs in their promoters.

Engineering a Decoy Substrate in Soybean to Enable Recognition of the Soybean Mosaic Virus NIa Protease.

In Arabidopsis, recognition of the AvrPphB effector protease from Pseudomonas syringae is mediated by the disease resistance (R) protein RPS5, which is activated by AvrPphB-induced cleavage of the Arabidopsis protein kinase PBS1. The recognition specificity of RPS5 can be altered by substituting the AvrPphB cleavage site within PBS1 with cleavage sequences for other proteases, including proteases from viruses. AvrPphB also activates defense responses in soybean (Glycine max), suggesting that soybean may contain an R protein analogous to RPS5. It was unknown, however, whether this response is mediated by cleavage of a soybean PBS1-like protein. Here, we show that soybean contains three PBS1 orthologs and that their products are cleaved by AvrPphB. Further, transient expression of soybean PBS1 derivatives containing a five-alanine insertion at their AvrPphB cleavage sites activated cell death in soybean protoplasts, demonstrating that soybean likely contains an AvrPphB-specific resistance protein that is activated by a conformational change in soybean PBS1 proteins. Significantly, we show that a soybean PBS1 decoy protein modified to contain a cleavage site for the soybean mosaic virus (SMV) NIa protease triggers cell death in soybean protoplasts when cleaved by this protease, indicating that the PBS1 decoy approach will work in soybean, using endogenous PBS1 genes. Lastly, we show that activation of the AvrPphB-dependent cell death response effectively inhibits systemic spread of SMV in soybean. These data also indicate that decoy engineering may be feasible in other crop plant species that recognize AvrPphB protease activity.

Interaction of Arabidopsis TGA3 and WRKY53 transcription factors on Cestrum yellow leaf curling virus (CmYLCV) promoter mediates salicylic acid-dependent gene expression in planta.

MAIN CONCLUSION: This paper highlighted a salicylic acid-inducible Caulimoviral promoter fragment from Cestrum yellow leaf curling virus (CmYLCV). Interaction of Arabidopsis transcription factors TGA3 and WRKY53 on CmYLCV promoter resulted in the enhancement of the promoter activity via NPR1-dependent salicylic acid signaling. Several transcriptional promoters isolated from plant-infecting Caulimoviruses are being presently used worldwide as efficient tools for plant gene expression. The CmYLCV promoter has been isolated from the Cestrum yellow leaf curling virus (Caulimoviruses) and characterized more than 12 years ago; also we have earlier reported a near-constitutive, pathogen-inducible CmYLCV promoter fragment (-329 to +137 from transcription start site; TSS) that enhances stronger (3×) expression than the previously reported fragments; all these fragments are highly efficient in monocot and dicot plants (Sahoo et al. Planta 240: 855-875, 2014). Here, we have shown that the full-length CmYLCV promoter fragment (-729 to +137 from TSS) is salicylic acid (SA) inducible. In this context, we have performed an in-depth study to elucidate the factors responsible for SA-inducibility of the CmYLCV promoter. We found that the as-1 1 and W-box1 elements (located at -649 and -640 from the TSS) of the CmYLCV promoter are required for SA-induced activation by recruiting Arabidopsis TGA3 and WRKY53 transcription factors. Consequently, as a nascent observation, we established the physical interaction between TGA3 and WYKY53; also demonstrated that the N-terminal domain of TGA3 is sufficient for the interaction with the full-length WRKY53. Such interaction synergistically activates the CmYLCV promoter activity in planta. Further, we found that activation of the CmYLCV promoter by SA through TGA3 and WRKY53 interaction depends on NPR1. Finally, the findings presented here provide strong support for the direct regulatory roles of TGA3 and WRKY53 in the SA and NPR1-dependent activation of a Caulimoviral promoter (CmYLCV).

Novel Synthetic Promoters from the Cestrum Yellow Leaf Curling Virus.

Constitutive promoters direct gene expression uniformly in most tissues and cells at all stages of plant growth and development; they confer steady levels of transgene expression in plant cells and hence their demand is high in plant biology. The gene silencing due to promoter homology can be avoided by either using diverse promoters isolated from different plant and viral genomes or by designing synthetic promoters. The aim of this chapter was to describe the basic protocols needed to develop and analyze novel, synthetic, nearly constitutive promoters from Cestrum yellow leaf curling virus (CmYLCV) through promoter/leader deletion and activating cis-sequence analysis. We also describe the methods to evaluate the strength of the promoters efficiently in various transient expression systems like agroinfiltration assay, gene-gun method, and assay in tobacco protoplasts. Besides, the detailed methods for developing transgenic plants (tobacco and Arabidopsis) for evaluation of the promoter using the GUS reporter gene are also described. The detailed procedure for electrophoretic mobility shift assay (EMSA) coupled with super-shift EMSA analysis are also described for showing the binding of tobacco transcription factor, TGA1a to cis-elements in the CmYLCV distal promoter region.

Overexpression of human peroxisomal enoyl-CoA delta isomerase2 HsPECI2, an ortholog of bamboo expressed during gregarious flowering alters salinity stress responses and polar lipid content in tobacco.

Peroxisomal enoyl-CoA delta isomerase2 (PECI2) is one of the key enzymes that has critical role in lipid metabolism and plant development during salt stress. Seven out of ten tobacco plants overexpressing human PECI2 (HsPECI2) with PTS1-sequence showed hypersensitivity to salt. Under salt-stress, T2 transformed plants (HsPECI2) displayed reduced primary root, delayed shoot-growth, and visibly smaller rosette leaves turning pale yellow as compared to the pKYLX71 vector control plant. Also, we found altered reactive oxygen species (ROS) levels and reduced catalase activity in 100mM sodium chloride (NaCl) treated HsPECI2 transformed plant compared with the pKYLX71 counterpart. ESI-MS/MS data showed that the polar lipids were differentially modulated upon salt treatment in HsPECI2 transformed and pKYLX71 plants as compared with the respective untreated counterpart. Notably, the levels of monogalactosyldiacylglycerol and phosphatidic acid varied significantly, whereas phosphatidylcholine, phosphatidylserine and digalactosyldiacylglycerol contents were moderately upregulated. In parallel, abscisic acid (ABA) responsiveness assay confirmed insensitivity of HsPECI2 transformed plant towards ABA. Overall our data proclaim that HsPECI2 play multifunctional role in normal development and response to salinity stress apart from its primary role in ß-oxidation.

Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells.

The gene Par-4 (Prostate Apoptosis Response 4) was originally identified in prostate cancer cells undergoing apoptosis and its product Par-4 showed cancer specific pro-apoptotic activity. Particularly, the SAC domain of Par-4 (SAC-Par-4) selectively kills cancer cells leaving normal cells unaffected. The therapeutic significance of bioactive SAC-Par-4 is enormous in cancer biology; however, its large scale production is still a matter of concern. Here we report the production of SAC-Par-4-GFP fusion protein coupled to translational enhancer sequence (5' AMV) and apoplast signal peptide (aTP) in transgenic Nicotiana tabacum cv. Samsun NN plants under the control of a unique recombinant promoter M24. Transgene integration was confirmed by genomic DNA PCR, Southern and Northern blotting, Real-time PCR, and Nuclear run-on assays. Results of Western blot analysis and ELISA confirmed expression of recombinant SAC-Par-4-GFP protein and it was as high as 0.15% of total soluble protein. In addition, we found that targeting of plant recombinant SAC-Par-4-GFP to the apoplast and endoplasmic reticulum (ER) was essential for the stability of plant recombinant protein in comparison to the bacterial derived SAC-Par-4. Deglycosylation analysis demonstrated that ER-targeted SAC-Par-4-GFP-SEKDEL undergoes O-linked glycosylation unlike apoplast-targeted SAC-Par-4-GFP. Furthermore, various in vitro studies like mammalian cells proliferation assay (MTT), apoptosis induction assays, and NF-κB suppression suggested the cytotoxic and apoptotic properties of plant-derived SAC-Par-4-GFP against multiple prostate cancer cell lines. Additionally, pre-treatment of MAT-LyLu prostate cancer cells with purified SAC-Par-4-GFP significantly delayed the onset of tumor in a syngeneic rat prostate cancer model. Taken altogether, we proclaim that plant made SAC-Par-4 may become a useful alternate therapy for effectively alleviating cancer in the new era.

Synthetic promoters in planta.

MAIN CONCLUSION: This paper reviews the importance, prospective and development of synthetic promoters reported in planta. A review of the synthetic promoters developed in planta would help researchers utilize the available resources and design new promoters to benefit fundamental research and agricultural applications. The demand for promoters for the improvement and application of transgenic techniques in research and agricultural production is increasing. Native/naturally occurring promoters have some limitations in terms of their induction conditions, transcription efficiency and size. The strength and specificity of native promoter can be tailored by manipulating its 'cis-architecture' by the use of several recombinant DNA technologies. Newly derived chimeric promoters with specific attributes are emerging as an efficient tool for plant molecular biology. In the last three decades, synthetic promoters have been used to regulate plant gene expression. To better understand synthetic promoters, in this article, we reviewed promoter structure, the scope of cis-engineering, strategies for their development, their importance in plant biology and the total number of such promoters (188) developed in planta to date; we then categorized them under different functional regimes as biotic stress-inducible, abiotic stress-inducible, light-responsive, chemical-inducible, hormone-inducible, constitutive and tissue-specific. Furthermore, we identified a set of 36 synthetic promoters that control multiple types of expression in planta. Additionally, we illustrated the differences between native and synthetic promoters and among different synthetic promoter in each group, especially in terms of efficiency and induction conditions. As a prospective of this review, the use of ideal synthetic promoters is one of the prime requirements for generating transgenic plants suitable for promoting sustainable agriculture and plant molecular farming.

Comparative analysis of synthetic DNA promoters for high-level gene expression in plants.

MAIN CONCLUSION: We have designed two near- constitutive and stress-inducible promoters (CmYLCV9.11 and CmYLCV4); those are highly efficient in both dicot and monocot plants and have prospective to substitute the CaMV 35S promoter. We performed structural and functional studies of the full-length transcript promoter from Cestrum yellow leaf curling virus (CmYLCV) employing promoter/leader deletion and activating cis-sequence analysis. We designed a 465-bp long CmYLCV9.11 promoter fragment (-329 to +137 from transcription start site) that showed enhanced promoter activity and was highly responsive to both biotic and abiotic stresses. The CmYLCV9.11 promoter was about 28-fold stronger than the CaMV35S promoter in transient and stable transgenic assays using ß-glucuronidase (GUS) reporter gene. The CmYLCV9.11 promoter also demonstrated stronger activity than the previously reported CmYLCV promoter fragments, CmpC (-341 to +5) and CmpS (-349 to +59) in transient systems like maize protoplasts and onion epidermal cells as well as transgenic systems. A good correlation between CmYLCV9.11 promoter-driven GUS-accumulation/enzymatic activities with corresponding uidA-mRNA level in transgenic tobacco plants was shown. Histochemical (X-Gluc) staining of transgenic seedlings, root and floral parts expressing the GUS under the control of CmYLCV9.11, CaMV35S, CmpC and CmpS promoters also support the above findings. The CmYLCV9.11 promoter is a constitutive promoter and the expression level in tissues of transgenic tobacco plants was in the following order: root > leaf > stem. The tobacco transcription factor TGA1a was found to bind strongly to the CmYLCV9.11 promoter region, as shown by Gel-shift assay and South-Western blot analysis. In addition, the CmYLCV9.11 promoter was regulated by a number of abiotic and biotic stresses as studied in transgenic Arabidopsis and tobacco plants. The newly derived CmYLCV9.11 promoter is an efficient tool for biotechnological applications.