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Summary Rice (Oryza sativa L.) is one of the most important food crops. Starch is the main substance of rice endosperm and largely determines the grain quality and yield. Starch biosynthesis in endosperm is very complex, requiring a series of enzymes which are also regulated by many transcription factors (TFs). But until now, the large‐scale regulatory network for rice endosperm starch biosynthesis has not been established. Here, we constructed a rice endosperm starch biosynthesis regulatory network comprised of 277 TFs and 15 starch synthesis enzyme‐encoding genes (SSEGs) using DNA affinity chromatography/pull‐down combined with liquid chromatography‐mass spectrometry (DNA pull‐down and LC–MS). In this regulatory network, each SSEG is directly regulated by 7–46 TFs. Based on this network, we found a new pathway ‘ABA‐OsABI5‐OsERF44‐SSEGs’ that regulates rice endosperm starch biosynthesis. We also knocked out five TFs targeting the key amylose synthesis enzyme gene OsGBSSI in japonica rice ‘Nipponbare’ background and found that all mutants had moderately decreased amylose content (AC) in endosperm and improved eating and cooking quality (ECQ). Notably, the knockout of OsSPL7 and OsB3 improves the ECQ without compromising the rice appearance quality, which was further validated in the indica rice ‘Zhongjiazao17’ background. In summary, this gene regulatory network for rice endosperm starch biosynthesis established here will provide important theoretical and practical guidance for the genetic improvement of rice quality.

Investigation of RNA- and DNA-binding proteins to a defined regulatory sequence, such as an AU-rich RNA and a DNA enhancer element, is important for understanding gene regulation through their interactions. For in vitro binding studies, an electrophoretic mobility shift assay (EMSA) was widely used in the past. In line with the trend toward using non-radioactive materials in various bioassays, end-labeled biotinylated RNA and DNA oligonucleotides can be more practical probes to study protein–RNA and protein–DNA interactions; thereby, the binding complexes can be pulled down with streptavidin-conjugated resins and identified by Western blotting. However, setting up RNA and DNA pull-down assays with biotinylated probes in optimum protein binding conditions remains challenging. Here, we demonstrate the step-by step optimization of pull-down for IRP (iron-responsive-element-binding protein) with a 5′-biotinylated stem-loop IRE (iron-responsive element) RNA, HuR, and AUF1 with an AU-rich RNA element and Nrf2 binding to an antioxidant-responsive element (ARE) enhancer in the human ferritin H gene. This study was designed to address key technical questions in RNA and DNA pull-down assays: (1) how much RNA and DNA probes we should use; (2) what binding buffer and cell lysis buffer we can use; (3) how to verify the specific interaction; (4) what streptavidin resin (agarose or magnetic beads) works; and (5) what Western blotting results we can expect from varying to optimum conditions. We anticipate that our optimized pull-down conditions can be applicable to other RNA- and DNA-binding proteins along with emerging non-coding small RNA-binding proteins for their in vitro characterization.

High‐molecular‐weight glutenin subunits (HMW‐GS), a major component of seed storage proteins (SSP) in wheat, largely determine processing quality. HMW‐GS encoded by GLU‐1 loci are mainly controlled at the transcriptional level by interactions between cis ‐elements and transcription factors (TFs). We previously identified a conserved cis ‐regulatory module CCRM1‐1 as the most essential cis ‐element for Glu‐1 endosperm‐specific high expression. However, the TFs targeting CCRM1‐1 remained unknown. Here, we built the first DNA pull‐down plus liquid chromatography‐mass spectrometry platform in wheat and identified 31 TFs interacting with CCRM1‐1. TaB3‐2A1 as proof of concept was confirmed to bind to CCRM1‐1 by yeast one hybrid and electrophoretic mobility shift assays. Transactivation experiments demonstrated that TaB3‐2A1 repressed CCRM1‐1‐driven transcription activity. TaB3‐2A1 overexpression significantly reduced HMW‐GS and other SSP, but enhanced starch content. Transcriptome analyses confirmed that enhanced expression of TaB3‐2A1 down‐regulated SSP genes and up‐regulated starch synthesis‐related genes, such as TaAGPL3 , TaAGPS2 , TaGBSSI , TaSUS1 and TaSUS5 , suggesting that it is an integrator modulating the balance of carbon and nitrogen metabolism. TaB3‐2A1 also had significant effects on agronomic traits, including heading date, plant height and grain weight. We identified two major haplotypes of TaB3‐2A1 and found that TaB3‐2A1‐Hap1 conferred lower seed protein content, but higher starch content, plant height and grain weight than TaB3‐2A1‐Hap2 and was subjected to positive selection in a panel of elite wheat cultivars. These findings provide a high‐efficiency tool to detect TFs binding to targeted promoters, considerable gene resources for dissecting regulatory mechanisms underlying Glu‐1 expression, and a useful gene for wheat improvement.

High NaCl (200 mM) increases the transcription of phospholipase Dδ (PLDδ) in roots and leaves of the salt-resistant woody species Populus euphratica. We isolated a 1138 bp promoter fragment upstream of the translation initiation codon of PePLDδ. A promoter–reporter construct, PePLDδ-pro::GUS, was introduced into Arabidopsis plants (Arabidopsis thaliana) to demonstrate the NaCl-induced PePLDδ promoter activity in root and leaf tissues. Mass spectrometry analysis of DNA pull-down-enriched proteins in P. euphratica revealed that PeGLABRA3, a basic helix–loop–helix transcription factor, was the target transcription factor for binding the promoter region of PePLDδ. The PeGLABRA3 binding to PePLDδ-pro was further verified by virus-induced gene silencing, luciferase reporter assay (LRA), yeast one-hybrid assay, and electrophoretic mobility shift assay (EMSA). In addition, the PeGLABRA3 gene was cloned and overexpressed in Arabidopsis to determine the function of PeGLABRA3 in salt tolerance. PeGLABRA3-overexpressed Arabidopsis lines (OE1 and OE2) had a greater capacity to scavenge reactive oxygen species (ROS) and to extrude Na+ under salinity stress. Furthermore, the EMSA and LRA results confirmed that PeGLABRA3 interacted with the promoter of AtPLDδ in transgenic plants. The upregulated AtPLDδ in PeGLABRA3-transgenic lines resulted in an increase in phosphatidic acid species under no-salt and saline conditions. We conclude that PeGLABRA3 activated AtPLDδ transcription under salt stress by binding to the AtPLDδ promoter region, conferring Na+ and ROS homeostasis control via signaling pathways mediated by PLDδ and phosphatidic acid.

The assimilatory pathway of the nitrogen cycle in the haloarchaeon Haloferax mediterranei has been well described and characterized in previous studies. However, the regulatory mechanisms involved in the gene expression of this pathway remain unknown in haloarchaea. This work focuses on elucidating the regulation at the transcriptional level of the assimilative nasABC operon (HFX_2002 to HFX_2004) through different approaches. Characterization of its promoter region using β-galactosidase as a reporter gene and site-directed mutagenesis has allowed us to identify possible candidate binding regions for a transcriptional factor. The identification of a potential transcriptional regulator related to nitrogen metabolism has become a real challenge due to the lack of information on haloarchaea. The investigation of protein–DNA binding by streptavidin bead pull-down analysis combined with mass spectrometry resulted in the in vitro identification of a transcriptional regulator belonging to the Lrp/AsnC family, which binds to the nasABC operon promoter (p.nasABC). To our knowledge, this study is the first report to suggest the AsnC transcriptional regulator as a powerful candidate to play a regulatory role in nasABC gene expression in Hfx. mediterranei and, in general, in the assimilatory nitrogen pathway.

During development of complex tissues such as the mammalian central nervous system (CNS), many genes act in concert to create sophisticated gene regulatory networks (GRNs) to instruct cell fate specification of the many cell types of the CNS. In this work, we have dissected a GRN that regulates a binary cell fate decision in the developing murine retina between a rod photoreceptor and a bipolar interneuron. This network converges upon Blimp1, a key transcription factor in rod versus bipolar cell fate decision. A cis-regulatory module (CRM) of Blimp1, B108, was identified by promoter bashing that faithfully recapitulates endogenous Blimp1 expression. Knock-out of B108 by CRISPR/Cas9 by in vivo electroporation in P0 pups resulted in Blimp1 KO phenotype. Otx2 and RORβ, potent transcription factors in retinal development, are required to activate Blimp1 expression via B108. Otx2 is in return repressed by Blimp1 in a negative feedback loop. Vsx2, a key TF for bipolar cells, was found to suppress Blimp1 via previously uncharacterized binding motif on B108 to ensure the proper ratio of rods to bipolar cells. Finally, we have shown Vsx2 and Blimp1 mRNAs may be asymmetrically segregated in dividing progenitors in the post-natal retina that sets up asymmetric cell fate in the siblings. Collectively these findings led to the identification of the GRN that regulates the decision rod vs bipolar fate. The sibling that retains Vsx2 mRNA suppresses Blimp1, maintains high Otx2 to become a bipolar cell. Whereas the sibling that retains Blimp1 suppresses Otx2 and Vsx2 to obtain a rod fate.

Over the past few decades, dozens of in vitro methods have been developed to map, investigate and validate protein–protein interactions. However, most of these approaches are time‐consuming and labour‐intensive or require specialised equipment or substantial amounts of purified proteins. Here, we describe a fast and versatile research protocol that is suitable for the in vitro analysis of the physical interaction between proteins or for mapping the binding surfaces. The principle of this method is based on the immobilisation of the protein/domain of interest to a carrier followed by its incubation with a labelled putative binding partner, which is generated by a coupled in vitro transcription/translation reaction. Interacting proteins are removed from the carrier, fractionated and visualised by SDS/PAGE autoradiography (or western blotting). This simple and cheap method can be easily carried out in every wet lab. The GST‐IVTT pull‐down assay is suitable for the rapid validation and mapping of functional protein interactions in vitro. The 35S‐labelled prey protein or its pieces (putative interactors) are produced by a single‐step IVTT (in vitro transcription/translation) reaction from various templates. Preys are mixed with immobilised GST (control) or GST‐tagged proteins of interest, and the binding is simply analysed by SDS/PAGE autoradiography.

Heterochromatic domains are enriched with repressive histone marks, including histone H3 lysine 9 methylation, written by lysine methyltransferases (KMTs). The pre-replication complex protein, origin recognition complex-associated (ORCA/LRWD1), preferentially localizes to heterochromatic regions in post-replicated cells. Its role in heterochromatin organization remained elusive. ORCA recognizes methylated H3K9 marks and interacts with repressive KMTs, including G9a/GLP and Suv39H1 in a chromatin context-dependent manner. Single-molecule pull-down assays demonstrate that ORCA-ORC (Origin Recognition Complex) and multiple H3K9 KMTs exist in a single complex and that ORCA stabilizes H3K9 KMT complex. Cells lacking ORCA show alterations in chromatin architecture, with significantly reduced H3K9 di- and tri-methylation at specific chromatin sites. Changes in heterochromatin structure due to loss of ORCA affect replication timing, preferentially at the late-replicating regions. We demonstrate that ORCA acts as a scaffold for the establishment of H3K9 KMT complex and its association and activity at specific chromatin sites is crucial for the organization of heterochromatin structure. The genetic material inside cells is contained within molecules of DNA. In animals and other eukaryotes, the DNA is tightly wrapped around proteins called histones to form a compact structure known as chromatin. There are two forms of chromatin: loosely packed chromatin tends to contain genes that are highly active in cells, while tightly packed chromatin—called heterochromatin—tends to contain less-active genes. How tightly DNA is packed in chromatin can be changed by adding small molecules known as methyl tags to individual histone proteins. Enzymes called KMTs are responsible for attaching these methyl tags to a specific site on the histones. Before a cell divides, it duplicates its DNA and these methyl tags, so that they can be passed onto the newly formed cells. This enables the new cells to ‘remember’ which genes were inactive or active in the original cell. A protein known as ORCA associates with heterochromatin, but it is not clear what role it plays in controlling the structure of chromatin. Giri et al. studied ORCA and the KMTs in human cells. The experiments show that ORCA recognizes the methyl tags and binds to the KMTs in regions of heterochromatin, but not in regions where the DNA is more loosely packed. Next, Giri et al. used a technique called single-molecule pull-down, which is able to identify individual proteins within a group. These experiments showed that several KMT enzymes can bind to a single ORCA protein at the same time. ORCA stabilizes the binding of KMTs to chromatin, which enables the KMTs to modify the histones within it. Cells lacking ORCA had fewer methyl tags on their histones, which altered the structure of the chromatin. This also affected the timing with which DNA copying takes place in cells before the cell divides. Giri et al.'s findings suggest that ORCA acts as a scaffold for the KMTs and that its activity at specific sites on chromatin is important for the organization of heterochromatin. The next step is to identify the exact regions in the genome where the timing of DNA copying is regulated by ORCA.

Amsacta moorei entomopoxvirus (AMEV) is a poxvirus that can only infect insects. This virus is an attractive research material because it is similar to smallpox virus. AMEV is one of many viruses that encode protein kinases that drive the host's cellular mechanisms, modifying immune responses to it, and regulating viral protein activity. We report here the functional characterization of a serine/threonine (Ser/Thr) protein kinase (PK) gene (ORF AMV197) of AMEV. Expression of the AMV197 gene in baculovirus expression system yielded a ~ 35.5 kDa protein. PK activity of expressed AMV197 was shown by standard PK assay. Substrate profiling of AMV197 protein by peptide microarray indicated that the expressed protein phosphorylated 81 of 624 substrates which belong to 28 families of PK substrates. While the hypothetical AMV197 protein phosphorylates Ser/Thr only, we demonstrated that the expressed PK also phosphorylates probes with tyrosine residues on the array which is a rare property among PKs. Pull-down assay of the AMV197 protein with the subcellular protein fractionations of Ld652 cells showed that it is using two cellular proteins (18 and 42 kDa) as novel putative substrates. Our results suggest that AMEV can regulate cellular mechanisms by phosphorylating cellular proteins through AMV197 PK. However, further experiments are needed to identify the exact role of this PK in the replication of AMEV.

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.