AGL16 regulates genome-wide gene expression and flowering time with partial dependency on SOC1 in Arabidopsis

Flowering transition is pivotal and tightly regulated by complex gene-regulatory-networks, in which AGL16 plays important roles. But the molecular function and binding property of AGL16 is not fully explored in vivo. With ChIP-seq and comparative transcriptomics approaches, we characterized the AGL16 targets spectrum and tested its close molecular and genetic interactions with SOC1, the key flowering integrator. AGL16 bound to promoters of more than 2000 genes via CArG-box motifs that were highly similar to that of SOC1. Being consistent with this, AGL16 formed protein complex and shared a common set of targets with SOC1. However, only very few genes showed differential expression in the agl16-1 loss-of-function mutant, whereas in the soc1-2 knockout background, AGL16 repressed and activated the expression of 375 and 182 genes, respectively, with more than a quarter of the DEGs were also bound by AGL16. AGL16 targeted potentially to about seventy flowering time genes involved in multiple pathways. Corroborating with these, AGL16 repressed the flowering time stronger in soc1-2 than in Col-0 background. These data reveals that AGL16 regulates gene expression and flowering time with a partial dependency on SOC1 activity. Moreover, AGL16 participated in the regulation of water loss and seed dormancy. Our study thus defines the AGL16 molecular spectrum and provides insights underlining the molecular coordination of flowering and environmental adaptation.

DOTFL1 affects the floral transition in orchid Dendrobium Chao Praya Smile

A major obstacle for orchid (Orchidaceae)breeding and production is a long juvenile phase before orchid reproductive development. The molecular basis for prolonged vegetative growth in orchids remains largely unclear despite many efforts to clarify the relevant mechanisms. In this study, we report functional characterization of Dendrobium Orchid TERMINAL FLOWER1 (DOTFL1), an ortholog of TFL1 in Arabidopsis (Arabidopsis thaliana), from the orchid Dendrobium Chao Praya Smile. DOTFL1 is highly expressed in pseudobulbs and the shoot apical meristem (SAM) before and during the floral transition, but is downregulated in inflorescence apices and open flowers. Ectopic expression of DOTFL1 rescues the early-flowering and terminal-flower phenotypes of tfl1-20 in Arabidopsis. Overexpression of DOTFL1 in Dendrobium orchids delays flowering and produces defective inflorescence meristems and flowers with vegetative traits, whereas knockdown of DOTFL1 accelerates flowering and perturbs the maintenance of the inflorescence meristem. Notably, DOTFL1 suppresses orchid flowering and associated pseudobulb formation during the floral transition. We further reveal that two orchid MADS-box transcription factors, Dendrobium Orchid SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (DOSOC1) and AGAMOUS-LIKE 24 (DOAGL24), could interact with each other and bind to the CArG-box motif at DOTFL1, implying a regulatory hierarchy similar to their counterparts in Arabidopsis. Taken together, our findings suggest that DOTFL1 promotes vegetative growth, modulates successive developmental events required for reproductive success in Dendrobium orchids, and may have evolved with a previously unknown role in controlling pseudobulb formation in the Orchidaceae family.

Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

Background Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. Results Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. Conclusions PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.

DNA-binding properties of the MADS-domain transcription factor SEPALLATA3 and mutant variants characterized by SELEX-seq

Key message We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. Abstract MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

Kindlin-2 Mediates Mechanical Activation of Cardiac Myofibroblasts

We identify the focal adhesion protein kindlin-2 as player in a novel mechanotransduction pathway that controls profibrotic cardiac fibroblast to myofibroblast activation. Kindlin-2 is co-upregulated with the myofibroblast marker α-smooth muscle actin (α-SMA) in fibrotic rat hearts and in human cardiac fibroblasts exposed to fibrosis-stiff culture substrates and pro-fibrotic TGF-β1. Stressing fibroblasts using ferromagnetic microbeads, stretchable silicone membranes, and cell contraction agonists all result in kindlin-2 translocation to the nucleus. Overexpression of full-length kindlin-2 but not of kindlin-2 missing a putative nuclear localization sequence (∆NLS kindlin-2) results in increased α-SMA promoter activity. Downregulating kindlin-2 with siRNA leads to decreased myofibroblast contraction and reduced α-SMA expression, which is dependent on CC(A/T)-rich GG(CArG) box elements in the α-SMA promoter. Lost myofibroblast features under kindlin-2 knockdown are rescued with wild-type but not ∆NLS kindlin-2, indicating that myofibroblast control by kindlin-2 requires its nuclear translocation. Because kindlin-2 can act as a mechanotransducer regulating the transcription of α-SMA, it is a potential target to interfere with myofibroblast activation in tissue fibrosis.

YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in ∼90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons

MADS-box transcription factors (TFs) are ubiquitous in eukaryotic organisms and play major roles during plant development. Nevertheless, their function in seed development remains largely unknown. Here, we show that the imprinted Arabidopsis thaliana MADS-box TF PHERES1 (PHE1) is a master regulator of paternally expressed imprinted genes, as well as of non-imprinted key regulators of endosperm development. PHE1 binding sites show distinct epigenetic modifications on maternal and paternal alleles, correlating with parental-specific transcriptional activity. Importantly, we show that the CArG-box-like DNA-binding motifs that are bound by PHE1 have been distributed by RC/Helitron transposable elements. Our data provide an example of the molecular domestication of these elements which, by distributing PHE1 binding sites throughout the genome, have facilitated the recruitment of crucial endosperm regulators into a single transcriptional network.

The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons

MADS-box transcription factors are ubiquitous in eukaryotic organisms and play major roles during plant development. Nevertheless, their function in seed development remains largely unknown. Here we show that the imprinted Arabidopsis thaliana MADS-box TF PHERES1 (PHE1) is a master regulator of paternally expressed imprinted genes, as well as of non-imprinted key regulators of endosperm development. PHE1 binding sites show distinct epigenetic modifications on maternal and paternal alleles, correlating with parental-specific transcriptional activity. Importantly, we show that the CArG-box-like DNA-binding motifs bound by PHE1 have been distributed by RC/Helitron transposable elements. Our data provide an example of molecular domestication of these elements, which by distributing PHE1 binding sites throughout the genome, have facilitated the recruitment of crucial endosperm regulators into a single transcriptional network.

BpAP1 directly regulates BpDEF to promote male inflorescence formation in Betula platyphylla × B. pendula

Flowering is a crucial process for plants that is under complex genetic control. AP1 acts as an organizer and a switch for the transition from vegetative to reproductive growth. In our previous study, we found that overexpression of BpAP1 significantly promoted the formation of male inflorescence in birch (Betula platyphlla × B. pendula). In this study, we aimed at investigating the molecular regulatory mechanism of BpAP1 during the process of male inflorescence formation in birch. We found that overexpression of BpAP1 affected the expression of many flowering-related genes, and had significant effect on B class MADS-box genes. A BpAP1-mediated gene regulatory network was constructed and B class gene BpDEF was finally predicted as a key target gene of BpAP1. Chromatin immunoprecipitation results indicated that BpAP1 could directly regulate BpDEF during the process of male inflorescence formation. Yeast one-hybrid assays and its validation in tobacco results suggested that BpAP1 regulated BpDEF via binding to a consensus DNA sequence known as CArG box. Gene function analysis of BpDEF indicated that BpDEF may function in sex-determination, and in particular specify the identity of male inflorescence in birch. Our results provide valuable clues for our understanding of the molecular mechanism of BpAP1 during the process of male inflorescence formation in birch.