Missense variants in ANKRD11 cause KBG syndrome by impairment of stability or transcriptional activity of the encoded protein

Purpose: Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized the clinical, molecular and functional spectra of ANKRD11 missense variants. Methods: We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants by in silico analyses and cell-based experiments. Results: We identified 29 individuals with (mostly de novo) ANKRD11 missense variants, who presented with syndromic neurodevelopmental disorders and were phenotypically similar to individuals with KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in Repression Domain 2. Cellularly, most variants caused reduced ANKRD11 stability. One variant resulted in decreased proteasome degradation and loss of ANKRD11 transcriptional activity. Conclusion: Our study indicates that pathogenic heterozygous missense variants in ANKRD11 cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges, as the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping.

The NAC transcription factor ClNAC68 positively regulates sugar content and seed development in watermelon by repressing ClINV and ClGH3.6

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in fruit ripening and quality. The watermelon genome encodes 80 NAC genes, and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues. Among these genes, ClNAC68 expression was significantly higher in flesh than in rind. However, the intrinsic regulatory mechanism of ClNAC68 in fruit ripening and quality is still unknown. In this study, we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus. Knockout of ClNAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh. Development was delayed, germination was inhibited, and the IAA content was significantly decreased in mutant seeds. Transcriptome analysis showed that the invertase gene ClINV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh, and expression of the indole-3-acetic acid-amido synthetase gene ClGH3.6 in the IAA signaling pathway was also induced in mutant seeds. EMSA and dual-luciferase assays showed that ClNAC68 directly bound to the promoters of ClINV and ClGH3.6 to repress their expression. These results indicated that ClNAC68 positively regulated sugar and IAA accumulation by repressing ClINV and ClGH3.6. Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development.

Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex

The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

Improving the Efficiency of Adventitious Shoot Induction and Somatic Embryogenesis via Modification of WUSCHEL and LEAFY COTYLEDON 1

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants.

NC2 complex is a key factor for the activation of catalase-3 transcription by regulating H2A.Z deposition

Negative cofactor 2 (NC2), including two subunits NC2α and NC2β, is a conserved positive/negative regulator of class II gene transcription in eukaryotes. It is known that NC2 functions by regulating the assembly of the transcription preinitiation complex. However, the exact role of NC2 in transcriptional regulation is still unclear. Here, we reveal that, in Neurospora crassa, NC2 activates catalase-3 (cat-3) gene transcription in the form of heterodimer mediated by histone fold (HF) domains of two subunits. Deletion of HF domain in either of two subunits disrupts the NC2α–NC2β interaction and the binding of intact NC2 heterodimer to cat-3 locus. Loss of NC2 dramatically increases histone variant H2A.Z deposition at cat-3 locus. Further studies show that NC2 recruits chromatin remodeling complex INO80C to remove H2A.Z from the nucleosomes around cat-3 locus, resulting in transcriptional activation of cat-3. Besides HF domains of two subunits, interestingly, C-terminal repression domain of NC2β is required not only for NC2 binding to cat-3 locus, but also for the recruitment of INO80C to cat-3 locus and removal of H2A.Z from the nucleosomes. Collectively, our findings reveal a novel mechanism of NC2 in transcription activation through recruiting INO80C to remove H2A.Z from special H2A.Z-containing nucleosomes.

Structure-function analysis of Arabidopsis TOPLESS reveals fundamental conservation of repression mechanisms across eukaryotes

SummaryThe plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli is poorly understood. Previously, we had used the N-terminus of Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle domain subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.

FLOWERING HTH1 is involved in CONSTANS-mediated flowering regulation in Arabidopsis

Flowering at the right time is essential for maximum reproductive fitness. In Arabidopsis thaliana, the CONSTANS (CO) protein facilitates the transition from the vegetative phase to the reproductive phase under long-day conditions. The formation of heterodimeric complexes between CO and DNA binding domain-containing transcription factors is important for the induction of day length-dependent flowering. Here, we report a myb-like helix turn helix (HTH) transcriptional regulator family protein as a new modulator of floral transition, which we have named FLOWERING HTH1 (FHTH1). We isolated FHTH1 as a CO-interacting protein by a yeast two-hybrid screen using an Arabidopsis transcription factor library. Our analysis showed that FHTH1 presented in the nucleus and the FHTH1-CO complex was formed in the same subcellular location. We also observed the expression of a FHTH1:GUS construct in the leaf vasculature, where CO exists. Transgenic plants overexpressing FHTH1 fused with the plant-specific repression domain SRDX showed a delayed flowering phenotype in long days, resembling the phenotype of the co mutant. Our results suggest that FHTH1 may contribute to CO-mediated photoperiodic flowering regulation.

Recruitment of the protein phosphatase-1 catalytic subunit to promoters by the dual-function transcription factor RFX1

SummeryRFX proteins are a family of conserved DNA binding proteins involved in various, essential cellular and developmental processes. RFX1 is a ubiquitously expressed, dual-activity transcription factor capable of both activation and repression of target genes.The exact mechanism by which RFX1 regulates its target is not known yet. In this work, we show that the C-terminal repression domain of RFX1 interacts with the Serine/Threonine protein phosphatase PP1c, and that interaction with RFX1 can target PP1c to specific sites in the genome. Given that PP1c was shown to de-phosphorylate several transcription factors, as well as the regulatory C-terminal domain of RNA Polymerase II the recruitment of PP1c to promoters may be a mechanism by which RFX1 regulates the target genes.