The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2.

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

The endoplasmic reticulum coat protein II transport machinery coordinates cellular lipid secretion and cholesterol biosynthesis.

Triglycerides and cholesterol are essential for life in most organisms. Triglycerides serve as the principal energy storage depot and, where vascular systems exist, as a means of energy transport. Cholesterol is essential for the functional integrity of all cellular membrane systems. The endoplasmic reticulum is the site of secretory lipoprotein production and de novo cholesterol synthesis, yet little is known about how these activities are coordinated with each other or with the activity of the COPII machinery, which transports endoplasmic reticulum cargo to the Golgi. The Sar1B component of this machinery is mutated in chylomicron retention disorder, indicating that this Sar1 isoform secures delivery of dietary lipids into the circulation. However, it is not known why some patients with chylomicron retention disorder develop hepatic steatosis, despite impaired intestinal fat malabsorption, and why very severe hypocholesterolemia develops in this condition. Here, we show that Sar1B also promotes hepatic apolipoprotein (apo) B lipoprotein secretion and that this promoting activity is coordinated with the processes regulating apoB expression and the transfer of triglycerides/cholesterol moieties onto this large lipid transport protein. We also show that although Sar1A antagonizes the lipoprotein secretion-promoting activity of Sar1B, both isoforms modulate the expression of genes encoding cholesterol biosynthetic enzymes and the synthesis of cholesterol de novo. These results not only establish that Sar1B promotes the secretion of hepatic lipids but also adds regulation of cholesterol synthesis to Sar1B's repertoire of transport functions.

The origin, global distribution, and functional impact of the human 8p23 inversion polymorphism.

Genomic inversions are an increasingly recognized source of genetic variation. However, a lack of reliable high-throughput genotyping assays for these structures has precluded a full understanding of an inversion's phylogenetic, phenotypic, and population genetic properties. We characterize these properties for one of the largest polymorphic inversions in man (the ∼4.5-Mb 8p23.1 inversion), a structure that encompasses numerous signals of natural selection and disease association. We developed and validated a flexible bioinformatics tool that utilizes SNP data to enable accurate, high-throughput genotyping of the 8p23.1 inversion. This tool was applied retrospectively to diverse genome-wide data sets, revealing significant population stratification that largely follows a clinal "serial founder effect" distribution model. Phylogenetic analyses establish the inversion's ancestral origin within the Homo lineage, indicating that 8p23.1 inversion has occurred independently in the Pan lineage. The human inversion breakpoint was localized to an inverted pair of human endogenous retrovirus elements within the large, flanking low-copy repeats; experimental validation of this breakpoint confirmed these elements as the likely intermediary substrates that sponsored inversion formation. In five data sets, mRNA levels of disease-associated genes were robustly associated with inversion genotype. Moreover, a haplotype associated with systemic lupus erythematosus was restricted to the derived inversion state. We conclude that the 8p23.1 inversion is an evolutionarily dynamic structure that can now be accommodated into the understanding of human genetic and phenotypic diversity.

Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity.

Recent studies have revealed an important role for hormones in plant immunity. We are now beginning to understand the contribution of crosstalk among different hormone signaling networks to the outcome of plant-pathogen interactions. Cytokinins are plant hormones that regulate development and responses to the environment. Cytokinin signaling involves a phosphorelay circuitry similar to two-component systems used by bacteria and fungi to perceive and react to various environmental stimuli. In this study, we asked whether cytokinin and components of cytokinin signaling contribute to plant immunity. We demonstrate that cytokinin levels in Arabidopsis are important in determining the amplitude of immune responses, ultimately influencing the outcome of plant-pathogen interactions. We show that high concentrations of cytokinin lead to increased defense responses to a virulent oomycete pathogen, through a process that is dependent on salicylic acid (SA) accumulation and activation of defense gene expression. Surprisingly, treatment with lower concentrations of cytokinin results in increased susceptibility. These functions for cytokinin in plant immunity require a host phosphorelay system and are mediated in part by type-A response regulators, which act as negative regulators of basal and pathogen-induced SA-dependent gene expression. Our results support a model in which cytokinin up-regulates plant immunity via an elevation of SA-dependent defense responses and in which SA in turn feedback-inhibits cytokinin signaling. The crosstalk between cytokinin and SA signaling networks may help plants fine-tune defense responses against pathogens.

CDKN2B expression in adipose tissue of familial combined hyperlipidemia patients.

The purpose of this study was to determine the core biological processes perturbed in the subcutaneous adipose tissue of familial combined hyperlipidemia (FCHL) patients. Annotation of FCHL and control microarray datasets revealed a distinctive FCHL transcriptome, characterized by gene expression changes regulating five overlapping systems: the cytoskeleton, cell adhesion and extracellular matrix; vesicular trafficking; lipid homeostasis; and cell cycle and apoptosis. Expression values for the cell-cycle inhibitor CDKN2B were increased, replicating data from an independent FCHL cohort. In 3T3-L1 cells, CDKN2B knockdown induced C/EBPα expression and lipid accumulation. The minor allele at SNP site rs1063192 (C) was predicted to create a perfect seed for the human miRNA-323b-5p. A miR-323b-5p mimic significantly reduced endogenous CDKN2B protein levels and the activity of a CDKN2B 3'UTR luciferase reporter carrying the rs1063192 C allele. Although the allele displayed suggestive evidence of association with reduced CDKN2B mRNA in the MuTHER adipose tissue dataset, family studies suggest the association between increased CDKN2B expression and FCHL-lipid abnormalities is driven by factors external to this gene locus. In conclusion, from a comparative annotation analysis of two separate FCHL adipose tissue transcriptomes and a subsequent focus on CDKN2B, we propose that dysfunctional adipogenesis forms an integral part of FCHL pathogenesis.

The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling.

Cytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial two-component phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

Musings from the Tribbles Research and Innovation Network.

This commentary integrates historical and modern findings that underpin our understanding of the cell-specific functions of the Tribbles (TRIB) proteins that bear on tumorigenesis. We touch on the initial discovery of roles played by mammalian TRIB proteins in a diverse range of cell-types and pathologies, for example, TRIB1 in regulatory T-cells, TRIB2 in acute myeloid leukaemia and TRIB3 in gliomas; the origins and diversity of TRIB1 transcripts; microRNA-mediated (miRNA) regulation of TRIB1 transcript decay and translation; the substantial conformational changes that ensue on binding of TRIB1 to the transcription factor C/EBPα; and the unique pocket formed by TRIB1 to sequester its C-terminal motif bearing a binding site for the E3 ubiquitin ligase COP1. Unashamedly, the narrative is relayed through the perspective of the Tribbles Research and Innovation Network, and its establishment, progress and future ambitions: the growth of TRIB and COP1 research to hasten discovery of their cell-specific contributions to health and obesity-related cancers.

A role for two-component signaling elements in the Arabidopsis growth recovery response to ethylene.

Previous studies indicate that the ability of Arabidopsis seedlings to recover normal growth following an ethylene treatment involves histidine kinase activity of the ethylene receptors. As histidine kinases can function as inputs for a two-component signaling system, we examined loss-of-function mutants involving two-component signaling elements. We find that mutants of phosphotransfer proteins and type-B response regulators exhibit a defect in their ethylene growth recovery response similar to that found with the loss-of-function ethylene receptor mutant etr1-7. The ability of two-component signaling elements to regulate the growth recovery response to ethylene functions independently from their well-characterized role in cytokinin signaling, based on the analysis of cytokinin receptor mutants as well as following chemical inhibition of cytokinin biosynthesis. Histidine kinase activity of the receptor ETR1 also facilitates growth recovery in the ethylene hypersensitive response, which is characterized by a transient decrease in growth rate when seedlings are treated continuously with a low dose of ethylene; however, this response was found to operate independently of the type-B response regulators. These results indicate that histidine kinase activity of the ethylene receptor ETR1 performs two independent functions: (a) regulating the growth recovery to ethylene through a two-component signaling system involving phosphotransfer proteins and type-B response regulators and (b) regulating the hypersensitive response to ethylene in a type-B response regulator independent manner.

Signaling via Histidine-Containing Phosphotransfer Proteins in Arabidopsis.

The Arabidopsis genome encodes a number of proteins with similarity to two-component phosphorelay signaling elements, including hybrid receptor histidine kinases, two classes of response regulator proteins (type-A and type-B ARRs) and a family of six histidine-containing phosphotransfer proteins (AHPs), five of which contain a conserved His residue that is required for phosphorelay signaling. The current model for cytokinin signaling includes a multistep phosphorelay: three histidine kinases and at least five type-B ARRs have been shown to act as positive regulators of cytokinin signaling, while a number of type-A ARRs, and AHP6, act as negative regulators of the pathway. In our recent Plant Cell paper, we provided genetic evidence that at least four AHPs can act as positive regulators of cytokinin signaling, affecting responses to cytokinin in the root and the shoot. In this addendum, we discuss the role of AHPs in cytokinin signaling and speculate on their potential interactions with other signaling pathways in Arabidopsis.

Cytokinin regulates type-A Arabidopsis Response Regulator activity and protein stability via two-component phosphorelay.

The plant hormone cytokinin regulates many aspects of growth and development. Cytokinin signaling involves His kinase receptors that perceive cytokinin and transmit the signal via a multistep phosphorelay similar to bacterial two-component signaling systems. The final targets of this phosphorelay are a set of Arabidopsis thaliana Response Regulator (ARR) proteins containing a receiver domain with a conserved Asp phosphorylation site. One class of these, the type-A ARRs, are negative regulators of cytokinin signaling that are rapidly transcriptionally upregulated in response to cytokinin. In this study, we tested the role of phosphorylation in type-A ARR function. Our results indicate that phosphorylation of the receiver domain is required for type-A ARR function and suggest that negative regulation of cytokinin signaling by the type-A ARRs most likely involves phosphorylation-dependent interactions. Furthermore, we show that a subset of the type-A ARR proteins are stabilized in response to cytokinin in part via phosphorylation. These studies shed light on the mechanism by which type-A ARRs act to negatively regulate cytokinin signaling and reveal a novel mechanism by which cytokinin controls type-A ARR function.

A subset of Arabidopsis AP2 transcription factors mediates cytokinin responses in concert with a two-component pathway.

The plant hormone cytokinin regulates numerous growth and developmental processes. A signal transduction pathway for cytokinin has been elucidated that is similar to bacterial two-component phosphorelays. In Arabidopsis, this pathway is comprised of receptors that are similar to sensor histidine kinases, histidine-containing phosphotransfer proteins, and response regulators (ARRs). There are two classes of response regulators, the type-A ARRs, which act as negative regulators of cytokinin responses, and the type-B ARRs, which are transcription factors that play a positive role in mediating cytokinin-regulated gene expression. Here we show that several closely related members of the Arabidopsis AP2 gene family of unknown function are transcriptionally up-regulated by cytokinin through this pathway, and we have designated these AP2 genes CYTOKININ RESPONSE FACTORS (CRFs). In addition to their transcriptional regulation by cytokinin, the CRF proteins rapidly accumulate in the nucleus in response to cytokinin, and this relocalization depends on the histidine kinases and the downstream histidine-containing phosphotransfer proteins, but is independent of the ARRs. Analysis of loss-of-function mutations reveals that the CRFs function redundantly to regulate the development of embryos, cotyledons, and leaves. Furthermore, the CRFs mediate a large fraction of the transcriptional response to cytokinin, affecting a set of cytokinin-responsive genes that largely overlaps with type-B ARR targets. These results indicate that the CRF proteins function in tandem with the type-B ARRs to mediate the initial cytokinin response. Thus, the evolutionarily ancient two-component system that is used by cytokinin branches to incorporate a unique family of plant-specific transcription factors.

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling.

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.

Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis.

CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.