PIF4 and PIF4-Interacting Proteins: At the Nexus of Plant Light, Temperature and Hormone Signal Integrations

Basic helix-loop-helix (bHLH) family transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is necessary for plant adaption to light or high ambient temperature. PIF4 directly associates with plenty of its target genes and modulates the global transcriptome to induce or reduce gene expression levels. However, PIF4 activity is tightly controlled by its interacting proteins. Until now, twenty-five individual proteins have been reported to physically interact with PIF4. These PIF4-interacting proteins act together with PIF4 and form a unique nexus for plant adaption to light or temperature change. In this review, we will discuss the different categories of PIF4-interacting proteins, including photoreceptors, circadian clock regulators, hormone signaling components, and transcription factors. These distinct PIF4-interacting proteins either integrate light and/or temperature cues with endogenous hormone signaling, or control PIF4 abundances and transcriptional activities. Taken together, PIF4 and PIF4-interacting proteins play major roles for exogenous and endogenous signal integrations, and therefore establish a robust network for plants to cope with their surrounding environmental alterations.

Single-stranded RNA oligonucleotides that recruit endogenous hnRNPA1 enable the targeted reduction of gene expression

Targeted gene knockdown has become one of the most powerful tools in molecular biology and holds substantial promise in therapeutic applications. While existing technologies such as siRNAs, CRISPRi, and ASOs effectively and specifically reduce gene expression, few can be used to first discover the genes that influence a particular phenotype and then directly transition to being used as oligonucleotide therapeutics. Thus, a tool that could help bridge the gap between target discovery and the development of therapeutic leads would benefit the scientific community. Here, we present hnRNPA1 recruiting oligonucleotides, or AROs, as single-stranded RNA (ssRNA) molecules that knockdown transcript levels of target genes. AROs target specific pre-mRNA transcripts via sequence homology and leverage the ubiquitous and abundant endogenous RNA-binding protein hnRNPA1 to degrade target transcripts. Using RT-qPCR, we show that AROs effectively knock down target genes when delivered via a plasmid and expressed using a Pol II promoter or when delivered directly as single-strand RNAs. Additionally, as proof of principle, we use a ssRNA ARO to knockdown KRT14 in squamous cell carcinoma and show reduced invasive potential. We believe AROs fill an important niche in the scientific toolbox by taking advantage of endogenous RNA binding machinery for RNA knockdowns.

Metformin and Pioglitazone Reduce Gene Expression of Inflammatory Factors in Insulin Resistant and Hypertrophied Adipocytes

Objective: In obesity, chronic low grade inflammation, created by induction of pro-inflammatory markers, causes adipocyte dysfunction in adipose tissue. Adipocytes dysfunction is associated with various diseases including insulin resistance and obesity. In obesity, inflammatory factors such as osteopontin (OPN), angiopoietin-like protein 2 (Angptl2) and transforming growth factor-β (TGF-β) are induced in adipose tissue. Metformin and pioglitazone that are used to modulate inflammation, but the relevant mechanism is poorly understood. This study aimed to investigate the effect of metformin and pioglitazone as anti-diabetic drugs, on gene expression of osteopontin, Angptl2 and TGF-β as inflammatory factors in insulin resistance and hypertrophied adipocyte in 3T3-L1 cell line model in vitro. Materials and Methods: In this experimental research, we differentiated3T3-L1 preadipocytes to adipocytes. The adipocytes treated in insulin resistance and hypertrophied conditions with metformin and pioglitazone and assayed gene expression of OPN, Angptl2 and TGF-β by Real-Time PCR. Data was analyzed by SPSS statistic software. Results: The results showed that expression of OPN, Angptl2, and TGF-β were increased significantly over 2-fold (P-value< 0.05) in insulin resistance and hypertrophied adipocytes compared to normal adipocytes. Pre- and co-treatment with metformin and pioglitazone led to reduced expression of Angptl2 and TGF-β. Only metformin significantly reduced the expression of Angptl2, TGF-β and OPN in hypertrophied adipocyte. Conclusion: These results support the proposal that metformin and pioglitazone reduce gene expression of inflammatory factors in insulin resistant and hypertrophied adipocytes.

Short residence times of DNA-bound transcription factors can reduce gene expression noise and increase the transmission of information in a gene regulation system

Gene expression noise is not just ubiquitous but also variable, and we still do not understand some of the most elementary factors that affect it. Among them is the residence time of a transcription factor (TF) on DNA, the mean time that a DNA-bound TF remains bound. Here, we use a stochastic model of transcriptional regulation to study how this residence time affects gene expression. We find that the effect of residence time on gene expression depends on the level of induction of the gene. At high levels of induction, residence time has no effect on gene expression. However, as the level of induction decreases, short residence times reduce gene expression noise. The reason is that fast on-off TF binding dynamics prevent long periods where proteins are predominantly synthesized or degraded, which can cause excessive fluctuations in gene expression. As a consequence, short residence times can help a gene regulation system acquire information about the cellular environment it operates in. Our predictions are consistent with the observation that experimentally measured residence times are usually modest and lie between seconds to minutes.

mRNA levels can be reduced by antisense oligonucleotides via no-go decay pathway

Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2′-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3′ end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.

Abstract 289: Non-viral Rna Delivery To The Endothelium

Dysfunctional vasculature contributes to more disease than any other tissue in the body1. Small interfering RNAs (siRNAs) have the potential to help elucidate the role of endothelial cells in vivo by durably silencing multiple genes simultaneously, but this requires efficient delivery, which has remained challenging in cell types besides hepatocytes. We have developed nanoparticles that deliver siRNA to endothelial cells with high specificity, thereby facilitating the silencing of multiple endothelial cell genes in vivo. These particles do not significantly reduce gene expression in hepatocytes or immune cells even at doses forty times greater than those required for endothelial gene silencing. Optimized formulations achieved the most durable non-liver silencing reported to date, and delivered siRNAs that modified endothelial function in mouse models of vascular permeability, emphysema, primary tumor growth, and metastasis. We believe the nanomaterial described here may improve the ability to study endothelial gene function in vivo, and be used to treat diseases caused by vascular dysfunction.