Bidirectional approaches for optogenetic regulation of gene expression in mammalian cells using Arabidopsis cryptochrome 2.

Optogenetic tools allow regulation of cellular processes with light, which can be delivered with spatiotemporal resolution. In previous work, we used cryptochrome 2 (CRY2) and CIB1, Arabidopsis proteins that interact upon light illumination, to regulate transcription with light in yeast. While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus. The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators. We used this knowledge to develop two different approaches to regulate cellular protein levels with light: a system using CRY2 and CIB1 to induce protein expression with light through stimulation of transcription, and a system using CRY2 and a LOV-fused degron to simultaneously block transcription and deplete protein levels with light. These tools will allow precise, bi-directional control of gene expression in a variety of cells and model systems.

HDAC6 contributes to pathological responses of heart and skeletal muscle to chronic angiotensin-II signaling.

Little is known about the function of the cytoplasmic histone deacetylase HDAC6 in striated muscle. Here, we addressed the role of HDAC6 in cardiac and skeletal muscle remodeling induced by the peptide hormone angiotensin II (ANG II), which plays a central role in blood pressure control, heart failure, and associated skeletal muscle wasting. Comparable with wild-type (WT) mice, HDAC6 null mice developed cardiac hypertrophy and fibrosis in response to ANG II. However, whereas WT mice developed systolic dysfunction upon treatment with ANG II, cardiac function was maintained in HDAC6 null mice treated with ANG II for up to 8 wk. The cardioprotective effect of HDAC6 deletion was mimicked in WT mice treated with the small molecule HDAC6 inhibitor tubastatin A. HDAC6 null mice also exhibited improved left ventricular function in the setting of pressure overload mediated by transverse aortic constriction. HDAC6 inhibition appeared to preserve systolic function, in part, by enhancing cooperativity of myofibrillar force generation. Finally, we show that HDAC6 null mice are resistant to skeletal muscle wasting mediated by chronic ANG-II signaling. These findings define novel roles for HDAC6 in striated muscle and suggest potential for HDAC6-selective inhibitors for the treatment of cardiac dysfunction and muscle wasting in patients with heart failure.

BET acetyl-lysine binding proteins control pathological cardiac hypertrophy.

Cardiac hypertrophy is an independent predictor of adverse outcomes in patients with heart failure, and thus represents an attractive target for novel therapeutic intervention. JQ1, a small molecule inhibitor of bromodomain and extraterminal (BET) acetyl-lysine reader proteins, was identified in a high throughput screen designed to discover novel small molecule regulators of cardiomyocyte hypertrophy. JQ1 dose-dependently blocked agonist-dependent hypertrophy of cultured neonatal rat ventricular myocytes (NRVMs) and reversed the prototypical gene program associated with pathological cardiac hypertrophy. JQ1 also blocked left ventricular hypertrophy (LVH) and improved cardiac function in adult mice subjected to transverse aortic constriction (TAC). The BET family consists of BRD2, BRD3, BRD4 and BRDT. BRD4 protein expression was increased during cardiac hypertrophy, and hypertrophic stimuli promoted recruitment of BRD4 to the transcriptional start site (TSS) of the gene encoding atrial natriuretic factor (ANF). Binding of BRD4 to the ANF TSS was associated with increased phosphorylation of local RNA polymerase II. These findings define a novel function for BET proteins as signal-responsive regulators of cardiac hypertrophy, and suggest that small molecule inhibitors of these epigenetic reader proteins have potential as therapeutics for heart failure.

Differential effects of the trans-18:1 isomer profile of partially hydrogenated vegetable oils on cholesterol and lipoprotein metabolism in male F1B hamsters.

Trans-fatty acid consumption from partially hydrogenated vegetable oil (PHVO) has been positively associated with multiple cardiovascular disease risk factors and events. This study was designed to examine the effects of trans-fatty acid isomer profile of PHVO on plasma lipids and lipoproteins and hepatic expression of key genes involved in cholesterol and fatty acid metabolism. Thirty-three male F(1)B strain Syrian Golden Hamsters were allocated to 1 of 3 hypercholesterolemic diets containing (5% by weight): 1) tristearin [control fat (CON)]; 2) partially hydrogenated high-oleic acid sunflower oil (PH-SUN); or 3) partially hydrogenated high-linoleic acid safflower oil (PH-SAF). PH-SUN contained more trans-4 to trans-10 18:1 compared with PH-SAF, which contained more trans-11 to trans-16 18:1. The addition of both PHVO to the diet increased plasma total cholesterol concentrations relative to CON, but only PH-SUN increased the plasma ratio of non-HDL:HDL cholesterol compared with CON. PH-SUN increased VLDL (total, large, and medium) and IDL particle concentrations while decreasing total, medium, and small HDL particle concentrations relative to CON. Both PHVO diets increased the hepatic cholesterol ester concentration, whereas the hepatic TG concentration was lower in PH-SUN compared with PH-SAF and CON. Levels of hepatic LDL receptor, HMG-CoA reductase, and sterol response element binding protein 1 mRNA were specifically reduced in the PH-SUN group compared to the CON group. Expression of SREBP1c was upregulated in both PHVO groups compared to CON, whereas only the PH-SAF group had higher levels of the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 compared to CON. These results indicate that differences in the trans-fatty acid profile of PHVO can differentially affect lipid and lipoprotein metabolism.

Photodimerization systems for regulating protein-protein interactions with light.

Optogenetic dimerizers are modular domains that can be utilized in a variety of versatile ways to modulate cellular biochemistry. Because of their modularity, many applications using these tools can be easily transferred to new targets without extensive engineering. While a number of photodimerizer systems are currently available, the field remains nascent, with new optimizations for existing systems and new approaches to regulating biological function continuing to be introduced at a steady pace.

Optical Control of Peroxisomal Trafficking.

The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times.