No abstract

Summary Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we reported a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a “histone H4 mimic” GK-X-GK motif that is di-acetylated by Tip60. The di-acetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructs an activated Twist/BRD4/P-TEFb/RNA-PolII complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.

Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass-tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue-specific manner during cell growth. In this study, we further extend this residue-specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc-responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild-type and zap1delta cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up-regulated more than four times in the zap1delta mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS-PAGE, the expression level of a novel protein was found to increase two-fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances.

Obesity and its associated comorbidities (e.g., diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually1 and are among the most prevalent and challenging conditions confronting the medical profession2,3. Neurotensin (NT), a 13-amino acid peptide predominantly localized in specialized enteroendocrine (EE) cells of the small bowel4 and released by fat ingestion5, facilitates fatty acid (FA) translocation in rat intestine6, and stimulates growth of various cancers7; the effects of NT are mediated through three known NT receptors (NTR1, 2 and 3)8. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality9; however, a role for NT as a causative factor in these diseases is unknown. Here, we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates FA absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3/sortilin. Consistent with the findings in mice, expression of NT in Drosophila midgut EE cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and suggest that NT may provide a prognostic marker of future obesity and a potential target for prevention and treatment.

Mutations in FUS have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. Little is known about the regulation of FUS subcellular localization or how the ALS mutations alter FUS function. Here we demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS point mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. PABP1 formed large cytoplasmic foci that co-localized with the mutant FUS inclusions. No such foci, which resemble stress granules, were observed in the presence of wild-type FUS. In addition, processing bodies, which are functionally related to stress granules, were adjacent to but not co-localized with the mutant FUS inclusions. Our results suggest that the ALS mutations in the C-terminal NLS of FUS can impair FUS nuclear localization and induce cytoplasmic mislocalization, inclusion formation, and potential perturbation of RNA metabolism.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3′ untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

SignificanceMutations in fused in sarcoma (FUS) contribute to a subset of familial amyotrophic lateral sclerosis (ALS). ALS-linked mutations cause a liquid–liquid phase separation of FUS protein, induce cytoplasmic inclusions in cells, and interfere with RNA metabolism pathways. Our proteomic analysis shows that proteins sequestered into inclusions are enriched in translation and RNA quality surveillance pathways. This study demonstrates that ALS mutations in FUS indeed suppress protein translation and affect the mRNA nonsense-mediated decay (NMD) pathway. NMD-promoting factors UPF1 and UPF3b increased, whereas the negative regulator UPF3a decreased in the cells of patients with ALS. The disruption in NMD regulation and suppression of protein biosynthesis likely contribute to neurodegeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron death.More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1 mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1-mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In this study, we identified a dynein subunit as a component of the mutant SOD1-containing high molecular weight complexes using proteomic techniques. We further demonstrated interaction and colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the onset of symptoms in the ALS animal models and increased over the disease progression. Motor neurons with long axons are particularly susceptible to defects in axonal transport. Our results demonstrate a direct "gain-of-interaction" between mutant SOD1 and dynein, which may provide insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal transport or other dynein functions. The aberrant interaction is potentially critical to the formation of mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration in ALS.