Lifelong persistence of nuclear RNAs in the mouse brain.

Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type-specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.

Direct reprogramming of human smooth muscle and vascular endothelial cells reveals defects associated with aging and Hutchinson-Gilford progeria syndrome.

Vascular dysfunctions are a common feature of multiple age-related diseases. However, modeling healthy and pathological aging of the human vasculature represents an unresolved experimental challenge. Here, we generated induced vascular endothelial cells (iVECs) and smooth muscle cells (iSMCs) by direct reprogramming of healthy human fibroblasts from donors of different ages and Hutchinson-Gilford Progeria Syndrome (HGPS) patients. iVECs induced from old donors revealed upregulation of GSTM1 and PALD1, genes linked to oxidative stress, inflammation and endothelial junction stability, as vascular aging markers. A functional assay performed on PALD1 KD VECs demonstrated a recovery in vascular permeability. We found that iSMCs from HGPS donors overexpressed bone morphogenetic protein (BMP)-4, which plays a key role in both vascular calcification and endothelial barrier damage observed in HGPS. Strikingly, BMP4 concentrations are higher in serum from HGPS vs. age-matched mice. Furthermore, targeting BMP4 with blocking antibody recovered the functionality of the vascular barrier in vitro, hence representing a potential future therapeutic strategy to limit cardiovascular dysfunction in HGPS. These results show that iVECs and iSMCs retain disease-related signatures, allowing modeling of vascular aging and HGPS in vitro.

Nup93 regulates breast tumor growth by modulating cell proliferation and actin cytoskeleton remodeling.

Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing LIMCH1, a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis.

Selective clearance of the inner nuclear membrane protein emerin by vesicular transport during ER stress.

The inner nuclear membrane (INM) is a subdomain of the endoplasmic reticulum (ER) that is gated by the nuclear pore complex. It is unknown whether proteins of the INM and ER are degraded through shared or distinct pathways in mammalian cells. We applied dynamic proteomics to profile protein half-lives and report that INM and ER residents turn over at similar rates, indicating that the INM's unique topology is not a barrier to turnover. Using a microscopy approach, we observed that the proteasome can degrade INM proteins in situ. However, we also uncovered evidence for selective, vesicular transport-mediated turnover of a single INM protein, emerin, that is potentiated by ER stress. Emerin is rapidly cleared from the INM by a mechanism that requires emerin's LEM domain to mediate vesicular trafficking to lysosomes. This work demonstrates that the INM can be dynamically remodeled in response to environmental inputs.

Predicting age from the transcriptome of human dermal fibroblasts.

Biomarkers of aging can be used to assess the health of individuals and to study aging and age-related diseases. We generate a large dataset of genome-wide RNA-seq profiles of human dermal fibroblasts from 133 people aged 1 to 94 years old to test whether signatures of aging are encoded within the transcriptome. We develop an ensemble machine learning method that predicts age to a median error of 4 years, outperforming previous methods used to predict age. The ensemble was further validated by testing it on ten progeria patients, and our method is the only one that predicts accelerated aging in these patients.

Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes.

Nuclear pore complexes have recently been shown to play roles in gene activation; however their potential involvement in metazoan transcription remains unclear. Here we show that the nucleoporins Sec13, Nup98, and Nup88, as well as a group of FG-repeat nucleoporins, bind to the Drosophila genome at functionally distinct loci that often do not represent nuclear envelope contact sites. Whereas Nup88 localizes to silent loci, Sec13, Nup98, and a subset of FG-repeat nucleoporins bind to developmentally regulated genes undergoing transcription induction. Strikingly, RNAi-mediated knockdown of intranuclear Sec13 and Nup98 specifically inhibits transcription of their target genes and prevents efficient reactivation of transcription after heat shock, suggesting an essential role of NPC components in regulating complex gene expression programs of multicellular organisms.

Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions.

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment.

Inhibition of the activity of SRC and Abl tyrosine protein kinases by the binding of the Wiskott-Aldrich syndrome protein.

The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible.