Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patients after diagnosis is ~1 yr. Here we show that the levels of H3K27 di-and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, we also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransfer-ase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover , the gain of H3K27me3 and Ezh2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.

Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Perlecan, a modular proteoglycan carrying primary heparan sulfate (HS) side chains, is a major component of blood vessel basement membranes. It sequesters growth factors such as fibroblast growth factor 2 (FGF-2) and regulates the ligand-receptor interactions on the cell surface, and thus it has been implicated in the control of angiogenesis. Both stimulatory and inhibitory effects of perlecan on FGF-2 signaling have been reported. To understand the in vivo function of HS carried by perlecan, the perlecan gene heparan sulfate proteoglycan 2 (Hspg2) was mutated in mouse by gene targeting. The HS at the NH 2 terminus of perlecan was removed while the core protein remained intact. Perlecan HS-deficient (Hspg2 ⌬3/⌬3 ) mice survived embryonic development and were apparently healthy as adults. However, mutant mice exhibited significantly delayed wound healing, retarded FGF-2-induced tumor growth, and defective angiogenesis. In the mouse corneal angiogenesis model, FGF-2-induced neovascularization was significantly impaired in Hspg2 ⌬3/⌬3 mutant mice. Our results suggest that HS in perlecan positively regulates the angiogenesis in vivo.

MMP14 encodes a membrane-tethered metalloproteinase MT1-MMP, capable of remodeling the extracellular matrix and modulating receptors on the cell surface. Loss of MT1-MMP results in craniofacial abnormalities. Here we show that MT1-MMP forms a complex with FGFR2 and ADAM9 in osteoblasts and proteolytically inactivates ADAM9, hence protecting FGFR2 from ADAM9-mediated ectodomain shedding on the cell surface. In Mmp14-/- osteoblasts, FGF-induced proliferation and downstream signaling are specifically compromised, in conjunction with ADAM9 upregulation and FGFR2 shedding. The retarded parietal growth in Mmp14-/- embryos starts at 15.5 dpc, attributable to the impaired FGFR2 signaling due to increased shedding mediated by ADAM9. Adam9 depletion completely rescues the defective FGFR2 signaling and largely restores calvarial bone growth in Mmp14-/- embryos. These data reveal a regulatory paradigm for FGRF2 signaling and identify MT1-MMP as a critical negative modulator of ADAM9 activity to maintain FGFR2 signaling in calvarial osteogenesis.

Toll-like receptor (TLR) signaling induces a rapid reorganization of the actin cytoskeleton in cultured mouse dendritic cells (DC), leading to enhanced antigen endocytosis and a concomitant loss of filamentous actin–rich podosomes. We show that as podosomes are lost, TLR signaling induces prominent focal contacts and a transient reduction in DC migratory capacity in vitro. We further show that podosomes in mouse DC are foci of pronounced gelatinase activity, dependent on the enzyme membrane type I matrix metalloprotease (MT1-MMP), and that DC transiently lose the ability to degrade the extracellular matrix after TLR signaling. Surprisingly, MMP inhibitors block TLR signaling–induced podosome disassembly, although stimulated endocytosis is unaffected, which demonstrates that the two phenomena are not obligatorily coupled. Podosome disassembly caused by TLR signaling occurs normally in DC lacking MT1-MMP, and instead requires the tumor necrosis factor α–converting enzyme ADAM17 (a disintegrin and metalloprotease 17), which demonstrates a novel role for this “sheddase” in regulating an actin-based structure.

Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA-binding proteins either rely on crosslinking-mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture e ciency. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of speci c lncRNAs in their native cellular context followed by LC-MS/MS identi cation. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.

Breast cancer is a heterogeneous disease, affecting over 3.5 million women worldwide, yet the functional role of cis-regulatory elements including super-enhancers in different breast cancer subtypes remains poorly characterized. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with a poor prognosis. Here we apply integrated epigenomic and transcriptomic profiling to uncover super-enhancer heterogeneity between breast cancer subtypes, and provide clinically relevant biological insights towards TNBC. Using CRISPR/Cas9-mediated gene editing, we identify genes that are specifically regulated by TNBC-specific super-enhancers, including FOXC1 and MET, thereby unveiling a mechanism for specific overexpression of the key oncogenes in TNBC. We also identify ANLN as a TNBC-specific gene regulated by super-enhancer. Our studies reveal a TNBC-specific epigenomic landscape, contributing to the dysregulated oncogene expression in breast tumorigenesis.

Tumor cells use membrane type 1 matrix metalloproteinase (MT1-MMP) for invasion and metastasis. However, the signaling mechanisms that underlie MT1-MMP regulation in cancer have remained unclear. Using a systematic gain-of-function kinome screen for MT1-MMP activity, we have here identified kinases that significantly enhance MT1-MMP activity in tumor cells. In particular, we discovered an MT1-MMP/FGF receptor-4 (FGFR4) membrane complex that either stimulates or suppresses MT1-MMP and FGFR4 activities, depending on a tumor progression-associated polymorphism in FGFR4. The FGFR4-R388 allele, linked to poor cancer prognosis, increased collagen invasion by decreasing lysosomal MT1-MMP degradation. FGFR4-R388 induced MT1-MMP phosphorylation and endosomal stabilization, and surprisingly, the increased MT1-MMP in return enhanced FGFR4-R388 autophosphorylation. A phosphorylation-defective MT1-MMP was stabilized on the cell surface, where it induced simultaneous FGFR4-R388 internalization and dissociation of cell-cell junctions. In contrast, the alternative FGFR4-G388 variant downregulated MT1-MMP, and the overexpression of MT1-MMP and particularly its phosphorylation-defective mutant vice versa induced FGFR4-G388 degradation. These results provide a mechanistic basis for FGFR4-R388 function in cancer invasion.