Targeted chemotherapy overcomes drug resistance in melanoma.

The emergence of drug resistance is a major obstacle for the success of targeted therapy in melanoma. Additionally, conventional chemotherapy has not been effective as drug-resistant cells escape lethal DNA damage effects by inducing growth arrest commonly referred to as cellular dormancy. We present a therapeutic strategy termed "targeted chemotherapy" by depleting protein phosphatase 2A (PP2A) or its inhibition using a small molecule inhibitor (1,10-phenanthroline-5,6-dione [phendione]) in drug-resistant melanoma. Targeted chemotherapy induces the DNA damage response without causing DNA breaks or allowing cellular dormancy. Phendione treatment reduces tumor growth of BRAFV600E-driven melanoma patient-derived xenografts (PDX) and diminishes growth of NRASQ61R-driven melanoma, a cancer with no effective therapy. Remarkably, phendione treatment inhibits the acquisition of resistance to BRAF inhibition in BRAFV600E PDX highlighting its effectiveness in combating the advent of drug resistance.

Neuronal differentiation requires BRAT1 complex to remove REST from chromatin.

Repressor element-1 silencing transcription factor (REST) is required for the formation of mature neurons. REST dysregulation underlies a key mechanism of neurodegeneration associated with neurological disorders. However, the mechanisms leading to alterations of REST-mediated silencing of key neurogenesis genes are not known. Here, we show that BRCA1 Associated ATM Activator 1 (BRAT1), a gene linked to neurodegenerative diseases, is required for the activation of REST-responsive genes during neuronal differentiation. We find that INTS11 and INTS9 subunits of Integrator complex interact with BRAT1 as a distinct trimeric complex to activate critical neuronal genes during differentiation. BRAT1 depletion results in persistence of REST residence on critical neuronal genes disrupting the differentiation of NT2 cells into astrocytes and neuronal cells. We identified BRAT1 and INTS11 co-occupying the promoter region of these genes and pinpoint a role for BRAT1 in recruiting INTS11 to their promoters. Disease-causing mutations in BRAT1 diminish its association with INTS11/INTS9, linking the manifestation of disease phenotypes with a defect in transcriptional activation of key neuronal genes by BRAT1/INTS11/INTS9 complex. Finally, loss of Brat1 in mouse embryonic stem cells leads to a defect in neuronal differentiation assay. Importantly, while reconstitution with wild-type BRAT1 restores neuronal differentiation, the addition of a BRAT1 mutant is unable to associate with INTS11/INTS9 and fails to rescue the neuronal phenotype. Taken together, our study highlights the importance of BRAT1 association with INTS11 and INTS9 in the development of the nervous system.

Predicted dynamical couplings of protein residues characterize catalysis, transport and allostery.

MOTIVATION: Protein function is intrinsically linked to native dynamics, but the systematic characterization of functionally relevant dynamics remains elusive besides specific examples. Here we exhaustively characterize three types of dynamical couplings between protein residues: co-directionality (moving along collinear directions), coordination (small fluctuations of the interatomic distance) and deformation (the extent by which perturbations applied at one residue modify the local structure of the other one), which we analytically compute through the torsional network model. RESULTS: We find that ligand binding sites are characterized by large within-site coordination and co-directionality, much larger than expected for generic sets of residues with equivalent sequence distances. In addition, catalytic sites are characterized by high coordination couplings with other residues in the protein, supporting the view that the overall protein structure facilitates the catalytic dynamics. The binding sites of allosteric effectors are characterized by comparably smaller coordination and higher within-site deformation than other ligands, which supports their dynamic nature. Allosteric inhibitors are coupled to the active site more frequently through deformation than through coordination, while the contrary holds for activators. We characterize the dynamical couplings of the sodium-dependent Leucine transporter protein (LeuT). The couplings between and within sites progress consistently along the transport cycle, providing a mechanistic description of the coupling between the uptake and release of ions and substrate, and they highlight qualitative differences between the wild-type and a mutant for which chloride is necessary for transport. AVAILABILITY AND IMPLEMENTATION: The program tnm is freely available at https://github.com/ugobas/tnm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BRAT1 associates with INTS11/INTS9 heterodimer to regulate key neurodevelopmental genes.

Integrator is a multi-subunits protein complex involved in regulation of gene expression. Several Integrator subunits have been found to be mutated in human neurodevelopmental disorders, suggesting a key role for the complex in the development of nervous system. BRAT1 is similarly linked with neurodegenerative diseases and neurodevelopmental disorders such as rigidity and multifocal-seizure syndrome. Here, we show that INTS11 and INTS9 subunits of Integrator complex interact with BRAT1 and form a trimeric complex in human HEK293T cells as well as in pluripotent human embryonal carcinoma cell line (NT2). We find that BRAT1 depletion disrupts the differentiation of NT2 cells into astrocytes and neural cells. Loss of BRAT1 results in inability to activate many neuronal genes that are targets of REST, a neuronal silencer. We identified BRAT1 and INTS11 co-occupying the promoter region of these genes and pinpoint a role for BRAT1 in recruiting INTS11 to their promoters. Disease-causing mutations in BRAT1 diminish its association with INTS11/INTS9, linking the manifestation of disease phenotypes with a defect in transcriptional activation of key neuronal genes by BRAT1/INTS11/INTS9 complex. Highlights: Integrator subunits INTS9 and INTS11 tightly interact with BRAT1 Depletion of BRAT1 causes a dramatic delay in human neural differentiation BRAT1 and INTS11 module targets the promoters of neural marker genes and co-regulates their expression. The recruitment of INTS11 to these sites is BRAT1-dependent. Pathogenic E522K mutation in BRAT1 disrupts its interaction with INTS11/INTS9 heterodimer.

The Integrator complex regulates microRNA abundance through RISC loading.

MicroRNA (miRNA) homeostasis is crucial for the posttranscriptional regulation of their target genes during development and in disease states. miRNAs are derived from primary transcripts and are processed from a hairpin precursor intermediary to a mature 22-nucleotide duplex RNA. Loading of the duplex into the Argonaute (AGO) protein family is pivotal to miRNA abundance and its posttranscriptional function. The Integrator complex plays a key role in protein coding and noncoding RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global destabilization of mature miRNAs. Enhanced ultraviolet cross-linking and immunoprecipitation of Integrator uncovered an association with duplex miRNAs before their loading onto AGOs. Tracing miRNA fate from biogenesis to stabilization by incorporating 4-thiouridine in nascent transcripts pinpointed a critical role for Integrator in miRNA assembly into AGOs.

The Integrator complex at the crossroad of coding and noncoding RNA.

Genomic transcription is fundamental to all organisms. In metazoans, the Integrator complex is required for endonucleolytic processing of noncoding RNAs, regulation of RNA polymerase II pause-release, and premature transcription attenuation at coding genes. Recent insights into the structural composition and evolution of Integrator subunits have informed our understanding of its biochemical functionality. Moreover, studies in multiple model organisms point to an essential function of Integrator in signaling response and cellular development, highlighting a key role in neuronal differentiation. Indeed, alterations in Integrator complex subunits have been identified in patients with neurodevelopmental diseases and cancer. Taken together, we propose that Integrator is a central regulator of transcriptional processes and that its evolution reflects genomic complexity in regulatory elements and chromatin architecture.

Integrator enforces the fidelity of transcriptional termination at protein-coding genes.

Integrator regulates the 3'-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3'-end processing and termination of a set of protein-coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de novo trimethylation of lysine-36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex beyond the canonical polyadenylation sites. 3' RNA sequencing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes features downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3'UTR. Together, this study identifies a subset of protein-coding transcripts whose 3' end processing requires the Integrator complex.