A combinatorial approach to uncover an additional Integrator subunit.

RNA polymerase II (RNAPII) controls expression of all protein-coding genes and most noncoding loci in higher eukaryotes. Calibrating RNAPII activity requires an assortment of polymerase-associated factors that are recruited at sites of active transcription. The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Integrator is known to be composed of 14 subunits that assemble and operate in a modular fashion. We employed proteomics and machine-learning structure prediction (AlphaFold2) to identify an additional Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS13/14/10 module and interfaces with the Int-PP2A module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes. Our study shows that omics approaches combined with AlphaFold2-based predictions provide additional insights into the molecular architecture of large and dynamic multiprotein complexes.

EGR1 is a gatekeeper of inflammatory enhancers in human macrophages.

Monocytes and monocyte-derived macrophages originate through a multistep differentiation process. First, hematopoietic stem cells generate lineage-restricted progenitors that eventually develop into peripheral, postmitotic monocytes. Second, blood-circulating monocytes undergo differentiation into macrophages, which are specialized phagocytic cells capable of tissue infiltration. While monocytes mediate some level of inflammation and cell toxicity, macrophages boast the widest set of defense mechanisms against pathogens and elicit robust inflammatory responses. Here, we analyze the molecular determinants of monocytic and macrophagic commitment by profiling the EGR1 transcription factor. EGR1 is essential for monopoiesis and binds enhancers that regulate monocytic developmental genes such as CSF1R However, differentiating macrophages present a very different EGR1 binding pattern. We identify novel binding sites of EGR1 at a large set of inflammatory enhancers, even in the absence of its binding motif. We show that EGR1 repressive activity results in suppression of inflammatory genes and is mediated by the NuRD corepressor complex.

Rapid and Scalable Profiling of Nascent RNA with fastGRO.

Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Unlike steady-state RNA-sequencing (RNA-seq), nascent RNA profiling mirrors real-time activity of RNA polymerases and provides an accurate readout of transcriptome-wide variations. Some species of nuclear RNAs (i.e., large intergenic noncoding RNAs [lincRNAs] and eRNAs) have a short half-life and can only be accurately gauged by nascent RNA techniques. Furthermore, nascent RNA-seq detects post-cleavage RNA at termination sites and promoter-associated antisense RNAs, providing insights into RNA polymerase II (RNAPII) dynamics and processivity. Here, we present a run-on assay with 4-thio ribonucleotide (4-S-UTP) labeling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques can. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5 × 106 cells.

Neural progenitors derived from Tuberous Sclerosis Complex patients exhibit attenuated PI3K/AKT signaling and delayed neuronal differentiation.

Tuberous Sclerosis Complex (TSC) is a disease caused by autosomal dominant mutations in the TSC1 or TSC2 genes, and is characterized by tumor susceptibility, brain lesions, seizures and behavioral impairments. The TSC1 and TSC2 genes encode proteins forming a complex (TSC), which is a major regulator and suppressor of mammalian target of rapamycin complex 1 (mTORC1), a signaling complex that promotes cell growth and proliferation. TSC1/2 loss of heterozygosity (LOH) and the subsequent complete loss of TSC regulatory activity in null cells causes mTORC1 dysregulation and TSC-associated brain lesions or other tissue tumors. However, it is not clear whether TSC1/2 heterozygous brain cells are abnormal and contribute to TSC neuropathology. To investigate this issue, we generated induced pluripotent stem cells (iPSCs) from TSC patients and unaffected controls, and utilized these to obtain neural progenitor cells (NPCs) and differentiated neurons in vitro. These patient-derived TSC2 heterozygous NPCs were delayed in their ability to differentiate into neurons. Patient-derived progenitor cells also exhibited a modest activation of mTORC1 signaling downstream of TSC, and a marked attenuation of upstream PI3K/AKT signaling. We further show that pharmacologic PI3K or AKT inhibition, but not mTORC1 inhibition, causes a neuronal differentiation delay, mimicking the patient phenotype. Together these data suggest that heterozygous TSC2 mutations disrupt neuronal development, potentially contributing to the disease neuropathology, and that this defect may result from dysregulated PI3K/AKT signaling in neural progenitor cells.

Role of Akt-independent mTORC1 and GSK3ß signaling in sublethal NMDA-induced injury and the recovery of neuronal electrophysiology and survival.

Glutamate-induced excitotoxicity, mediated by overstimulation of N-methyl-D-aspartate (NMDA) receptors, is a mechanism that causes secondary damage to neurons. The early phase of injury causes loss of dendritic spines and changes to synaptic activity. The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway has been implicated in the modulation and regulation of synaptic strength, activity, maturation, and axonal regeneration. The present study focuses on the physiology and survival of neurons following manipulation of Akt and several downstream targets, such as GSK3ß, FOXO1, and mTORC1, prior to NMDA-induced injury. Our analysis reveals that exposure to sublethal levels of NMDA does not alter phosphorylation of Akt, S6, and GSK3ß at two and twenty four hours following injury. Electrophysiological recordings show that NMDA-induced injury causes a significant decrease in spontaneous excitatory postsynaptic currents at both two and twenty four hours, and this phenotype can be prevented by inhibiting mTORC1 or GSK3ß, but not Akt. Additionally, inhibition of mTORC1 or GSK3ß promotes neuronal survival following NMDA-induced injury. Thus, NMDA-induced excitotoxicity involves a mechanism that requires the permissive activity of mTORC1 and GSK3ß, demonstrating the importance of these kinases in the neuronal response to injury.