Co-transcriptional splicing facilitates transcription of gigantic genes.

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing.

MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons.

Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown, and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed an unbiased, high-throughput screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we found that therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies.

A maternally programmed intergenerational mechanism enables male offspring to make piRNAs from Y-linked precursor RNAs in Drosophila.

In animals, PIWI-interacting RNAs (piRNAs) direct PIWI proteins to silence complementary targets such as transposons. In Drosophila and other species with a maternally specified germline, piRNAs deposited in the egg initiate piRNA biogenesis in the progeny. However, Y chromosome loci cannot participate in such a chain of intergenerational inheritance.

SARS-CoV-2 infection of human pluripotent stem cell-derived vascular cells reveals smooth muscle cells as key mediators of vascular pathology during infection.

Although respiratory symptoms are the most prevalent disease manifestation of infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), nearly 20% of hospitalized patients are at risk for thromboembolic events. This prothrombotic state is considered a key factor in the increased risk of stroke, which is observed clinically during both acute infection and long after symptoms clear. Here we develop a model of SARS-CoV-2 infection using human-induced pluripotent stem cell-derived endothelial cells (ECs), pericytes (PCs), and smooth muscle cells (SMCs) to recapitulate the vascular pathology associated with SARS-CoV-2 exposure.

Targeted therapies prime oncogene-driven lung cancers for macrophage-mediated destruction.

Macrophage immune checkpoint inhibitors, such as anti-CD47 antibodies, show promise in clinical trials for solid and hematologic malignancies. However, the best strategies to use these therapies remain unknown and ongoing studies suggest they may be most effective when used in combination with other anticancer agents. Here, we developed a novel screening platform to identify drugs that render lung cancer cells more vulnerable to macrophage attack, and we identified therapeutic synergy exists between genotype-directed therapies and anti-CD47 antibodies.

Fragile X Syndrome Patient-Derived Neurons Developing in the Mouse Brain Show FMR1-Dependent Phenotypes.

Background: Fragile X syndrome (FXS) is characterized by physical abnormalities, anxiety, intellectual disability, hyperactivity, autistic behaviors, and seizures. Abnormal neuronal development in FXS is poorly understood. Data on patients with FXS remain scarce, and FXS animal models have failed to yield successful therapies.

Characterizing Protein-Ligand Binding Using Atomistic Simulation and Machine Learning: Application to Drug Resistance in HIV-1 Protease.

Over the past several decades, atomistic simulations of biomolecules, whether carried out using molecular dynamics or Monte Carlo techniques, have provided detailed insights into their function. Comparing the results of such simulations for a few closely related systems has guided our understanding of the mechanisms by which changes such as ligand binding or mutation can alter the function. The general problem of detecting and interpreting such mechanisms from simulations of many related systems, however, remains a challenge.

Molecular Determinants of Epistasis in HIV-1 Protease: Elucidating the Interdependence of L89V and L90M Mutations in Resistance.

Protease inhibitors have the highest potency among antiviral therapies against HIV-1 infections, yet the virus can evolve resistance. Darunavir (DRV), currently the most potent Food and Drug Administration-approved protease inhibitor, retains potency against single-site mutations. However, complex combinations of mutations can confer resistance to DRV.

Constrained Mutational Sampling of Amino Acids in HIV-1 Protease Evolution.

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease.

Synonymous Mutations at the Beginning of the Influenza A Virus Hemagglutinin Gene Impact Experimental Fitness.

The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones.

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness.

Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bodies.

This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies.

Chromatin dynamics regulate mesenchymal stem cell lineage specification and differentiation to osteogenesis.

Multipotent mesenchymal stromal cells (MSCs) are critical for regeneration of multiple tissues. Epigenetic mechanisms are fundamental regulators of lineage specification and cell fate, and as such, we addressed the question of which epigenetic modifications characterize the transition of nascent MSCs to a tissue specific MSC-derived phenotype. By profiling the temporal changes of seven histone marks correlated to gene expression during proliferation, early commitment, matrix deposition, and mineralization stages, we identified distinct epigenetic mechanisms that regulate transcriptional programs necessary for tissue-specific phenotype development.

Structural Basis of the Disorder in the Tandem Zinc Finger Domain of the RNA-Binding Protein Tristetraprolin.

Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d.

Genome-Wide Studies Reveal that H3K4me3 Modification in Bivalent Genes Is Dynamically Regulated during the Pluripotent Cell Cycle and Stabilized upon Differentiation.

Stem cell phenotypes are reflected by posttranslational histone modifications, and this chromatin-related memory must be mitotically inherited to maintain cell identity through proliferative expansion. In human embryonic stem cells (hESCs), bivalent genes with both activating (H3K4me3) and repressive (H3K27me3) histone modifications are essential to sustain pluripotency. Yet, the molecular mechanisms by which this epigenetic landscape is transferred to progeny cells remain to be established.

Genome-wide co-occupancy of AML1-ETO and N-CoR defines the t(8;21) AML signature in leukemic cells.

Background: Many leukemias result from chromosomal rearrangements. The t(8;21) chromosomal translocation produces AML1-ETO, an oncogenic fusion protein that compromises the function of AML1, a transcription factor critical for myeloid cell differentiation. Because of the pressing need for new therapies in the treatment of acute myleoid leukemia, we investigated the genome-wide occupancy of AML1-ETO in leukemic cells to discover novel regulatory mechanisms involving AML-ETO bound genes.

The bone-specific Runx2-P1 promoter displays conserved three-dimensional chromatin structure with the syntenic Supt3h promoter.

Three-dimensional organization of chromatin is fundamental for transcriptional regulation. Tissue-specific transcriptional programs are orchestrated by transcription factors and epigenetic regulators. The RUNX2 transcription factor is required for differentiation of precursor cells into mature osteoblasts.

Epigenetic landscape during osteoblastogenesis defines a differentiation-dependent Runx2 promoter region.

Runx2 is a developmentally regulated gene in vertebrates and is essential for bone formation and skeletal homeostasis. The induction of runx2-P1 isoform transcripts is a hallmark of early osteoblastogenesis. Although previous in vitro studies have defined a minimal Runx2-P1 promoter sequence with well-characterized functional elements, several lines of evidence suggest that transcription of the Runx2-P1 isoform relies on elements that extend beyond the previously defined P1 promoter boundaries.

Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis.

Background: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed.

Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has become the dominant technique for mapping transcription factor (TF) binding regions genome-wide. We performed an integrative analysis centered around 457 ChIP-seq data sets on 119 human TFs generated by the ENCODE Consortium. We identified highly enriched sequence motifs in most data sets, revealing new motifs and validating known ones.

Functional analysis of transcription factor binding sites in human promoters.

Background: The binding of transcription factors to specific locations in the genome is integral to the orchestration of transcriptional regulation in cells. To characterize transcription factor binding site function on a large scale, we predicted and mutagenized 455 binding sites in human promoters. We carried out functional tests on these sites in four different immortalized human cell lines using transient transfections with a luciferase reporter assay, primarily for the transcription factors CTCF, GABP, GATA2, E2F, STAT, and YY1.

A machine learning approach for the prediction of protein surface loop flexibility.

Proteins often undergo conformational changes when binding to each other. A major fraction of backbone conformational changes involves motion on the protein surface, particularly in loops. Accounting for the motion of protein surface loops represents a challenge for protein-protein docking algorithms.

A combined experimental and theoretical study of ion solvation in liquid N-methylacetamide.

Most current biomolecular simulations are based on potential energy functions that treat the electrostatic energy as a sum of pairwise Coulombic interactions between effective fixed atomic charges. This approximation, in which many-body induced polarization effects are included in an average way, is expected to be satisfactory for a wide range of systems, but less accurate for processes involving the transfer and partition of ions among heterogeneous environments. The limitations of these potential energy functions are perhaps most obvious in studies of ion permeation through membrane channels.

Simulating Monovalent and Divalent Ions in Aqueous Solution Using a Drude Polarizable Force Field.

An accurate representation of ion solvation in aqueous solution is critical for meaningful computer simulations of a broad range of physical and biological processes. Polarizable models based on classical Drude oscillators are introduced and parametrized for a large set of monoatomic ions including cations of the alkali metals (Li(+), Na(+), K(+), Rb(+) and Cs(+)) and alkaline earth elements (Mg(2+), Ca(2+), Sr(2+) and Ba(2+)) along with Zn(2+) and halide anions (F(-), Cl(-), Br(-) and I(-)). The models are parameterized, in conjunction with the polarizable SWM4-NDP water model [Lamoureux et al.