Integrative dissection of gene regulatory elements at base resolution.

Although vast numbers of putative gene regulatory elements have been cataloged, the sequence motifs and individual bases that underlie their functions remain largely unknown. Here, we combine epigenetic perturbations, base editing, and deep learning to dissect regulatory sequences within the exemplar immune locus encoding CD69. We converge on a ∼170 base interval within a differentially accessible and acetylated enhancer critical for CD69 induction in stimulated Jurkat T cells.

Chromatin complex dependencies reveal targeting opportunities in leukemia.

Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs.

Long-range phasing of dynamic, tissue-specific and allele-specific regulatory elements.

Epigenomic maps identify gene regulatory elements by their chromatin state. However, prevailing short-read sequencing methods cannot effectively distinguish alleles, evaluate the interdependence of elements in a locus or capture single-molecule dynamics. Here, we apply targeted nanopore sequencing to profile chromatin accessibility and DNA methylation on contiguous ~100-kb DNA molecules that span loci relevant to development, immunity and imprinting.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect.

Diverse Chemical Scaffolds Enhance Oligodendrocyte Formation by Inhibiting CYP51, TM7SF2, or EBP.

Small molecules that promote oligodendrocyte formation have been identified in "drug repurposing" screens to nominate candidate therapeutics for diseases in which myelin is lost, including multiple sclerosis. We recently reported that many such molecules enhance oligodendrocyte formation not by their canonical targets but by inhibiting a narrow range of enzymes in cholesterol biosynthesis. Here we identify enhancers of oligodendrocyte formation obtained by screening a structurally diverse library of 10,000 small molecules.

Orthologous CRISPR-Cas9 enzymes for combinatorial genetic screens.

Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes.

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest.

Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains.

Adaptive Chromatin Remodeling Drives Glioblastoma Stem Cell Plasticity and Drug Tolerance.

Glioblastoma, the most common and aggressive malignant brain tumor, is propagated by stem-like cancer cells refractory to existing therapies. Understanding the molecular mechanisms that control glioblastoma stem cell (GSC) proliferation and drug resistance may reveal opportunities for therapeutic interventions. Here we show that GSCs can reversibly transition to a slow-cycling, persistent state in response to targeted kinase inhibitors.

Depletion of Olig2 in oligodendrocyte progenitor cells infected by Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis virus (TMEV) infects the central nervous system of mice and causes a demyelinating disease that is a model for multiple sclerosis. During the chronic phase of the disease, TMEV persists in oligodendrocytes and macrophages. Lack of remyelination has been attributed to insufficient proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), but the molecular mechanisms remain unknown.

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo.

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes.

NPTX1 regulates neural lineage specification from human pluripotent stem cells.

Neural induction is the first fundamental step in nervous system formation. During development, a tightly regulated niche modulates transient extracellular signals to influence neural lineage commitment. To date, however, the cascade of molecular events that sustain these signals in humans is not well understood.

Generation and characterization of epiblast stem cells from blastocyst-stage mouse embryos.

Mouse epiblast stem cells (EpiSCs) are pluripotent embryonic cells that can be used to interrogate developmental transitions that occur during gastrulation. EpiSCs can also be robustly differentiated into functional somatic and germ cell derivatives making them a useful tool for studying development and regenerative medicine. Typically, mouse EpiSCs are isolated from the early postimplantation epiblast around 5.

Transcription factor-mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells.

Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to induced oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs.

Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells.

Myelin-related disorders such as multiple sclerosis and leukodystrophies, for which restoration of oligodendrocyte function would be an effective treatment, are poised to benefit greatly from stem cell biology. Progress in myelin repair has been constrained by difficulties in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs, but current differentiation strategies are poorly reproducible and generate heterogenous populations of cells.

Isolation of epiblast stem cells from preimplantation mouse embryos.

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively.