Dual genome-wide coding and lncRNA screens in neural induction of induced pluripotent stem cells.

Human chromosomes are pervasively transcribed, but systematic understanding of coding and lncRNA genome function in cell differentiation is lacking. Using CRISPR interference (CRISPRi) in human induced pluripotent stem cells, we performed dual genome-wide screens - assessing 18,905 protein-coding and 10,678 lncRNA loci - and identified 419 coding and 201 lncRNA genes that regulate neural induction. Integrative analyses revealed distinct properties of coding and lncRNA genome function, including a 10-fold enrichment of lncRNA genes for roles in differentiation compared to proliferation. Further, we applied Perturb-seq to obtain granular insights into neural induction phenotypes. While most coding hits stalled or aborted differentiation, lncRNA hits were enriched for the genesis of diverse cellular states, including those outside the neural lineage. In addition to providing a rich resource (danlimlab.shinyapps.io/dualgenomewide) for understanding coding and lncRNA gene function in development, these results indicate that the lncRNA genome regulates lineage commitment in a manner fundamentally distinct from coding genes.

Universal prediction of cell-cycle position using transfer learning.

BACKGROUND: The cell cycle is a highly conserved, continuous process which controls faithful replication and division of cells. Single-cell technologies have enabled increasingly precise measurements of the cell cycle both as a biological process of interest and as a possible confounding factor. Despite its importance and conservation, there is no universally applicable approach to infer position in the cell cycle with high-resolution from single-cell RNA-seq data. RESULTS: Here, we present tricycle, an R/Bioconductor package, to address this challenge by leveraging key features of the biology of the cell cycle, the mathematical properties of principal component analysis of periodic functions, and the use of transfer learning. We estimate a cell-cycle embedding using a fixed reference dataset and project new data into this reference embedding, an approach that overcomes key limitations of learning a dataset-dependent embedding. Tricycle then predicts a cell-specific position in the cell cycle based on the data projection. The accuracy of tricycle compares favorably to gold-standard experimental assays, which generally require specialized measurements in specifically constructed in vitro systems. Using internal controls which are available for any dataset, we show that tricycle predictions generalize to datasets with multiple cell types, across tissues, species, and even sequencing assays. CONCLUSIONS: Tricycle generalizes across datasets and is highly scalable and applicable to atlas-level single-cell RNA-seq data.

High-altitude populations need special considerations for COVID-19.

The atmospheric pressure that decreases with altitude affects lung physiology. However, these changes in physiology are not usually considered in ventilator design and testing. We argue that high altitude human populations require special attention to access the international supply of ventilators.

Precocious neuronal differentiation and disrupted oxygen responses in Kabuki syndrome.

Chromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.

A ketogenic diet rescues hippocampal memory defects in a mouse model of Kabuki syndrome.

Kabuki syndrome is a Mendelian intellectual disability syndrome caused by mutations in either of two genes (KMT2D and KDM6A) involved in chromatin accessibility. We previously showed that an agent that promotes chromatin opening, the histone deacetylase inhibitor (HDACi) AR-42, ameliorates the deficiency of adult neurogenesis in the granule cell layer of the dentate gyrus and rescues hippocampal memory defects in a mouse model of Kabuki syndrome (Kmt2d+/ßGeo). Unlike a drug, a dietary intervention could be quickly transitioned to the clinic. Therefore, we have explored whether treatment with a ketogenic diet could lead to a similar rescue through increased amounts of beta-hydroxybutyrate, an endogenous HDACi. Here, we report that a ketogenic diet in Kmt2d+/ßGeo mice modulates H3ac and H3K4me3 in the granule cell layer, with concomitant rescue of both the neurogenesis defect and hippocampal memory abnormalities seen in Kmt2d+/ßGeo mice; similar effects on neurogenesis were observed on exogenous administration of beta-hydroxybutyrate. These data suggest that dietary modulation of epigenetic modifications through elevation of beta-hydroxybutyrate may provide a feasible strategy to treat the intellectual disability seen in Kabuki syndrome and related disorders.

More than hypomyelination in Pol-III disorder.

The 4H syndrome (hypomyelination, hypodontia, hypogonadotropic hypogonadism) is a newly recognized leukodystrophy. The classical form is characterized by the association of hypomyelination, abnormal dentition, and hypogonadotropic hypogonadism, but the recent identification of 2 genes responsible for the syndrome demonstrates that these 3 main characteristics can be variably combined among "Pol-III (polymerase III)-related leukodystrophies." The pathophysiology of this group of diseases is still to be elucidated, and there are no neuropathologic descriptions of brain tissue. We report the clinical, neuroradiologic, and neuropathologic findings of a patient affected by 4H syndrome with confirmed POLR3A mutations. We found a marked loss of oligodendrocytes, varying in severity in different brain regions, and accompanied by severe loss of myelin, moderately severe loss of axons, and patchy perivascular regions of better preserved white matter. There was relatively mild white matter astrogliosis and microgliosis. A macrophage reaction involving viable normal-appearing oligodendroglia suggests the possibility of an immunologic process in this disorder. Cortical laminar astrogliosis and mineralization of Layers I and II in particular were present. Thus, despite the uniformly hypomyelinating pattern seen on magnetic resonance imaging, neuropathologic examination reveals a complex heterogeneous leukodystrophy with prominent neuroaxonal and glial involvement in this disorder.

Extended access to cocaine self-administration results in reduced glutamate function within the medial prefrontal cortex.

Previous studies have shown that brief access to cocaine yields an increase in D2 receptor binding in the medial prefrontal cortex (mPFC), but that extended access to cocaine results in normalized binding of D2 receptors (i.e. the D2 binding returned to control levels). Extended-access conditions have also been shown to produce increased expression of the NR2 subunit of the N-Methyl-D-aspartate receptor in the mPFC. These results implicate disrupted glutamate and dopamine function within this area. Therefore, in the present study, we monitored glutamate and dopamine content within the mPFC during, or 24 hours after, cocaine self-administration in animals that experienced various amounts of exposure to the drug. Naïve subjects showed decreased glutamate and increased dopamine levels within the mPFC during cocaine self-administration. Exposure to seven 1-hour daily cocaine self-administration sessions did not alter the response to self-administered cocaine, but resulted in decreased basal dopamine levels. While exposure to 17 1-hour sessions also resulted in reduced basal dopamine levels, these animals showed increased dopaminergic, but completely diminished glutamatergic, response to self-administered cocaine. Finally, exposure to 17 cocaine self-administration sessions, the last 10 of which being 6-hour sessions, resulted in diminished glutamatergic response to self-administered cocaine and reduced basal glutamate levels within the mPFC while normalizing (i.e. causing a return to control levels) both the dopaminergic response to self-administered cocaine as well as basal dopamine levels within this area. These data demonstrate directly that the transition to escalated cocaine use involves progressive changes in dopamine and glutamate function within the mPFC.

Mutations of POLR3A encoding a catalytic subunit of RNA polymerase Pol III cause a recessive hypomyelinating leukodystrophy.

Leukodystrophies are a heterogeneous group of inherited neurodegenerative disorders characterized by abnormal white matter visible by brain imaging. It is estimated that at least 30% to 40% of individuals remain without a precise diagnosis despite extensive investigations. We mapped tremor-ataxia with central hypomyelination (TACH) to 10q22.3-23.1 in French-Canadian families and sequenced candidate genes within this interval. Two missense and one insertion mutations in five individuals with TACH were uncovered in POLR3A, which codes for the largest subunit of RNA polymerase III (Pol III). Because these families were mapped to the same locus as leukodystrophy with oligodontia (LO) and presented clinical and radiological overlap with individuals with hypomyelination, hypodontia and hypogonadotropic hypogonadism (4H) syndrome, we sequenced this gene in nine individuals with 4H and eight with LO. In total, 14 recessive mutations were found in 19 individuals with TACH, 4H, or LO, establishing that these leukodystrophies are allelic. No individual was found to carry two nonsense mutations. Immunoblots on 4H fibroblasts and on the autopsied brain of an individual diagnosed with 4H documented a significant decrease in POLR3A levels, and there was a more significant decrease in the cerebral white matter compared to that in the cortex. Pol III has a wide set of target RNA transcripts, including all nuclear-coded tRNA. We hypothesize that the decrease in POLR3A leads to dysregulation of the expression of certain Pol III targets and thereby perturbs cytoplasmic protein synthesis. This type of broad alteration in protein synthesis is predicted to occur in other leukoencephalopathies such as hypomyelinating leukodystrophy-3, caused by mutations in aminoacyl-tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1).