The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/ brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

The C-terminal repeat domain (CTD), an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II, serves as a flexible binding scaffold for numerous nuclear factors; which factors bind is determined by the phosphorylation patterns on the CTD repeats. Changes in phosphorylation patterns, as polymerase transcribes a gene, are thought to orchestrate the association of different sets of factors with the transcriptase and strongly influence functional organization of the nucleus. In this review we appraise what is known, and what is not known, about patterns of phosphorylation on the CTD of RNA polymerases II at the beginning, the middle, and the end of genes; the proposal that doubly phosphorylated repeats are present on elongating polymerase is explored. We discuss briefly proteins known to associate with the phosphorylated CTD at the beginning and ends of genes; we explore in more detail proteins that are recruited to the body of genes, the diversity of their functions, and the potential consequences of tethering these functions to elongating RNA polymerase II. We also discuss accumulating structural information on phosphoCTD-binding proteins and how it illustrates the variety of binding domains and interaction modes, emphasizing the structural flexibility of the CTD. We end with a number of open questions that highlight the extent of what remains to be learned about the phosphorylation and functions of the CTD.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knock-out, RNA interference knock-down, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Microglia are resident immune cells of the CNS that are activated by infection, neuronal injury and inflammation. Here we utilize flow cytometry and deep RNA sequencing of acutely isolated spinal cord microglia to define their activation in vivo. Analysis of resting microglia identified 29 genes that distinguish microglia from other CNS cells and peripheral macrophages/monocytes. We then analyzed molecular changes in microglia during neurodegenerative disease activation using the SOD1G93A mouse model of ALS. We find that SOD1G93A microglia are not derived from infiltrating monocytes, and that both potentially neuroprotective and toxic factors are concurrently up-regulated, including Alzheimer’s disease genes. Mutant microglia differed from SOD1WT, LPS activated microglia, and M1/M2 macrophages, that define an ALS-specific phenotype. Concurrent mRNA/FACS analysis revealed post-transcriptional regulation of microglia surface receptors, and T cell-associated changes in the transcriptome. These results provide insights into microglia biology and establish a resource for future studies of neuroinflammation.

Histone methylation and the enzymes that mediate it are important regulators of chromatin structure and gene transcription. In particular, the histone H3 lysine 36 (K36) methyltransferase Set2 has recently been shown to associate with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), implying that this enzyme has an important role in the transcription elongation process. Here we show that a novel domain in the C terminus of Set2 is responsible for interaction between Set2 and RNAPII. This domain, termed the Set2 Rpb1 interacting (SRI) domain, is encompassed by amino acid residues 619 to 718 in Set2 and is found to occur in a number of putative Set2 homologs from Schizosaccharomyces pombe to humans. Unexpectedly, BIACORE analysis reveals that the SRI domain binds specifically, and with high affinity, to CTD repeats that are doubly modified (serine 2 and serine 5 phosphorylated), indicating that Set2 association across the body of genes requires a specific pattern of phosphorylated RNAPII. Deletion of the SRI domain not only abolishes Set2-RNAPII interaction but also abolishes K36 methylation in vivo, indicating that this interaction is required for establishing K36 methylation on chromatin. Using 6-azauracil (6AU) as an indicator of transcription elongation defects, we found that deletion of the SRI domain conferred a strong resistance to this compound, which was identical to that observed with set2 deletion mutants. Furthermore, yeast strains carrying set2 alleles that are catalytically inactive or yeast strains bearing point mutations at K36 were also found to be resistant to 6AU. These data suggest that it is the methylation by Set2 that affects transcription elongation. In agreement with this, we have determined that deletion of SET2, its SRI domain, or amino acid substitutions at K36 result in an alteration of RNAPII occupancy levels over transcribing genes. Taken together, these data indicate K36 methylation, established by the SRI domain-mediated association of Set2 with RNAPII, plays an important role in the transcription elongation process.Successful synthesis of mRNA by RNA polymerase II (RNAPII) requires tight regulation of the initiation, elongation, and termination processes of transcription. The process of transcription elongation is affected in part by the binding of regulatory factors to the phosphorylated C-terminal domain (CTD) of the RNAPII subunit Rpb1. Recent studies have highlighted an important role for histone methylation in the process of transcription elongation. In particular, studies have shown that the Set1 and Set2 methyltransferases, which catalyze methylation of histone H3 lysines 4 (K4) and 36 (K36), respectively, are associated with RNAPII at different stages of the transcription elongation cycle. While Set1 associates with RNAPII via the Paf1 transcription elongation complex in a manner that is dependent on the serine 5 (Ser5)-phosphorylated CTD, Set2 is recruited to RNAPII in a manner that is dependent on the CTD and the Ctk1 kinase (CTDK-I) that effects...

Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 59 end and higher in the middle and at the 39 end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1 ts mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 4 CDK9 and Ctk1 4 CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1. The C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) is an intrinsically unstructured extension of the enzyme's largest subunit, RPB1. The CTD is composed of a tandem array of seven amino acid repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ; the number of heptad repeats varies from organism to organism and appears to correlate with genomic complexity (Corden 1990). Although dispensable for polymerase activity in vitro, the CTD is conserved throughout evolution and is essential to life (Zehring et al. 1988). The CTD's function is to coordinate transcription with nuclear processes such as mRNA processing and chromatin modification; it fulfills this role by serving as a selective binding scaffold for nuclear factors (for review, see Phatnani and Greenleaf 2006

SummaryGrowing evidence implicates the importance of glia, particularly astrocytes, in neurological and psychiatric diseases. Here, we describe a rapid and robust method for the differentiation of highly pure populations of replicative astrocytes from human induced pluripotent stem cells (hiPSCs), via a neural progenitor cell (NPC) intermediate. We evaluated this protocol across 42 NPC lines (derived from 30 individuals). Transcriptomic analysis demonstrated that hiPSC-astrocytes from four individuals are highly similar to primary human fetal astrocytes and characteristic of a non-reactive state. hiPSC-astrocytes respond to inflammatory stimulants, display phagocytic capacity, and enhance microglial phagocytosis. hiPSC-astrocytes also possess spontaneous calcium transient activity. Our protocol is a reproducible, straightforward (single medium), and rapid (<30 days) method to generate populations of hiPSC-astrocytes that can be used for neuron-astrocyte and microglia-astrocyte co-cultures for the study of neuropsychiatric disorders.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.