Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats ...(CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.
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IJS, NUK, SBMB, UL, UM, UPUK
The role of non-neuronal cells in Alzheimer's disease progression has not been fully elucidated. Using single-nucleus RNA sequencing, we identified a population of disease-associated astrocytes in an ...Alzheimer's disease mouse model. These disease-associated astrocytes appeared at early disease stages and increased in abundance with disease progression. We discovered that similar astrocytes appeared in aged wild-type mice and in aging human brains, suggesting their linkage to genetic and age-related factors.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Single-cell RNA sequencing (RNA-Seq) provides rich information about cell types and states. However, it is difficult to capture rare dynamic processes, such as adult neurogenesis, because isolation ...of rare neurons from adult tissue is challenging and markers for each phase are limited. Here, we develop Div-Seq, which combines scalable single-nucleus RNA-Seq (sNuc-Seq) with pulse labeling of proliferating cells by 5-ethynyl-2'-deoxyuridine (EdU) to profile individual dividing cells. sNuc-Seq and Div-Seq can sensitively identify closely related hippocampal cell types and track transcriptional dynamics of newborn neurons within the adult hippocampal neurogenic niche, respectively. We also apply Div-Seq to identify and profile rare newborn neurons in the adult spinal cord, a noncanonical neurogenic region. sNuc-Seq and Div-Seq open the way for unbiased analysis of diverse complex tissues.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
The extent of microglial heterogeneity in humans remains a central yet poorly explored question in light of the development of therapies targeting this cell type. Here, we investigate the population ...structure of live microglia purified from human cerebral cortex samples obtained at autopsy and during neurosurgical procedures. Using single cell RNA sequencing, we find that some subsets are enriched for disease-related genes and RNA signatures. We confirm the presence of four of these microglial subpopulations histologically and illustrate the utility of our data by characterizing further microglial cluster 7, enriched for genes depleted in the cortex of individuals with Alzheimer's disease (AD). Histologically, these cluster 7 microglia are reduced in frequency in AD tissue, and we validate this observation in an independent set of single nucleus data. Thus, our live human microglia identify a range of subtypes, and we prioritize one of these as being altered in AD.
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of ...the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.
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•METTL3, METTL14, WTAP, and KIAA1429 are required for mRNA methylation•Methylation maps, upon depletion of WTAP, reveal two classes of methylation•WTAP-dependent sites are mostly static and correlate with mRNA stability•Thousands of WTAP-independent sites are present at the first transcribed nucleotide
N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Epigenetic information can be inherited through the mammalian germline and represents a plausible transgenerational carrier of environmental information. To test whether transgenerational inheritance ...of environmental information occurs in mammals, we carried out an expression profiling screen for genes in mice that responded to paternal diet. Offspring of males fed a low-protein diet exhibited elevated hepatic expression of many genes involved in lipid and cholesterol biosynthesis and decreased levels of cholesterol esters, relative to the offspring of males fed a control diet. Epigenomic profiling of offspring livers revealed numerous modest (∼20%) changes in cytosine methylation depending on paternal diet, including reproducible changes in methylation over a likely enhancer for the key lipid regulator Ppara. These results, in conjunction with recent human epidemiological data, indicate that parental diet can affect cholesterol and lipid metabolism in offspring and define a model system to study environmental reprogramming of the heritable epigenome.
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► Diet consumed by male mice can affect gene expression in offspring ► Offspring of animals on low-protein diet upregulate cholesterol biosynthesis genes ► Paternal diet affects hepatic levels of cholesterol esters in offspring ► Response is partially linked to methylation changes at a putative enhancer of Pparα
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Single-nucleus RNA-seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate or are frozen, and opens the way to human genetics studies, ...clinical trials, and precise cell atlases of large organs. However, such applications are currently limited by batch effects, processing, and costs. Here, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter recovered profiles. We develop DemuxEM, a computational tool that detects inter-sample multiplets and assigns singlets to their sample of origin, and validate its accuracy using sex-specific gene expression, species-mixing and natural genetic variation. Our approach will facilitate tissue atlases of isogenic model organisms or from multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.
Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene ...expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of
cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected
cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.
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► Gene expression profiles for 38 states of human hematopoietic differentiation ► Expression organized in modules: lineage-specific or reused across lineages ► Transcription factors target each other in densely interconnected circuits ► Dozens of regulators of hematopoiesis identified and functionally validated
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The mechanisms that confer cognitive resilience to Alzheimer’s Disease (AD) are not fully understood. Here, we describe a neural circuit mechanism underlying this resilience in a familial AD ...mouse model. In the prodromal disease stage, interictal epileptiform spikes (IESs) emerge during anesthesia in the CA1 and mPFC regions, leading to working memory disruptions. These IESs are driven by inputs from the thalamic nucleus reuniens (nRE). Indeed, tonic deep brain stimulation of the nRE (tDBS-nRE) effectively suppresses IESs and restores firing rate homeostasis under anesthesia, preventing further impairments in nRE-CA1 synaptic facilitation and working memory. Notably, applying tDBS-nRE during the prodromal phase in young APP/PS1 mice mitigates age-dependent memory decline. The IES rate during anesthesia in young APP/PS1 mice correlates with later working memory impairments. These findings highlight the nRE as a central hub of functional resilience and underscore the clinical promise of DBS in conferring resilience to AD pathology by restoring circuit-level homeostasis.
Cells regulate gene expression using a complex network of signaling pathways, transcription factors and promoters. To gain insight into the structure and function of these networks, we analyzed gene ...expression in single- and multiple-mutant strains to build a quantitative model of the Hog1 MAPK-dependent osmotic stress response in budding yeast. Our model reveals that the Hog1 and general stress (Msn2/4) pathways interact, at both the signaling and promoter level, to integrate information and create a context-dependent response. This study lays out a path to identifying and characterizing the role of signal integration and processing in other gene regulatory networks.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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