The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universal responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in a number of Cre-driver lines, including novel Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.

DNA methylation is essential for normal development [1][2][3] and has been implicated in many pathologies including cancer 4,5 . Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing 6 and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that 'weak' CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during

Cell-type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of 4 knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0, and archaerhodopsin Arch-ER2. All 4 transgenes mediate Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent, and inducible nature of our ChR2 mice represents a significant advancement over previous lines, whereas the Arch-ER2 and eNpHR3.0 mice are the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre-driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

DNA methylation is a defining feature of mammalian cellular identity and essential for normal development1,2. Most cell types, except germ cells and pre-implantation embryos3–5, display relatively stable DNA methylation patterns with 70–80% of all CpGs being methylated6. Despite recent advances we still have a too limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in depth analysis of 42 whole genome bisulfite sequencing (WGBS) data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, a majority of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription factor binding sites (TFBS), which allow identification of key lineage specific regulators. In addition, differentially methylated regions (DMRs) often harbor SNPs associated with cell type related diseases as determined by GWAS. The results also highlight the general inefficiency of WGBS as 70–80% of the sequencing reads across these data sets provided little or no relevant information regarding CpG methylation. To further demonstrate the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore our selected DMRs can serve as a starting point to help guide novel, more effective reduced representation approaches to capture the most informative fraction of CpGs as well as further pinpoint putative regulatory elements.

Summary An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems, and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.

SUMMARY 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 PPARα. 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.

SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

SummaryDNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methyl cytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and to date no base-resolution maps exist to support and refine it. Here, we generated genome-scale DNA methylation maps in mouse gametes and through post-implantation embryogenesis. We find that the oocyte already exhibits global hypomethylation, most prominently at specific families of long interspersed element-1 and long terminal repeat retro-elements, which are disparate between gametes and resolve to lower methylation values in zygote. Surprisingly, the oocyte contributes a unique set of Differentially Methylated Regions (DMRs), including many CpG Island promoter regions, that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and resolve to hypermethylation after the blastocyst stage. Our data provide a complete genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.