text: Oakland Breathmobile rolls relief into Bay Area schools By Sam Harnett • Jun 15, 2016 Used under CC from Ben Dalton // cropped and resized Listen Listening... / 6:58 Industrial pollution in West Oakland and all throughout Alameda County can cause major problems for kids who live there. Aimed at addressing this issue, a mobile asthma clinic called the Breathmobile travels around to schools in the area. Sound healing By Truc Nguyen • Jun 14, 2016 Truc Nguyen Listen Listening... / 5:34 Eric Cetnarski works as a sound healer in Oakland. Sound healing can be many things: from listening to our favorite songs to using our voices. The audacity of 'Tourettes Without Regrets,' Oakland's underground variety show By Lindsey Lee Keel • Jun 6, 2016 "Tourettes Without Regrets - July 4, 2013" by Flickr user Oakland Local, used under CC BY-NC-ND 2.0 / Resized and cropped Listen Listening... / 6:36 Jamie DeWolf, founder and host of tonight's performance, steps up to the microphone. The stage he stands on juts out into the center of the Oakland Metro Operahouse, practically surrounded by the eager crowd. Weekend Go-Tos for June 3, 2016 By Ashleyanne Krigbaum • Jun 3, 2016 popfestsf.com Our suggestions for what to do in the Bay Area this weekend span the cinematic, the aerodynamic, and the pop-tastic. Cannabis News Roundup: Pot coming to wine country? By Steven Short • May 20, 2016 Used under CC BY / Cropped Oakland tries to right wrongs with dispensary rules … Police fund opposition to recreation measure … Health … Opinion … and more. LEGALIZATION & LEGISLATION Reformed criminals, certain neighborhoods, win preference in Oakland dispensary plan // East Bay Express Cannabis News Roundup: Oakland dominates the news By Steven Short • May 13, 2016 Used under license from Creative Commons New state agency wants input … Calaveras officials and public clash over Green Rush … Pot prices plummet with legalization … and more Cannabis News Roundup: Justice Dept. drops Harborside case By Steven Short • May 6, 2016 Used under license from Creative Commons. AUMA qualifies for November … Former S.F. sheriff now cannabis consultant … Sex and pot … and more. LEGALIZATION & LEGISLATION AUMA qualifies for November ballot // L.A. Times Meet Oakland's Birdman — before he's gone By Liza Veale • May 4, 2016 Listen Listening... / 8:14 In Oakland, just past Jack London Square, there's a zone along the water that's a little wild. It's past the new condos and great restaurants, past the wholesale produce warehouses that open for business before dawn, and down the channel from Lake Merritt's newly landscaped park. It's an in-between space. But change is coming from both sides. Can Oakland hotels help catch sex traffickers? By Shereen Adel • Apr 21, 2016 used under CC0 license Listen Listening... / 7:28 In recent years it has become more and more common for arrangements to buy and sell sex to be made online and behind closed doors. A lot of the time it's not among consenting adults — it's forced prostitution or sex trafficking. Yemeni family straddles border between Oakland and homeland By Mary Rees • Apr 14, 2016 Listen Listening... / 10:15 There's been a lot of news about refugees from Syria, Afghanistan and Sudan and their attempts to enter Europe, but we hear far less about people trying to escape Yemen, where a war has been going on for just over a year now. Daily news roundup for Monday, April 11, 2016 By Tammy Drummond • Apr 11, 2016 "Warriors Blimp 2" by Flickr user Ardail Smith. Used under CC BY-NC-SA 2.0 / cropped and resized Here's what's happening in the Bay Area as curated by KALW news: Warriors tie Bulls' mark with 72nd Win // East Bay Times Weekend Go-Tos for April 8, 2016 By Ashleyanne Krigbaum • Apr 8, 2016 sfcherryblossom.org The weekend is upon us, and we've wrangled a few suggestions of events and things worth doing around the Bay Area. Cannabis News Roundup: Cannabis for menstrual cramps By Steven Short • Apr 8, 2016 Used via Google Images/noncommercial reuse. DEA looks at reclassifying cannabis … Grownups just say no … Harborside leaves Oregon … "Heavy users = losers" … Events, opinion and more. LEGALIZATION & LEGISLATION DEA to consider reclassification of cannabis by mid-year // Huffington Post Daily News Roundup for Thursday, April 7, 2016 By Shereen Adel • Apr 7, 2016 By Shereen Adel Here's What's Happening in the Bay Area as Curated by KALW News: In wake of mural fight, Oakland developer agrees to hundreds of thousands of dollars in concessions // San Francisco Business Journal Oakland body cameras offer glimpses of officer-involved deaths By Holly McDede • Mar 17, 2016 Photo courtesy of Ana Biocini Listen Listening... / 9:45 Oakland Tribune crime reporter David DeBolt still remembers when his paper got the call from the Oakland Police Department. They wanted local reporters like him to come to police headquarters to watch police body camera footage of two recent officer-involved shootings. LISTEN Kamau Right Now! #4: Live from Oakland's Impact Hub By Matt Martin • Mar 17, 2016 Dan Roth On Thursday, March 17th, Kamau Right Now! was broadcast live from Impact Hub in downtown Oakland. Joining W. Kamau Bell were social justice leader Lateefah Simon, journalist and hip hop historian Dave "Davey D." Cook, poet-comedian Bucky Sinister. Plus, an "awkward talk" with Oakland Mayor Libby Schaaf and music from Bhi Bhiman. Daily news roundup for Wednesday, March 16, 2016 By Luisa Cardoza • Mar 16, 2016 Here's what's happening in the Bay Area, as curated by KALW News: Bay Area reservoirs still far from full: Conservation experts hope that drought lessons last // San Mateo Daily Journal Weekend Go-Tos for March 11, 2016 By Ashleyanne Krigbaum • Mar 11, 2016 Courtesy of Negativland We have a few suggestions for you on what to do around our Bay Area this weekend. Oakland negotiates with developers and community members to sell public land By Liza Veale • Mar 10, 2016 Image courtesy Thompson Dorfman Listen Listening... / 10:11 For decades, development investors didn't want to touch Oakland. In 1997, one economist called the city an "ugly duckling in a bay of swans." But these days, Oakland is one of the "hottest," or fastest-growing, real estate markets in the country. Daily News Roundup for Monday March 7, 2016 By Geraldine Ah-Sue • Mar 7, 2016 "Red" by Flickr user marcin ejsmont. Used under CC BY-NC-ND 2.0 / cropped and resized Here's what's happening in the Bay Area, as curated by KALW news: Sunset Magazine relocates to Oakland, setting sights on younger, urban readers // Oakland North "Guests were met with the soft and periodic sound of whizzing traffic on one side and ferry boats on the other as they made their way up the steps into the new Oakland offices of Sunset Magazine in Jack London Square. A smiling security guard opened the door with a small whoosh and let them enter the 110,000-square foot office space with white cubicles and office chairs on one end and tables for drinks, hors d'oeuvres and party bags on the other." Weekend Go-Tos for March 4, 2016 By Ashleyanne Krigbaum • Mar 4, 2016 whiteelephantsale.org Sure, it's going to be stormy outside this weekend, but we've lined up some unique and fun, indoor events to check out this weekend in the Bay Area. How a tiny shipping container community is causing a big fuss in Oakland By Chris Hambrick • Feb 23, 2016 Chris Hambrick Listen Listening... / 8:51 Here in the Bay Area, rents are rising, and housing inventory is shrinking. It's forcing many here to decide -- either you have to leave the area completely, or you have to get creative about your housing situation. A poem from Youth Radio: "Rose Flower" By Darelle Brown • Feb 22, 2016 "PICT0294" by Flickr user Sorin Florea. Used under CC BY-NC-ND 2.0 / Resized and cropped Listen Listening... / 1:53 Darelle Brown is a participant in Remix Your Life, an arts program run by Youth Radio. Brown grew up in Oakland, and he imagines what it's like to be a rose pushing it's way up through his city's soil. Weekend Go-Tos for February 19, 2016 By Ashleyanne Krigbaum • Feb 19, 2016 Happy Friday, everybody! We have a few recommendations of what to do around the Bay Area this weekend. Weekend Go-Tos for February 12, 2016 By Ashleyanne Krigbaum • Feb 12, 2016 Check out our suggestions of unique events happening around the Bay Area this weekend. Today's Local Music: Ira Marlowe By Steven Short • Feb 11, 2016 Courtesy of Ira Marlowe The husky voice you're hearing now belongs to singer/songwriter Ira Marlowe. The San Francisco Chronicle has called him "Oakland's sweet little secret." Ira Marlowe will perform solo at The Monkey House in Berkeley on Saturday (02.13) It's a small place, so arrange for tickets ahead of time. StoryCorps: Dancing despite disability By Tammerlin Drummond • Feb 10, 2016 Listen Listening... / 3:27 Twenty eight years ago, Bonnie Lewkowicz got a phone call from someone wanting to know what she thought about the idea of disabled people dancing. That question set her on an improbable odyssey. Weekend Go-Tos for February 5, 2016 By Ashleyanne Krigbaum • Feb 5, 2016 Looking for something fun and unique to do this weekend? Check out our suggestions for the weekend of February 5, 2016. Daily news roundup for Monday, February 1, 2016 By Tammy Drummond • Feb 1, 2016 "Super Bowl 50," by Flickr user Charlie Lyons-Pardue. Used under CC BY-NC 2.0 / resized and cropped Here's what's happening in the Bay Area, as curated by KALW news: Super Bowl 50's crazy ticket prices drop slightly, but Niner fans still left out // Mercury News Audiograph's Sound of the Week: The Alley's Rod Dibble By Colin Peden & Colin Peden • Jan 28, 2016 Colin Peden Listen Listening... / 6:13 All week long, we've been playing this sound and asking you to guess what exactly it is and where exactly in the Bay Area we recorded it. Pages« first ‹ previous 1 2 3 4 5 6 7 8 9 … next › last »


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The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. While many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified over 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising over 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders.
The telencephalon houses the cerebral cortex and basal ganglia, structures that are pivotal for human brain functions (Wilson and Rubenstein, 2000). Impaired telencephalic development and function are associated with major neurological and neuropsychiatric disorders including mental deficiency, cerebral palsy, epilepsy, schizophrenia and autism (Lewis and Sweet, 2009; Walsh et al., 2008a). Significant progress has been made towards defining spatially resolved gene expression patterns in the developing and adult mouse and human brain on a genomic scale (Diez-Roux et al., 2011; Gong et al., 2003; Gray et al., 2004; Lein et al., 2007; Portales-Casamar et al.; Visel et al., 2004; Zeng et al., 2012). In contrast, the distant-acting gene regulatory sequences that are critical for orchestrating the spatial and temporal expression of genes in the developing and adult brain remain poorly defined, despite evidence from large-scale human genetic studies demonstrating the contribution of regulatory sequences to a wide spectrum of human traits and disorders (Durbin et al., 2010; Maurano et al., 2012), and despite anecdotal direct evidence for a critical requirement for enhancers in brain development (Kurokawa et al., 2004; Shim et al., 2012).
Unlike protein-coding genes, enhancers involved in specific biological processes are difficult to identify because they reside in the vast and poorly characterized non-coding portion of the genome, and can be located hundreds of thousands of base pairs away from the promoters of the target genes they regulate (Lettice et al., 2003). The introduction of enhancer prediction methods based on extreme evolutionary conservation (Nobrega et al., 2003; Pennacchio et al., 2006; Visel et al., 2008) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) (Visel et al., 2009a) increased the efficiency of identifying enhancers. Importantly, ChIP-seq experiments performed directly on tissues can provide accurate predictions of the broad general anatomical region in which an enhancer is active (Visel et al., 2009a). Nevertheless, the spatial resolution of these methods is limited and detailed in vivo studies are required to precisely define the activity patterns of enhancers at high resolution.
To address the need for an improved understanding of the cis-regulatory architecture and gene networks active during telencephalic development, we combined sequence conservation- and ChIP-seq-based enhancer prediction with large-scale histological activity analysis of human telencephalon enhancers in transgenic mice. We demonstrate how the high-resolution neuroanatomical annotation of enhancer activities can be used to develop computational sequence classifiers for enhancers active in different subregions of the telencephalon. We also directly compare the genome-wide enhancer architecture active in the mouse and human cortex using ChIP-seq from these tissues, and provide examples of downstream applications for enhancers identified through this work.
To generate a genome-wide set of forebrain enhancer candidate sequences, we collected forebrain tissue from embryonic day [e]11.5 mouse embryos and performed tissue-ChIP-seq using an antibody for the enhancer-associated protein p300. Results were analyzed alongside previously described data to increase sampling depth (see Supplemental Methods). Genome-wide enrichment analysis led to the identification of 4,425 non-coding regions genome-wide that are distal from transcription start sites and significantly enriched in p300 binding in the e11.5 forebrain (Suppl. Table S1). Since p300 was previously shown to be associated with active tissue-specific enhancers (Blow et al., 2010; Visel et al., 2009a), these sequences were predicted to be distant-acting forebrain enhancers. As a complementary approach to identify candidate enhancers, we also used extreme sequence conservation in conjunction with genomic location. Thus, we scrutinized sequences under extreme evolutionary constraint (Siepel et al., 2005; Visel et al., 2008) in the genomic vicinity of 79 genes with a known role in forebrain development or function (Suppl. Table S2), and identified 231 additional candidate forebrain enhancer sequences (Suppl. Table S3). Combined, these two datasets comprised a total of 4,656 noncoding sequence elements that we hypothesized to be enriched in forebrain enhancers.
To validate candidate telencephalon enhancer sequences and define their in vivo activities in more detail, we selected 329 elements predicted to be enhancers by conservation and/or ChIP-seq for experimental testing (Suppl. Table S4). Nearly all of these selected elements were located near genes with a known function in the forebrain (Suppl. Table S2). In order to focus on the most conserved core regulatory architecture of mammalian telencephalon development, only ChIP-seq peaks that were detectably conserved between the human and mouse genome were tested. Regardless of the identification method, all tested sequences showed evidence of significant evolutionary constraint (phastCons scores ranging from 415 to 931, median 798; Suppl. Table S4). The selected candidate enhancer sequences were amplified from human genomic DNA, cloned into an enhancer reporter vector (Hsp68-LacZ), and used to generate transgenic mice by pronuclear injection (see Methods). Transgenic embryos were stained for reporter gene (LacZ) activity at e11.5 and reporter expression was annotated using established reproducibility criteria (Pennacchio et al., 2006). Only elements that drove expression in the forebrain in at least three embryos, each of them corresponding to an independent transgenic integration event, were considered as reproducible forebrain enhancers. In total, 105 of 329 (32%) candidate sequences tested were reproducible forebrain enhancers at e11.5, of which 36 showed reproducible expression exclusively in the forebrain (Suppl. Table S4). For comparison, in previous transgenic assays of p300 binding sites in two different non-neuronal tissues, limb buds and heart, only 4/155 (2.6%) tested sequences had reproducible forebrain enhancer activity at e11.5 (Blow et al., 2010; Visel et al., 2009a). Enhancer candidate sequences that overlapped p300 ChIP-seq peaks were more enriched in verifiable in vivo forebrain enhancers than extremely conserved sequences that showed no evidence of p300 binding (58% compared to 23%, Suppl. Table S4). Selected examples of reproducible forebrain enhancers whose in vivo activity was confirmed in transgenic mice are shown in Fig. 1 and whole-mount images for all validated enhancers are accessible online through the Vista Enhancer Browser (Visel et al., 2007).
To define the precise spatial expression patterns of telencephalic enhancers active at e11.5, we performed high-resolution analysis on a set of 145 enhancers (Suppl. Table S5). These sequences were selected from the 105 forebrain enhancers discovered in the present study and from complementary sets of forebrain enhancers identified at whole-mount resolution in previous enhancer screens (Pennacchio et al., 2006; Visel et al., 2008; Visel et al., 2009a). For each enhancer, a full set of contiguous coronal paraffin sections (average: 220 sections) was obtained. Full-resolution digital images of over 32,000 sections are available through the Vista Enhancer Browser (Visel et al., 2007). Selected sections of patterns driven by different enhancers in subregions of the pallium and subpallium are shown in Figures 2 and and3,3, illustrating the diversity of spatial specificities observed.
In addition to the spatial activity patterns of all 145 enhancers studied at e11.5, we also examined the temporal activities of a subset of these enhancers at later prenatal stages of telencephalon development (Figures 2S and 3F-G). These temporal comparisons showed that the spatial patterns of enhancer activity were largely constant. In two cases, enhancers active in subregions of the subpallium at e11.5 displayed characteristic features of subpallial cell populations (interneurons) that tangentially migrate to the pallium. At e13.5, these cells had just arrived in the ventrolateral pallium (hs692 and hs799), and by e15.5 they were in the dorsal pallium (hs799, arrowheads in Fig. 3F-G). These results support that enhancers regulate both spatial and temporal aspects of telencephalic gene expression in patterns consistent with the biology of these regions and cell types.
To facilitate analysis by computational methods, we devised a standardized neuroanatomical annotation scheme for the e11.5 stage of telencephalon development (Fig. 2A and and3C,3C, Suppl. Fig. S1, Suppl. Table S5). All telencephalon enhancer activity patterns examined in this study were annotated using this standardized annotation scheme, in some cases complemented by descriptions that further subdivide the standardized domains or are restricted to subsets of cells (Suppl. Table S5). The standardized annotations assigned to each enhancer through this annotation effort enable computational analysis of enhancer activity patterns, as well as a comparison to expression patterns of their presumptive target genes at this stage of development.
To test whether the telencephalon enhancers examined at high resolution generally recapitulate the spatial expression patterns of their presumptive target genes, we compared their LacZ reporter activities to RNA in situ-hybridization data. For example, the Arx gene is expressed both in subpallial and pallial regions, with increasing expression in pallial regions from e11.5 to e13.5 (Fig. 4A). We found that there are at least four distant-acting telencephalic enhancers in this extended locus, two of which drive subpallial and two of which drive pallial expression, indicating that developmental Arx regulation is more complex than initially suggested (Colasante et al., 2008). In addition, comparison of other genes with well-established roles in telencephalon development (Lef1, Wnt8b, Gsx2, Nr2f1) to nearby enhancers also revealed examples of spatially concordant enhancer activity and RNA expression (Fig. 4B-E). A recurring feature of these comparisons is the restriction of individual enhancer activities to subregions of the respective gene expression patterns, supporting the modular structure of telencephalic enhancer architecture. For instance, hs687 activity in the LGE matches Gsx2 RNA expression, while the latter is also expressed in the MGE and hs1172 activity in the pallium matches Nr2f1 RNA expression, while the gene is also expressed in the subpallium.
To assess whether these illustrative examples are representative of a general congruence between enhancer activity patterns and the expression of nearby genes, we performed a quantitative correlation analysis across the available data set (see Supplemental Experimental Procedures for details). Overall, we found a highly significant correlation between the activity patterns of enhancers and telencephalic expression patterns of nearby annotated genes (P=0.0003, Mann-Whitney test, Fig. 4F). In addition to the high-resolution comparisons of enhancer and gene activity patterns, we also examined whether the genome-wide set of 4,425 forebrain enhancer candidate sequences identified by ChIP-seq from embryonic mouse forebrain tissue is associated with genes with known functions in the telencephalon. Unbiased genome-wide assessment (McLean et al., 2010) showed highly significant enrichment in genes that cause forebrain-related phenotypes when deleted in mouse models (Suppl. Table S6). These observations support on a genomic scale that the large set of forebrain candidate enhancers predicted by ChIP-seq in this study is enriched near genes that are involved in telencephalon development.
A large set of telencephalon enhancers, analyzed at high spatial resolution and annotated to a standardized scheme, offers the possibility to examine sequence features that are associated with in vivo activity in different telencephalic subregions. To explore this regulatory code, we trained a Random Forests (RF) classifier (Breiman, 2001; Bureau et al., 2005; Cummings and Segal, 2004; Lunetta et al., 2004) to discriminate between enhancers active in 1. pallium only, 2. pallium and subpallium (compound pattern), or 3. subpallium only, and random genomic sequences (see Fig. 5 and Supplemental Methods). Classification is based on the presence or absence of combinations of sequence motifs matching known transcription factor binding sites (Bryne et al., 2008; Matys et al., 2006). The five most relevant motifs distinguishing the three classes of enhancers and their respective importance are shown in Fig. 5B (for additional motifs, see Suppl. Fig. S2 and Suppl. Table S8). We did not observe any single motif that was sufficient to accurately discriminate between the different classes of enhancers, suggesting that only the combinatorial binding of multiple transcription factors determines the observed spatial regulatory activity. The majority of the most discriminatory motifs (at least 60% of the top 15 motifs characterizing enhancers active in each of the telencephalic subregions considered) correspond to predicted binding sites for homeodomain-containing transcription factors, consistent with the known critical role of these proteins in telencephalon development (Hebert and Fishell, 2008). Suppl. Fig. S3 summarizes the enrichment of the 15 most relevant motifs for enhancer activity in the three different telencephalic subregions considered. Despite possible ambiguities associated with computational transcription factor binding site predictions, the RF classifier accurately predicts approximately 80% of the sequences (see Supplemental Methods, Suppl. Table S9). Sequence motifs with high quantitative importance for discriminating between different classes of telencephalon enhancers are overall more conserved in evolution compared to non-important motifs, supporting their functional relevance (Suppl. Fig. S4).
These computational predictions of relevant sequence motifs provide a starting point for experimental studies aimed at understanding the transcription factor binding site content of telencephalon enhancers in more detail. To illustrate the value of a large set of enhancers with known sequences and activity patterns for studying genetic dependencies in telencephalon development, we tested a subset of subpallial enhancers for their direct regulation by two major subpallial transcription factors, Dlx2 and Ascl1 (see Supplemental Methods). In a cell-based luciferase assay, we observed that Dlx2 and/or Ascl1 significantly increased reporter expression when co-transfected with 13 of 20 tested enhancers (Fig. 5C). Of note, these enhancers are located near several genes with known roles in subpallium development and the results are consistent with previous studies demonstrating that Dlx2 regulates the expression of Arx, Meis2 and Sp8, and that Ascl1 regulates the expression of Sox4 (Castro et al., 2011; Colasante et al., 2008; Long et al., 2009). Considering the expected complexity of the spectrum of transcription factors binding to different subsets of telencephalon enhancers (Fig. 5B, Suppl. Table S8), complementary scalable methods will be required to experimentally validate all binding sites within each of the enhancers identified. Our cell-based studies of a small subset of these sequences highlight, however, that the combined knowledge of the genomic location, the spatial activity, and the upstream transcription factors of discrete, distant-acting regulatory sequences generates hypotheses that are directly testable in genetic in vivo systems.
Our large-scale transgenic testing and high-resolution analysis of telencephalon enhancers focused on sequences that are highly conserved in evolution, with the goal to characterize the most conserved core regulatory architecture of mammalian telencephalon development. However, epigenomic methods also enable the systematic discovery of poorly conserved and lineage-specific enhancers (Schmidt et al., 2010). To explore possible differences between human and mouse telencephalon enhancers in more detail, we determined the genome-wide occupancy of the enhancer-associated proteins p300/CBP in human fetal (gestational week 20) cortex (Fig. 6A,B). ChIP-seq analysis identified 2,275 peaks (candidate enhancers) genome-wide that were located at least 2.5kb from the nearest transcript start site. Comparison with transcriptome data from human fetal cortex tissue revealed a 2.7-fold enrichment in candidate enhancers within 2.5-20kb of the transcript start sites of genes highly expressed in fetal human cortex (P < 1e-14, binomial distribution), with significant enrichment up to 220kb away from promoters (P < 0.001, binomial distribution, Fig. 6C). In contrast, no enrichment of p300/CBP binding sites was observed near genes highly expressed in other tissues. Similar to candidate enhancers predicted from mouse e11.5 forebrain, unsupervised statistical enrichment analysis of functional gene annotations (McLean et al., 2010) showed significant association with genes implicated in nervous system-related phenotypes (Suppl. Table S6). While many extremely conserved non-coding sequences in the human genome are enhancers active in the developing nervous system (Pennacchio et al., 2006), we observed that a third (36.5%) of ChIP-seq-predicted human brain candidate enhancers are under weak (phastCons < 350) or no detectable evolutionary constraint, suggesting that subsets of human brain enhancers may not be functionally conserved in mice.
At gestational week 20, the human cortex is considerably further developed than the mouse pallium at embryonic day 11.5, and instead corresponds broadly to early postnatal stages in mouse (Clancy et al., 2007). To enable a direct experimental comparison between the two species, we performed p300/CBP ChIP-seq on mouse postnatal (P0) cortex tissue. Using identical methods as for human tissue, we identified 1,132 candidate enhancers (distal ChIP-seq peaks). The majority (58%) of human-derived peaks showed significant or suggestive (sub-significant) enrichment in ChIP-seq reads at the orthologous site in the mouse genome (Fig. 6D). The remaining 42% either showed no enrichment in the orthologous mouse region or were not alignable to the mouse genome. While the lower sequencing coverage in the mouse data set may lead to an underestimation of mouse-compared to human-specific peaks (compare Fig. 6D/E), the presence of 307 peaks in non-alignable regions of the human genome (Fig. 6D) supports that a non-negligible proportion of human brain enhancers emerged in evolution after the divergence of primates and rodents from their last common ancestor.
Similar to the large collection of telencephalon enhancers identified and characterized at e11.5, ChIP-seq peaks derived from human fetal cortex are expected to include enhancers with a variety of in vivo activity patterns. To illustrate this, we examined the in vivo activities of candidate enhancers from human fetal cortex in postnatal transgenic mice. Two examples of such enhancers driving reproducible expression in a minimum of three independent transgenic animals are shown in Fig. 6F-K. Consistent with the ChIP-seq prediction, both enhancers were active in the cortex (red arrows), as well as additional, but distinct and reproducible regions of the telencephalon.
To illustrate the value of the genome-wide sets of human and mouse candidate enhancers for the interpretation of human genetic datasets, we compared the genomic position of these sequences with different catalogs of regions in the human genome implicated in neurodevelopmental, neurological or neuropsychiatric diseases. We intersected the genome-wide sets of candidate enhancers identified in the three different ChIP-seq experiments a) with lead single nucleotide polymorphisms (SNPs) from genome-wide association studies of relevant traits (Hindorff et al., 2009), b) with catalogs of syndromic microdeletions and -duplications (Firth et al., 2009), and c) with a set of autism-associated rare copy number variants (Marshall et al., 2008; Szatmari et al., 2007). Fourteen lead SNPs from genome-wide association studies, including SNPs associated with attention deficit hyperactivity disorder, bipolar disease and schizophrenia were found to be located within predicted forebrain enhancers. Moreover, 381 enhancers mapped within recurrent microdeletions or -duplications associated with neurological phenotypes, and 421 enhancers overlapped copy number variants present in autism cases, but not healthy controls. While further experimental studies will be required to examine possible causal roles of variants affecting enhancer sequences, the genome-wide sets of candidate enhancers identified from human and mouse brain tissue through this study provide a starting point to explore the role of telencephalon enhancers in human diseases.
The enhancers described in our high-resolution atlas can be used as molecular reagents to drive in vivo expression of reporter or effector genes to specific telencephalic subregions of interest, owing to the reproducibility of their activity patterns (Fig. 7A). To illustrate some of the resulting applications, we coupled enhancer hs1006, associated with the WNT8B gene, to a minimal Hsp68 promoter, followed by a tamoxifen-inducible Cre recombinase (CreERT2), an internal ribosomal entry site, and a green fluorescent protein (GFP) reporter (Fig. 7B). In stable transgenic mouse lines generated with this construct, termed CT2IG-hs1006, GFP expression at e11.5 was indistinguishable from LacZ reporter expression (Fig. 7A/B). GFP expression in these stable lines facilitates a temporally resolved mapping of enhancer activity. A comparison of GFP activity at e12.5, e15.5, and e17.5 with Wnt8b RNA expression indicated that enhancer activity spatially coincides with Wnt8b gene expression, supporting that this enhancer controls region-specific expression of the gene over an extended period of prenatal telencephalon development.
Since expression of the compound effector/reporter transcript in CT2IG-hs1006 mice faithfully resembled Wnt8b expression across multiple stages of development, the chemically inducible CreERT2 recombinase can be used for spatially and temporally highly restricted genomic recombineering applications, such as neuronal fate mapping studies. To demonstrate this, we crossed CT2IG-hs1006 mice with Rosa26-LacZ mice (Fig. 7B) (Indra et al., 1999). Tamoxifen induction of CreERT2 in pregnant compound CT2IG-hs1006:Rosa26-LacZ mice at e10.5 leads to recombination only in the small proportion of pallial cells in which the enhancer is active at this time point. LacZ staining at later stages revealed the spatial fate of cells in which the enhancer was active at e10.5. For example, hs1006-driven e10.5→ e12.5 fate mapping marked pallial cell populations with a distribution that is clearly distinct from hs1006 activity at this time point (compare e12.5 patterns in Figures 7C and and7D).7D). These data highlight the utility of these enhancers to precisely drive gene expression in the developing brain and their value as a rich resource for a diversity of uses.
This work provides a comprehensive resource for basic studies of telencephalon enhancers. Our targeted screen identified the genomic location of thousands of candidate enhancers putatively active in the embryonic forebrain. The mapping and annotation of the activity patterns of nearly 150 human telencephalon enhancers at histological resolution in transgenic mice provides insight into the regulatory architecture of individual genes that are required for forebrain development and will facilitate studies of molecular genetic pathways by identifying the genomic regions to which upstream transcription factors bind.
Our analysis revealed several cases of enhancers that drive similar patterns and are associated with the same gene (e.g. Fig. 4A) in a manner reminiscent of the 'shadow enhancers' observed in invertebrate models (Frankel et al., 2010; Hong et al., 2008). The data provided through this work will support the identification of minor spatial activity differences between such enhancers, as well as the functional exploration of their apparent redundancies. It is also remarkable that a large proportion of enhancers examined in this study drove patterns that were at least partially different from all other enhancers examined, highlighting the complexity of the developing forebrain, as well as the regulatory sequence code orchestrating its development.
The motif-based classifiers derived from enhancers active in different subregions of the telencephalon demonstrate the value of systematically annotated enhancer activity data sets for computational studies aimed at deciphering the correlation between the transcription factor binding sites present in an enhancer and its precise spatial activity pattern. Beyond such functional genomic studies, the enhancers identified and characterized in this work provide a comprehensive set of molecular reagents that can be used to target gene expression to defined subregions of the developing brain, or to defined cell states when differentiating stem cells in vitro. This will enable tissue-specific homologous recombination and deletion strategies or expression of reporter and selectable genes, as illustrated in Fig. 7.
Finally, results from this study are expected to enable and facilitate the functional genomic exploration of the role of enhancers in human brain disorders. There is accumulating evidence that non-coding sequence variants, as well as copy number variation in coding and non-coding portions of the genome have important impacts on a wide spectrum of disorders including bipolar, schizophrenia, autism, intellectual disability and epilepsy (Cooper et al., 2011; Durbin et al., 2010; International Schizophrenia Consortium, 2008; Malhotra et al., 2011; Sebat et al., 2007; Vacic et al., 2011; Visel et al., 2009b; Walsh et al., 2008b). However, the functional interpretation of non-coding sequence or copy number variants remains a major challenge and few potentially causative connections linking neurological traits to molecular variation in enhancers have been identified (e.g., (Poitras et al., 2010)). Thus, the systematic mapping and high-resolution analysis of telencephalon enhancers through this work is expected to provide functional genomic insights to guide studies that will mechanistically relate individual non-coding sequence and copy number variants to brain disorders.
ChIP-seq on forebrain tissue isolated from e11.5 CD-1 strain mouse embryos, using an antibody directed against p300, was performed according to previously described procedures (Visel et al., 2009a). For human tissue ChIP-seq and the matched mouse postnatal cortex data set, an anti-acCBP/p300 pan-specific antibody was used (May et al., 2011).
Enhancer candidate regions were analyzed in transgenic mouse embryos as previously described (Kothary et al., 1988; Pennacchio et al., 2006). Paraffin sections were prepared according to standard protocols. Serial sets of sections were digitally photographed and uploaded to the Vista Enhancer Browser (http://enhancer.lbl.gov).
A previously described Cre-ERT2 construct (Feil et al., 1997) was modified to allow Cre recombinase expression to be driven by the hs1006 enhancer (Fig. 7B). For fate mapping, CT2IG-hs1006 mice were crossed with Rosa26-LacZ reporter mice (Soriano, 1999).
Dlx2 and Ascl1 were selected for luciferase reporter assays due to their well-established roles in subpallial development and because they are representatives of two major groups of transcription factors found among the top motifs of the subpallium classifier (see Supplemental Experimental Procedures). P19 cells were grown by previously described methods (Farah et al., 2000).
Images of whole-mount-stained embryos and full sets of e11.5 coronal brain sections are available through the Vista Enhancer Browser, http://enhancer.lbl.gov. All enhancer reporter vectors described in this study are freely available from the authors. In addition, archived surplus transgenic embryos for many constructs can be made available upon request for complementary studies. The genome-wide set of ChIP-seq peaks derived from mouse e11.5 forebrain is provided in Suppl. Table S1. Raw data and additional ChIP-seq data sets from postnatal mouse and fetal human cortex are available from GEO under accession number GSE42881.
The authors thank Julian Golder and Noah Efron for help with digital image acquisition and data processing; Bing Ren and Zirong Li for help with chromatin immunoprecipitation from embryonic mouse tissue; Inna Dubchak, Simon Minovitsky and Alexandre Poliakov for web site support; staff at San Francisco General Hospital Women's Options Center for their consideration in allowing us to access donated fetal tissue. A.V. and L.A.P. were supported by NINDS grant R01NS062859A and by NHGRI grant R01HG003988. J.L.R.R. was supported by the Nina Ireland, Weston Havens Foundation, NINDS grant R01NS34661, NIMH grant R01MH081880, and NIMH grant R37MH049428. J.L.R.R. and A.R.K. were supported by CIRM RB2-1602. G.M. and S.N.S. were supported by T32 GM007449, K.P. was supported by T32 GMO7618, R.H. by F32 MH081431 and O.G. by NARSAD. A.R.K. was supported by NINDS grant R01NS075998. I.O. was supported by the Intramural Research Program of the NIH, National Library of Medicine. Research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC02-05CH11231, University of California.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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