Regulatory sharing between estrogen receptor α bound enhancers.

The human genome encodes an order of magnitude more gene expression enhancers than promoters, suggesting that most genes are regulated by the combined action of multiple enhancers. We have previously shown that neighboring estrogen-responsive enhancers exhibit complex synergistic contributions to the production of an estrogenic transcriptional response. Here we sought to determine the molecular underpinnings of this enhancer cooperativity. We generated genetic deletions of four estrogen receptor α (ER) bound enhancers that regulate two genes and found that enhancers containing full estrogen response element (ERE) motifs control ER binding at neighboring sites, while enhancers with pre-existing histone acetylation/accessibility confer a permissible chromatin environment to the neighboring enhancers. Genome engineering revealed that two enhancers with half EREs could not compensate for the lack of a full ERE site within the cluster. In contrast, two enhancers with full EREs produced a transcriptional response greater than the wild-type locus. By swapping genomic sequences, we found that the genomic location of a full ERE strongly influences enhancer activity. Our results lead to a model in which a full ERE is required for ER recruitment, but the presence of a pre-existing permissible chromatin environment can also be needed for estrogen-driven gene regulation to occur.

ZEBrA: Zebra finch Expression Brain Atlas-A resource for comparative molecular neuroanatomy and brain evolution studies.

An in-depth understanding of the genetics and evolution of brain function and behavior requires a detailed mapping of gene expression in functional brain circuits across major vertebrate clades. Here we present the Zebra finch Expression Brain Atlas (ZEBrA; www.zebrafinchatlas.org, RRID: SCR_012988), a web-based resource that maps the expression of genes linked to a broad range of functions onto the brain of zebra finches. ZEBrA is a first of its kind gene expression brain atlas for a bird species and a first for any sauropsid. ZEBrA's >3,200 high-resolution digital images of in situ hybridized sections for ~650 genes (as of June 2019) are presented in alignment with an annotated histological atlas and can be browsed down to cellular resolution. An extensive relational database connects expression patterns to information about gene function, mouse expression patterns and phenotypes, and gene involvement in human diseases and communication disorders. By enabling brain-wide gene expression assessments in a bird, ZEBrA provides important substrates for comparative neuroanatomy and molecular brain evolution studies. ZEBrA also provides unique opportunities for linking genetic pathways to vocal learning and motor control circuits, as well as for novel insights into the molecular basis of sex steroids actions, brain dimorphisms, reproductive and social behaviors, sleep function, and adult neurogenesis, among many fundamental themes.

Sufficiency analysis of estrogen responsive enhancers using synthetic activators.

Multiple regulatory regions bound by the same transcription factor have been shown to simultaneously control a single gene's expression. However, it remains unclear how these regulatory regions combine to regulate transcription. Here, we test the sufficiency of promoter-distal estrogen receptor α-binding sites (ERBSs) for activating gene expression by recruiting synthetic activators in the absence of estrogens. Targeting either dCas9-VP16(10x) or dCas9-p300(core) to ERBS induces H3K27ac and activates nearby expression in a manner similar to an estrogen induction, with dCas9-VP16(10x) acting as a stronger activator. The sufficiency of individual ERBSs is highly correlated with their necessity, indicating an inherent activation potential that is associated with the binding of RNA polymerase II and several transcription factors. By targeting ERBS combinations, we found that ERBSs work independently to control gene expression when bound by synthetic activators. The sufficiency results contrast necessity assays that show synergy between these ERBSs, suggesting that synergy occurs between ERBSs in terms of activator recruitment, whereas directly recruiting activators leads to independent effects on gene expression.

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines.

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor α-Bound Enhancers.

Multiple regulatory regions have the potential to regulate a single gene, yet how these elements combine to affect gene expression remains unclear. To uncover the combinatorial relationships between enhancers, we developed Enhancer-interference (Enhancer-i), a CRISPR interference-based approach that uses 2 different repressive domains, KRAB and SID, to prevent enhancer activation simultaneously at multiple regulatory regions. We applied Enhancer-i to promoter-distal estrogen receptor α binding sites (ERBS), which cluster around estradiol-responsive genes and therefore may collaborate to regulate gene expression. Targeting individual sites revealed predominant ERBS that are completely required for the transcriptional response, indicating a lack of redundancy. Simultaneous interference of different ERBS combinations identified supportive ERBS that contribute only when predominant sites are active. Using mathematical modeling, we find strong evidence for collaboration between predominant and supportive ERBS. Overall, our findings expose a complex functional hierarchy of enhancers, where multiple loci bound by the same transcription factor combine to fine-tune the expression of target genes.

Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites.

Transcription factors (TFs) bind to thousands of DNA sequences in mammalian genomes, but most of these binding events appear to have no direct effect on gene expression. It is unclear why only a subset of TF bound sites are actively involved in transcriptional regulation. Moreover, the key genomic features that accurately discriminate between active and inactive TF binding events remain ambiguous. Recent studies have identified promoter-distal RNA polymerase II (RNAP2) binding at enhancer elements, suggesting that these interactions may serve as a marker for active regulatory sequences. Despite these correlative analyses, a thorough functional validation of these genomic co-occupancies is still lacking. To characterize the gene regulatory activity of DNA sequences underlying promoter-distal TF binding events that co-occur with RNAP2 and TF sites devoid of RNAP2 occupancy using a functional reporter assay, we performed cis-regulatory element sequencing (CRE-seq). We tested more than 1000 promoter-distal CCAAT/enhancer-binding protein beta (CEBPB)-bound sites in HepG2 and K562 cells, and found that CEBPB-bound sites co-occurring with RNAP2 were more likely to exhibit enhancer activity. CEBPB-bound sites further maintained substantial cell-type specificity, indicating that local DNA sequence can accurately convey cell-type-specific regulatory information. By comparing our CRE-seq results to a comprehensive set of genome annotations, we identified a variety of genomic features that are strong predictors of regulatory element activity and cell-type-specific activity. Collectively, our functional assay results indicate that RNAP2 occupancy can be used as a key genomic marker that can distinguish active from inactive TF bound sites.

An optimized protocol for high-throughput in situ hybridization of zebra finch brain.

In situ hybridization (ISH) is a sensitive technique for documenting the tissue distribution of mRNAs. Advanced nonradioactive ISH methods that are based on the use of digoxigenin (DIG)-labeled probes and chromogenic detection have better spatial resolution than emulsion autoradiography techniques and, when paired with high-resolution digital imaging, allow for large-scale profiling of gene expression at cellular resolution within a histological context. However, technical challenges restrict the number of genes that can be investigated in a small laboratory setting. This protocol describes an optimized, low-cost, small-footprint, high-throughput ISH procedure to detect gene expression patterns in 10-µm brain sections from zebra finches. It uses DIG-labeled riboprobes synthesized from cDNA templates available through the Songbird Neurogenomics Consortium. The method is compatible with high-resolution digital imaging; it produces images with low background and a resolution approaching that of immunohistochemical methods. Approximately 180 slides can be processed each week using this protocol, but it can be scaled to accommodate a broad range of tissues from which cryosections can be obtained.

Genomics analysis of potassium channel genes in songbirds reveals molecular specializations of brain circuits for the maintenance and production of learned vocalizations.

BACKGROUND: A fundamental question in molecular neurobiology is how genes that determine basic neuronal properties shape the functional organization of brain circuits underlying complex learned behaviors. Given the growing availability of complete vertebrate genomes, comparative genomics represents a promising approach to address this question. Here we used genomics and molecular approaches to study how ion channel genes influence the properties of the brain circuitry that regulates birdsong, a learned vocal behavior with important similarities to human speech acquisition. We focused on potassium (K-)Channels, which are major determinants of neuronal cell excitability. RESULTS: We identified 107 K-Channel finch genes, including 6 novel genes common to non-mammalian vertebrate lineages. Twenty human genes are absent in songbirds, birds, or sauropsids, or unique to mammals, suggesting K-Channel properties may be lineage-specific. We also identified specific family members with insertions/deletions and/or high dN/dS ratios compared to chicken, a non-vocal learner. In situ hybridization revealed that while most K-Channel genes are broadly expressed in the brain, a subset is selectively expressed in song nuclei, representing molecular specializations of the vocal circuitry. CONCLUSIONS: Together, these findings shed new light on genes that may regulate biophysical and excitable properties of the song circuitry, identify potential targets for the manipulation of the song system, and reveal genomic specializations that may relate to the emergence of vocal learning and associated brain areas in birds.

Maternal care and altered social phenotype in a recently collected stock of Astatotilapia burtoni cichlid fish.

For over 30 years, the African cichlid fish, Astatotilapia burtoni, has been an important model system for studying the mechanisms underlying socially mediated behavioral change, with the focus being the dominance behavior of males. A recently collected wild-stock (WS) of this species invigorates interest in parallel studies of females' behavior. Here, we describe a robust 'good-mother' phenotype, increased maternal affiliation in fry, and subtle differences in males' behavior that are exhibited by this new stock. While the females of both the laboratory-stock (LS) and the WS brood the developing fry in their buccal cavity, only the WS continues to provide maternal care after initial release of the fry while the LS engage in filial cannibalism. We show that weight loss during starvation, either during brooding or with restriction of food, is greater in the LS than in the WS; thus, the observed behavioral differences may be tied to metabolic differences. The WS also exhibits a robust androgen response to challenge during the maternal care phase. Given the increasing power of genomic tools available for this species, the comparison of these two stocks will offer the opportunity to investigate the genetic and genomic basis of behavioral differences.