We are interested in identifying behaviorally regulated genes in vocal nuclei of the songbird brain. We focus on vocal learning, also a substrate for human speech and a behavior that is regulated. We are identifying genes active in the songbird brain using RNA seq, ChIP-seq and microarrays. Combining these platforms has given us increased knowledge for the molecular basis of learned vocal communication.
To best reflect the complexity of the transcriptome landscape during vocal learning, we created multiple cDNA libraries. We made 21 libraries, included 5 subtraction libraries, with the brains of 60 zebra finches in multiple developmental, pathological, and behavioral states. For a detailed description see the table on the songbirdtranscriptome.net website. From these libraries, we picked over 18,000 clones that we sequenced from both 5’ and 3’ ends. After removing contaminants and failed reactions approximately 13,700 cDNAs were placed into approximately 4,700 clusters (presumptive unique genes) containing approximately 6,100 subclusters (presumptive splice variants). These clusters and subclusters were machine-annotated and followed with multiple rounds of human-curated annotations. The clones were amplified and used to create a spotted cDNA array containing approximately 18,000 amplicons. Then, in both silent and singing birds, we isolated four vocal nuclei [AreaX, the robust nucleus of the arcopallium (RA), the high vocal center (HVC), and the Lateral magnocellular nucleus of the anterior nidopallium (LMAN)]. and found 33 singing regulated genes that we verified by in situ hybridization.
Transcripts from two other independant research groups (Li et al 2007: Replogie et al 2008) were later added to our songbirdtransciptome.net database, increasing the number of singing regulated genes to study. After we added these, the database contained 91,586 transcripts which were clustered, subclustered, and annotated.
We created a Zebra Finch Agilent Oligoarray which comprises of 43,488 60-mer oligos representing transcripts identified by three major research groups (Wada et al 2006; Li et al 2007: Replogie et al 2008). The array contains oligonucleotides representing 10,092 of 18,581 genes predicted in the zebra finch genome by ENSEMBL (v60). The array has performed reliably within and across several bird species. It is available for use either from the Duke University Microarray Center or directly from Agilent (AMADID 021323). For more information about the measured quality of the array in several scenarios, please see Zebrafinch Oligoarray QC.
We used the Agilent array to look at song-induced changes in gene expression in area X over a 7 hour time course. We identified 807 genes that had significant changes in gene expression during singing (figure 5, Nature 2010). We grouped these different temporal expression profiles into 20 clusters. In collaboration with the Sinha Lab, we performed a genome wide scan for transcription factor binding sites. We found that these transcription-factor-binding motifs were enriched in the promoters of late responding genes.
We expanded this study to 3 more song nuclei (HVC, RA, & LMAN) for changes in gene expression due to singing over the same 7 hour time course. We found, in at least one nuclei, that ~1,800 genes were up or down regulated during singing and found 20 genes that were up or down regulated in all four song nuclei.
Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is a method to detect where proteins are bound to the genome. And regions of the genome where histone H3 is acetylated are where enhancers are active. We used ChIP-Seq to identify regions where the acetylated form of histone H3 (H3K27ac) is bound using an antibody to H3K27ac Further, we looked at differences between RA vs Area X to identify different regions of the genome that are active in both these song nuclei.
This allowed us to look for regions of the genome in both the RA and Area X song nuclei that are active as a result of singing.
[Current and former lab members involved in this project: Mukta, Jason, Osceola, Andreas, James, & Lindsey (rotation student).]