Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells

Abstract

SummaryCell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates.

A conserved transcriptional network regulates lamina development in the Drosophila visual system

“…S4), and all available information on ey function, including that from ey mutants (Callaerts et al, 2001), expression of dominant-negative ey forms (Morante et al, 2011) or RNAi-mediated ey and toy knockdowns (our unpublished data), is compatible with these genes not playing a direct role in lamina development, at least during larval stages. Also, neither tsh nor its paralog tio is expressed in the OPC NE (Southall et al, 2013; data not shown), although cells of the OPC NE are ready to respond to tsh expression by proliferating and blocking lamina differentiation (supplementary material Fig. S5).…”

“…However, there are several profound differences. Neither ey nor toy, the two Pax6 genes positioned at the top of the genetic hierarchy of eye development, is expressed during lamina development (Callaerts et al, 2001;Morante et al, 2011;Southall et al, 2013) (supplementary material Fig. S4), and all available information on ey function, including that from ey mutants (Callaerts et al, 2001), expression of dominant-negative ey forms (Morante et al, 2011) or RNAi-mediated ey and toy knockdowns (our unpublished data), is compatible with these genes not playing a direct role in lamina development, at least during larval stages.…”

TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins

“…It is called TRIBE, T argets of R NA-binding proteins I dentified B y E diting, and is particularly well-suited to identify target RNAs within small numbers of cells. TRIBE is conceptually similar to the DNA-oriented ‘targeted DamID’ (van Steensel and Henikoff, 2000, Southall et al, 2013) and entails in vivo expression of a fusion protein between an RBP and the catalytic domain of the RNA-editing enzyme ADAR. The transcriptome is then sequenced to identify novel editing events introduced by the RPB-ADARcd.…”

iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

“…The PCR products are hybridized to microarrays or used directly for deep sequencing. In summary, DamID requires relatively low input material and processing time, is cost-effective and accurately reflects ChIP results (Southall et al, 2013). …”