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

Southall, Tony D.; Gold, Katrina S.; Egger, Boris; Davidson, Catherine M.; Caygill, Elizabeth E.; Marshall, Owen J.; Brand, Andrea H.

doi:10.1016/j.devcel.2013.05.020

Cited by 236 publications

(422 citation statements)

References 79 publications

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“…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al, 2001), expression of dominant-negative ey forms (Morante et al, 2011) or RNAi-mediated ey and toy knockdowns (our unpublished data), is compatible with these genes not playing a direct role in lamina development, at least during larval stages.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).…”

Section: Discussionmentioning

confidence: 96%

“…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al, 2001), expression of dominant-negative ey forms (Morante et al, 2011) or RNAi-mediated ey and toy knockdowns (our unpublished data), is compatible with these genes not playing a direct role in lamina development, at least during larval stages.…”

Section: Discussionmentioning

confidence: 99%

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

2014

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The visual system of insects is a multilayered structure composed externally by the compound eye and internally by the three ganglia of the optic lobe: lamina, medulla and the lobula complex. The differentiation of lamina neurons depends heavily on Hedgehog (Hh) signaling, which is delivered by the incoming photoreceptor axons, and occurs in a wave-like fashion. Despite the primary role of lamina neurons in visual perception, it is still unclear how these neurons are specified from neuroepithelial (NE) progenitors. Here we show that a homothorax (hth)-eyes absent (eya)-sine oculis (so)-dachshund (dac) gene regulatory cassette is involved in this specification. Lamina neurons differentiate from NE progenitors that express hth, eya and so. One of the first events in the differentiation of lamina neurons is the upregulation of dac expression in response to Hh signaling. We show that this dac upregulation, which marks the transition from NE progenitors into lamina precursors, also requires Eya/So, the expression of which is locked in by mutual feedback. dac expression is crucial for lamina differentiation because it ensures repression of hth, a negative regulator of single-minded, and thus dac allows further lamina neuron differentiation. Therefore, the specification of lamina neurons is controlled by coupling the cell-autonomous hth-eya-so-dac regulatory cassette to Hh signaling.

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

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

2014

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“…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.…”

Section: Introductionmentioning

confidence: 99%

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

McMahon

Rahman

Jin

et al. 2016

Cell

200

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RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of a RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (Targets of RNA-binding proteins Identified By Editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1 and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.

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“…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). …”

Section: Introductionmentioning

confidence: 99%

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

et al. 2016

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DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.

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

Cited by 236 publications

References 79 publications

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

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

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

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

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