This Title All WIREs
How to cite this WIREs title:
WIREs Dev Biol
Impact Factor: 3.883

Dam it's good! DamID profiling of protein‐DNA interactions

Full article on Wiley Online Library:   HTML PDF

Can't access this content? Tell your librarian.

The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin‐binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein–DNA interactions are important in the adult, where their perturbation can lead to disruption of homeostasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein–DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin‐binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology. WIREs Dev Biol 2016, 5:25–37. doi: 10.1002/wdev.205 This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Technologies > Analysis of Proteins Technologies > Analysis of the Transcriptome
Schematic illustrating cell type‐specific chromatin profiling with targeted DamID (TaDa). (a) Dam can be expressed in individual cell populations e.g., individual neuronal populations (green) within the CNS (gray). (b) In cells in which Gal4 is expressed (green), the bicistronic transcript is expressed at high levels, leading to low‐level translation of the secondary open reading frame (ORF) containing the Dam‐fusion. Where Gal4 is not expressed (grey), there is minimal expression of the bicistronic transcript. (c) Multiple uses for Tada depending on Dam‐fusion protein. RNA polymerase II (PolII)—Dam‐fusions can be used to generate a transcriptional profile for an individual population of cells. Transcription factor (TF)—Dam‐fusions may highlight differences in TF binding between cell types. Chromatin proteins (e.g., Polycomb—Pc), can be used to highlight differences in chromatin state between cell types.
[ Normal View | Magnified View ]
DNA adenine methyltransferase identification (DamID) applications. (a) Dam fused to a transcription factor (TF) with a known binding site can be used to methylate proximal DNA sequences in both cis and trans. (b) Dam fused to a histone modifier (e.g., a histone deacetylase—HDAC) or chromatin remodeler can be used to give an indication of chromatin state. (c) Dam fused to proteins which do not directly interact with DNA can methylate proximal loci. (d) Dam fused to an insulator protein will methylate all proximal and interacting sequences. (e) Targeting of Dam in a locus‐specific manner using sequence‐specific gene targeting tools, e.g., Gal4/upstream activation sequence (UAS) can be used to detect methylation in trans. (f) Dam‐fusions with proteins that make up the nuclear environment (e.g., lamins), can be used to determine interactions with the nuclear lamina or other nuclear compartments.
[ Normal View | Magnified View ]
Schematic illustrating DNA adenine methyltransferase identification (DamID) experimental pipeline. (a) Dam only or Dam fused to a protein of interest (POI) (blue) is expressed in a suitable cell type or transgenic organism. (b) Genomic DNA is extracted. DNA obtained includes N6‐adenine methylation sites (Me) catalyzed by Dam. (c) Genomic DNA is digested by the methylation sensitive restriction enzyme, DpnI. (d) Digested fragments are amplified by polymerase chain reaction (PCR). (e) Representative output indicating chromatin binding of a protein of interest at an individual locus. Vertical bars indicate the log2 ratio of Dam‐fusion/Dam only.
[ Normal View | Magnified View ]

Browse by Topic

Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics
Technologies > Analysis of Proteins
Technologies > Analysis of the Transcriptome