SILAC-MS Profiling of Reconstituted Human Chromatin Platforms for the Study of Transcription and RNA Regulation

DNA
packaged into chromatin is the core structure of the human
genome. Nearly all eukaryotic genome regulation must interface with
this genomic structure, and modification of the chromatin can influence
molecular mechanisms that regulate the underlying DNA. Many processes
are governed by regulated stepwise assembly mechanisms that build
complex machinery on chromatin to license a specific activity such
as transcription. Transcriptional activators drive the initial steps
of gene expression, regulated in part by chromatin. Here we describe
tools to study the stepwise assembly of protein complexes on chromatin
in a highly controlled manner using reconstituted human chromatin
platforms and quantitative proteomic profiling. We profile the early
steps in transcriptional activation and highlight the potential for
understanding the multiple ways chromatin can influence transcriptional
regulation. We also describe modifications of this approach to study
the activity of a long noncoding RNA to act as a dynamic scaffold
for proteins to be recruited to chromatin. This approach has the potential
to provide a more comprehensive understanding of important macromolecular
complex assembly that occurs on the human genome. The reconstituted
nature of the chromatin substrate offers a tunable system that can
be trapped at specific substeps to understand how chromatin interfaces
with genome regulation machinery.