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

Abstract

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.

Keywords: Mediator; RNA tethering; RepA; SILAC; TDP-43; Xist; activator; chromatin; lncRNA; transcription activation.

Figure 1. Profiling regulated protein complex assembly on reconstituted chromatin platforms.

A) Assembly of protein complexes on chromatin regulates many genomic processes. B) Scheme for SILAC labeling and profiling of the interactions of nuclear proteins with specific reconstituted chromatin substrates. C) DNA template used in all chromatin profiling experiments in the current study: a biotinylated PCR product containing an adenovirus E4 promoter with upstream Gal4 binding sites and LexA sequences flanked on each side by 601 nucleosome positioning sequences. D) General chromatin assembly scheme. E. coli-purified human histones, assembled into octamers were enzymatically assembled into chromatin using the DNA template described above using histone chaperone and nucleosome spacing factors. E) Limited micrococcal nuclease digestion of assembled chromatin demonstrating nucleosome-protection of DNA in a regular array.

Figure 2. SILAC-based profiling of early steps in transcriptional activation by Gal4-VP16.

A) Scheme of chromatin substrates compared by SILAC-mass spectrometry. B) SILAC profile of proteins enriched by Gal4-VP16 binding to chromatin. Replicate experiments with swapped, labeled nuclear extracts were compared. Mediator subunits are highlighted in red. C) Analysis of Mediator subunits identified in the SILAC profiling of GV enrichment on chromatin. (left) Subunits of Mediator subcomplexes listed with indicated identification in each SILAC experiment. (right) Cryo-EM structure of core Mediator from (permission will be obtained upon manuscript acceptance) with appropriate color-coding. D) Additional selected chromatin and transcription-associated proteins identified as enriched with Gal4-VP16 in either heavy (pink) or light (blue) sample only.

Figure 3. Profiling of a long noncoding RNA involved in mammalian X chromosome inactivation.

A) Dosage compensation in mammals is dependent on the lncRNA Xist for silencing of one X chromosome (ChrX) to an inactive state (Xi). (bottom) domain structure for Xist lncRNA, depicting the repeat regions A-E. Expanded view of the RepA transcript, which can be transcribed independently of full-length Xist. RepA harbors critical RNA regions important for chromosome silencing. B) Schematic of the two types of chromatin platforms profiled by SILAC-MS. RNAs are tethered to reconstituted chromatin via a LexA-PP7 fusion protein that binds LexA DNA sites on the chromatin and also binds PP7 aptamer-tagged in vitro transcribed RNAs. The control long noncoding RNA in this experiment is derived from the antisense sequence of the luciferase mRNA, which is similar in length and GC content to RepA. C) Profile of replicated SILAC-MS experiments relative to enrichment in the RepA-containing chromatin samples. D) Expanded view from C (red box) highlighted the specifically RepA-enriched proteins. E) Western validation for RepA enrichment of TDP-43 and PDCD7. Note, the most enriched band in the PDCD7 blot likely corresponds to a low-abundance multiply-ubiquitinated form identified in previous surveys of ubiquitnation sites^–. F) RNA immunoprecipitation of TDP-43 validating its interaction with RepA in MCF7 female breast cancer cells expressing Xist.