Quantitative proteomic analysis of the interactome of mammalian S/MAR (scaffold/matrix attachment region) elements​

Abstract: Matrix Attachment Regions (MARs) are eukaryotic regulatory DNA sequences that have been shown to play a key role in gene expression regulation and are believed to spatially organize chromatin within the nucleus by mediating binding of DNA to the nuclear matrix (NM), an operationally defined sub-nuclear entity. In the context of chromatin, MARs exhibit potent gene anti-silencing and expression augmentation activities. Due to these properties, MARs found many biotechnological applications in addition to their key application in constructing stably replicating episomal vectors that could provide (MAR-dependent) long-term heterologous transgene expression without integrating into the host DNA. Despite the well-documented function of MARs, their precise mode of action is still enigmatic, a fact that can mainly be attributed to our limited knowledge concerning MAR binding proteins.
In order to investigate the mammalian MAR interactome, we combined DNA oligo pull-down assays with, the well-established SILAC-based proteomics method for DNA-protein interaction screening (“reverse ChIP”). In this approach, functional MARs are immobilized on magnetic beads and subsequently used for pull-down assays employing HeLa nuclear extract and solubilized chromatin fractions. Additionally, the process of nuclear chromatin pellet solubilization was optimized in order to make this fraction compatible with affinity purification on DNA baits. Enriched proteins are identified and quantified by nanoLC-MS/MS. For bait design we focus on human interferon ß gene MAR as a model, because it harbors a well characterized base pair unwinding region (BUR), which is deemed to be essential for MAR function. With the aim to uncover basic principles of MAR function we have focused in this study on three interferon ß MAR subfragment baits that have previously been characterized (literature). These consist of a short bait of 156bp (156bp MAR fragment), which has the minimal sequence exhibiting affinity towards NM preparations as well as of two long baits, namely the 739bp MAR sequence construct and the tetrameric version of the short bait (tetramer of 155bp MAR sequence). The latter two were previously shown to be sufficient for stable episomal vector replication and propagation. As a control, we utilize MARs bearing a mutated BUR sequence (leading to the loss of MAR biological activity) or WT sequences with a reversed DNA sequence polarity. The latter exhibits the same GC/AT content with a concomitant loss of virtually all WT-specific transcription factor (TF) binding sites. This approach helps us to determine whether a specific MAR-protein interaction is BUR-dependent or BUR-independent.
The initial ”reverse ChIP” screen, resulted in the identification of 89 and 46 potential MAR binders from nuclear extract and solubilized nuclear pellet, respectively. In order to validate the potential MAR interactors, a parallel reaction monitoring approach (PRM) was employed to perform a second screen. PRM represents a targeted hypothesis-driven methodology that is mainly used for confirming diagnosis-relevant biomarkers or potential candidates derived from differential protein display assays (e.g. quantitative shotgun proteomics). Totally, sixty five percent of putative MAR binders underwent the validation process using the PRM approach. Among four validated BUR-dependent MAR binders, the transcription factors (TFs) NFKB1 (p105) and ELF2 were identified as common proteins binding to all three WT human interferon ß gene MAR (subfragment) baits. Notably, despite the specific interaction of several other TFs, only NFKB1 and ELF2 binding is shared between the biologically fully active MAR constructs (the 739bp and tetrameric bait). Additionally, the TF YY1, which is a facultative member of the INO80 chromatin remodeling complex, was the only common candidate enriched on those fragments in a BUR-independent MAR manner (out of a total of 32 confirmed BUR-independent MAR binders).
Generally, a high number of multifunctional TFs were found among all the confirmed human interferon ß gene MAR binders but many exhibited selective specificity towards only one of the (three) WT MAR subfragments studied in this work. A common hallmark of these proteins is their ability to either bind to repressed chromatin or to maintain an open chromatin state, potentially enabling the assembly of the RNA polymerase II transcription machinery. For example, four other INO80 chromatin remodeling complex members got validated as BUR-independent tetramer bait specific interactors, and it could be envisioned that their MAR binding is facilitated by YY1 and possibly stabilized by the concatemerization of the construct.
These findings lead us to speculate that the biological function of MARs could be mediated by the recruitment of certain TFs (possessing a vast spectrum of different biological activities) that might serve as platforms for the assembly of proteins with more specific activities rather than the recruitment of one universal “key” trans-acting factor. Finally, in the context of MAR biological activity, the biophysical effects of its AT-rich sequence context together with the BUR feature (independent of trans-acting factors) in maintaining an open chromatin structure awaits future studies

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2018

Classification and Topics

• Keyword: Matrix Attachment Region

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2019
• Creator: Puchalska, Monika
• Contributor: Mittler, Gerhard ; Albert-Ludwigs-Universität Freiburg. Fakultät für Biologie

Further information

• DOI: 10.6094/UNIFR/149956
• URN: urn:nbn:de:bsz:25-freidok-1499567
• Rights: Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 25.03.2025, 1:49 PM CET