Over 40% of proteins in any eukaryotic genome encode intrinsically disordered regions (IDRs) that do not adopt defined tertiary structures. Certain IDRs perform critical functions, but discovering them is non‐trivial as the biological context determines their function. We present IDR‐Screen, a framework to discover functional IDRs in a high‐throughput manner by simultaneously assaying large numbers of DNA sequences that code for short disordered sequences. Functionality‐conferring patterns in their protein sequence are inferred through statistical learning. Using yeast HSF1 transcription factor‐based assay, we discovered IDRs that function as transactivation domains (TADs) by screening a random sequence library and a designed library consisting of variants of 13 diverse TADs. Using machine learning, we find that segments devoid of positively charged residues but with redundant short sequence patterns of negatively charged and aromatic residues are a generic feature for TAD functionality. We anticipate that investigating defined sequence libraries using IDR‐Screen for specific functions can facilitate discovering novel and functional regions of the disordered proteome as well as understand the impact of natural and disease variants in disordered segments.

The stress response in yeast cells is regulated by at least two classes of transcription activators-HSF and Msn2/4, which differentially affect promoter chromatin remodeling. We demonstrate that the deletion of SNF2, an ATPase activity-containing subunit of the chromatin remodeling SWI/SNF complex, eliminates histone displacement, RNA polymerase II recruitment, and heat shock factor (HSF) binding at the HSP12 promoter while delaying these processes at the HSP82 and SSA4 promoters. Out of the three promoters, the double deletion of MSN2 and MSN4 eliminates both chromatin remodeling and HSF binding only at the HSP12 promoter, suggesting that Msn2/4 activators are primary determinants of chromatin disassembly at the HSP12 promoter. Unexpectedly, during heat shock the level of Msn2/4 at the HSP12 promoter declines. This is likely a result of promoter-targeted Msn2/4 degradation associated with transcription complex assembly. While histone displacement kinetic profiles bear clear promoter specificity, the kinetic profiles of recovery from heat shock for all analyzed genes display an equal or even higher nucleosome return rate, which is to some extent delayed by the deletion of SNF2.The stress response in yeast cells is regulated by at least two types of transcriptional activators: heat shock factor (HSF) and the partially redundant Msn2 and Msn4 (Msn2/4) activators (7,19). The HSF system is highly conserved in its overall composition and function from yeast to humans (61). HSF binds to the major groove of DNA in heat shock promoter elements (HSEs), which are also conserved from yeast to humans (61). The activity of HSF is regulated via several distinct pathways. These include a monomer-trimer transition (45), phosphorylation, and other posttranslational modifications (30, 31, 54), as well as repression by molecular chaperones interacting with HSF, thus blocking their own production (45, 60) and forming a self-regulatory loop.The Msn2/4 system is more specific to Saccharomyces cerevisiae. The Msn2 and Msn4 factors recognize and bind stress response element (STRE) sequences (44) in promoter regions of a large array of genes partially overlapping the HSF-regulated array (3,7,22,46). The Msn2 factor seems to have a more pronounced role, since mutants lacking only Msn2 have an already distinct decrease in STRE-regulated transcription; however, only MSN2 and MSN4 double deletions exhibit pleiotropic stress sensitivity (19). Under stress, Msn2/4 accumulate in the nucleus within a few minutes (24, 32). The Msn2/4 factors are regulated by efficient and oscillatory nuclear transport (32), hyperphosphorylation upon stress (21, 24), and degradation associated with transcription initiation (37, 38). While the HSF system is actively involved in chromatin remodeling events at gene promoters, the role of the Msn2/4 system in these processes is poorly understood.Chromatin remodeling varies in intensity and intermediate states between genes. The most evident and intense examples of chromatin remodeling are the changes taking place at ...

Chromatin remodeling at promoters of activated genes spans from mild histone modifications to outright displacement of nucleosomes in trans. Factors affecting these events are not always clear. Our results indicate that histone H3 acetylation associated with histone displacement differs drastically even between promoters of such closely related heat shock genes as HSP12, SSA4, and HSP82. The HSP12 promoter, with the highest level of histone displacement, showed the highest level of H3 acetylation, while the SSA4 promoter, with a lower histone displacement, showed only modest H3 acetylation. Moreover, for the HSP12 promoter, the level of acetylated H3 is temporarily increased prior to nucleosome departure. Individual promoters in strains expressing truncated versions of heat shock factor (HSF) showed that deletion of either one of two activating regions in HSF led to the diminished histone displacement and correspondingly lower H3 acetylation. The deletion of both regions simultaneously severely decreased histone displacement for all promoters tested, showing the dependence of these processes on HSF. The level of histone H3 acetylation at individual promoters in strains expressing truncated HSF also correlated with the extent of histone displacement. The beginning of chromatin remodeling coincides with the polymerase II loading on heat shock gene promoters and is regulated either by HSF binding or activation of preloaded HSF.Chromatin changes at promoters of eukaryotic genes play an essential role in regulation of transcription. These changes can range from posttranslational modifications of single amino acid residues in histones to more widespread histone modifications and finally to nucleosome displacement from promoters. Although assembly of the transcription initiation complex is antagonized by the presence of nucleosomes at gene promoters, full nucleosome displacement from the promoter is not always observed. Nucleosome displacement events are usually accompanied by posttranslational modifications of histones. These posttranslational modifications, often occurring in a cascade manner influencing one another, lie at the foundation of the "histone code" hypothesis (25,46). The most heavily characterized histone modifications are acetylation of lysines produced by the action of histone acetyltransferase (HAT)-containing complexes, such as SAGA, ADA, NuA3, NuA4, and others. The histone acetylation often leads to the loss of some histone-DNA bonds and to the formation of a distinct chromatin surface recognized by chromatin-remodeling coactivators bearing bromodomains (10, 22). These coactivators often belong to the class of ATP-dependent chromatin remodeling complexes, which include such multisubunit complexes as SWI/ SNF, RSC, ISWI, and others. These chromatin remodeling complexes use the energy released from ATP hydrolysis to destabilize and finally push away promoter nucleosomes either in cis along the DNA (15, 31) or in trans, completely detaching histones from DNA (5, 28). It is not always clear if histone m...

Chromatin remodeling is an essential part of transcription initiation. We show that at heat shock gene promoters functional interactions between individual ATP-dependent chromatin remodeling complexes play critical role in both nucleosome displacement and Pol II recruitment. Using HSP12, HSP82 and SSA4 gene promoters as reporters, we demonstrated that while inactivation of SNF2, a critical ATPase of the SWI/SNF complex, primarily affects the HSP12 promoter, depletion of STH1- a SNF2 homolog from the RSC complex reduces histone displacement and abolishes the Pol II recruitment at all three promoters. From these results, we conclude that redundancy between SWI/SNF and RSC complexes is only partial and likely is affecting different chromatin remodeling steps. While inactivation of other individual ATP-dependent chromatin remodeling complexes negligibly affects reporter promoters, combinatorial inactivation of SNF2 and ISW1 has a synergistic effect by diminishing histone loss during heat induction and eliminating Pol II recruitment. Importantly, it also eliminates preloading of HSF on HSP82 and SSA4 promoters before heat shock and diminishes HSF binding during heat shock. These observations suggest that prior action of chromatin remodeling complexes is necessary for the activator binding.