Grammar rules and exceptions for the language of transcriptional activation domains.

Transcriptional activation domains (ADs) of gene activators have remained enigmatic for decades as short, extremely variable, and structurally disordered sequences. Using a rational design and high throughput experimentation, we determine the grammar rules and exceptions for the language of ADs. According to identified rules, billions of highly active ADs can be composed of balanced amounts of acidic/aromatic amino acids, with either mixed composition of aromatic residues, or using only one aromatic residue mixed with acidic residues.

High-throughput discovery of functional disordered regions: investigation of transactivation domains.

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.

Nucleosome distortion as a possible mechanism of transcription activation domain function.

After more than three decades since the discovery of transcription activation domains (ADs) in gene-specific activators, the mechanism of their function remains enigmatic. The widely accepted model of direct recruitment by ADs of co-activators and basal transcriptional machinery components, however, is not always compatible with the short size yet very high degree of sequence randomness and intrinsic structural disorder of natural and synthetic ADs. In this review, we formulate the basis for an alternative and complementary model, whereby sequence randomness and intrinsic structural disorder of ADs are necessary for transient distorting interactions with promoter nucleosomes, triggering promoter nucleosome translocation and subsequently gene activation.

High-throughput screening system for inhibitors of human Heat Shock Factor 2.

Development of novel anti-cancer drug leads that target regulators of protein homeostasis is a formidable task in modern pharmacology. Finding specific inhibitors of human Heat Shock Factor 1 (hHSF1) has proven to be a challenging task, while screening for inhibitors of human Heat Shock Factor 2 (hHSF2) has never been described. We report the development of a novel system based on an in vivo cell growth restoration assay designed to identify specific inhibitors of human HSF2 in a high-throughput format.

ASF1 and the SWI/SNF complex interact functionally during nucleosome displacement, while FACT is required for nucleosome reassembly at yeast heat shock gene promoters during sustained stress.

Histone chaperones are an integral part of the transcription regulatory machinery. We investigated the involvement of histone chaperones and their functional interactions with ATP-dependent chromatin remodeling complexes in the regulation of yeast heat shock genes. Strong functional interaction between the histone chaperone ASF1 and the ATP-dependent chromatin remodeling complex SWI/SNF is exhibited in synergistic diminishment of nucleosome displacement during heat shock in the ΔASF1/ΔSNF2 strain in comparison to individual ASF1 or SNF2 inactivation.

Detection of transcriptional activators, co-activators, and chromatin remodeling by chromatin immunoprecipitation coupled with real-time PCR.

Investigation of DNA-protein interactions is a key approach in understanding mechanisms of gene regulation. The method described allows detection of dynamic DNA-protein interactions occurring at gene promoters in living cells during the time scale of seconds and minutes. The combination of chromatin immunoprecipitation with real-time PCR allows for detection of changes in activator and co-activator content of any promoter during transcriptional activation.

Functional interplay between chromatin remodeling complexes RSC, SWI/SNF and ISWI in regulation of yeast heat shock genes.

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.

[Alternative ways of stress regulation in cells of Saccharomyces cerevisiae: transcriptional activators Msn2 and Msn4].

Cell response to stress at the transcriptional level is characterized by the rapid expression of a large set of genes. In yeast Saccharomyces cerevisiae this gene activation is determined by the action of two types of activators--HSF and partially redundant Msn2 and Msn4. While HSF activation mechanisms are relatively well established during the last decade, the mechanisms of regulation by Msn2/4 started to clarify only recently.

Different requirements of the SWI/SNF complex for robust nucleosome displacement at promoters of heat shock factor and Msn2- and Msn4-regulated heat shock genes.

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.

Displacement of histones at promoters of Saccharomyces cerevisiae heat shock genes is differentially associated with histone H3 acetylation.

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.