Biology subjects

Donczew, R.

Publications and source records attributed to Donczew, R..

7 recordsLinked to original sources

bioRxiv2025

The extra-terminal domain drives the role of BET proteins in transcription

BET proteins facilitate the transcription of most eukaryotic genes, yet the specific mechanisms underlying their function remain incompletely understood. As chromatin readers, BET proteins use tandem bromodomains to recognize acetylated lysine residues on histones and other protein partners. However, recent evidence indicates that bromodomain activity alone does not account for the full spectrum of BET protein functions, underscoring the importance of additional conserved domains. Here, we systematically evaluated all conserved domains of BET proteins and identified the extra-terminal (ET) domain as essential for cell viability, genome-wide transcription, and BET chromatin occupancy. Moreover, we demonstrate that the ET domain exerts these effects by acting as a central hub for interactions with multiple transcriptional regulators. Our findings advance the current understanding of BET protein biology and reveal potential mechanisms by which cells can evade bromodomain inhibition under pathological conditions.

molecular biology↗

bioRxiv2024

An end-to-end workflow to study newly synthesized mRNA following rapid protein depletion in Saccharomyces cerevisiae

Accurate regulation of gene transcription by RNA polymerase II is essential for the growth and development of eukaryotic cells. Although significant progress has been made in understanding the mechanisms that regulate transcription, many questions remain unanswered. Defining the direct effects of transcriptional regulators is critically important to answering these questions. An effective approach for identifying the direct targets of transcriptional regulators is combining rapid protein depletion and quantification of newly transcribed RNA. The auxin-inducible degron (AID) system and thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) are powerful methods to rapidly degrade a target protein and directly quantify newly transcribed RNA, respectively. Both methods have been widely applied to study transcriptional regulation. To address unresolved questions in transcription, we engineered an end-to-end workflow in Saccharomyces cerevisiae to deplete proteins of interest using the AID system and measure newly transcribed RNA using SLAM-seq. We provide a step-by-step protocol to support rapid implementation and demonstrate that the workflow can help define the direct effects of transcriptional regulators using the BET proteins Bdf1 and Bdf2 as a test case. This workflow will help address outstanding questions underlying the molecular basis of transcription and other biological processes in S. cerevisiae and other systems.

molecular biology↗

bioRxiv2023

Surprising connections between DNA binding and function for the near-complete set of yeast transcription factors

DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes. How TFs locate their DNA targets and how multiple TFs cooperate to regulate individual genes is still unclear. Most yeast TFs are thought to regulate transcription via binding to upstream activating sequences, situated within a few hundred base pairs upstream of the regulated gene. While this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way with the large set of yeast TFs. Here, we have integrated information on the binding and expression targets for the near-complete set of yeast TFs. While we found many instances of functional TF binding sites in upstream regulatory regions, we found many more instances that do not fit this model. In many cases, rapid TF depletion affects gene expression where there is no detectable binding of that TF to the upstream region of the affected gene. In addition, for most TFs, only a small fraction of bound TFs regulates the nearby gene, showing that TF binding does not automatically correspond to regulation of the linked gene. Finally, we found that only a small percentage of TFs are exclusively strong activators or repressors with most TFs having dual function. Overall, our comprehensive mapping of TF binding and regulatory targets have both confirmed known TF relationships and revealed surprising properties of TF function.

genomics↗

bioRxiv2021

Mediator is broadly recruited to gene promoters via a Tail-independent mechanism

Mediator (MED) is a conserved factor with important roles in both basal and activated transcription. It is believed that MED plays a direct role in transcriptional regulation at most genes by functionally bridging enhancers and promoters. Here, we investigate the genome-wide roles of yeast MED by rapid depletion of its activator-binding domain (Tail) and monitoring changes in nascent transcription. We find that MED Tail and activator-mediated MED recruitment regulate only a small subset of genes. At most genes, MED bypasses the UAS and is directly recruited to promoters to facilitate transcription initiation. Our results define three classes of genes that differ in PIC assembly pathways and the requirements for MED Tail, SAGA, TFIID and BET factors Bdf1/2. We also find that the depletion of the MED middle module subunit Med7 mimics inactivation of Tail, suggesting a functional link. Our combined results have broad implications for the roles of MED, other coactivators, and mechanisms of transcriptional regulation at different gene classes.

molecular biology↗

bioRxiv2021

BET family members Bdf1/2 modulate global transcription initiation and elongation in Saccharomyces cerevisiae

Human bromodomain-containing BET family members are promising targets for therapy of cancer and immunoinflammatory diseases, but their mechanisms of action and functional redundancies are poorly understood. Bdf1/2, yeast homologues of the human BET factors, were previously proposed to target transcription factor TFIID to acetylated histone H4, analogous to bromodomains that are present within the largest subunit of metazoan TFIID. We investigated the genome-wide roles of Bdf1/2 and found that their important contributions to transcription extend beyond TFIID function, as transcription of many genes is more sensitive to Bdf1/2 than to TFIID depletion. Bdf1/2 co-occupy the majority of yeast promoters and affect preinitiation complex formation through recruitment of TFIID, Mediator and basal transcription factors to chromatin. Surprisingly, we discovered that hypersensitivity of genes to Bdf1/2 depletion results from combined defects in transcription initiation and processive elongation, a striking functional similarity to human BET proteins, most notably Brd4. Our results establish Bdf1/2 as critical for yeast transcription and provide important mechanistic insights into the function of BET proteins in all eukaryotes.

molecular biology↗

bioRxiv2021

An improved ChEC-seq method accurately maps the genome-wide binding of transcription coactivators and sequence-specific transcription factors

Mittal and colleagues have raised questions about mapping transcription factor locations on DNA using the MNase-based ChEC-seq method (Mittal et al., 2021). Partly due to this concern, we modified the experimental conditions of the MNase cleavage step and subsequent computational analyses, resulting in more stringent conditions for mapping protein-DNA interactions (Donczew et al., 2020). The revised method (dx.doi.org/10.17504/protocols.io.bizgkf3w) answers questions raised by Mittal et al. and, without changing earlier conclusions, identified widespread promoter binding of the transcription coactivators TFIID and SAGA at active genes. The revised method is also suitable for accurately mapping the genome-wide locations of DNA sequence-specific transcription factors.

genomics↗

bioRxiv2019

Two separate roles for the transcription coactivator SAGA and a set of genes redundantly regulated by TFIID and SAGA

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes - genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these "coactivator-redundant" genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set. We suggest that this overlapping function is linked to TBP-DNA recruitment. The remaining 87% of expressed genes that we term "TFIID-dependent" are highly sensitive to rapid TFIID depletion and insensitive to rapid SAGA depletion. Genome-wide mapping of SAGA and TFIID found binding of both factors at many genes independent of gene class. DNA analysis suggests that the distinction between the gene classes is due to multiple components rather than any single regulatory factor or promoter sequence motif.

molecular biology↗