Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

SCORe: SARS-CoV-2 Omicron Variant RBD-Binding DNA Aptamer for Multiplexed Rapid Detection and Pseudovirus Neutralization.

During the COVID-19 (coronavirus disease 2019) pandemic, several SARS-CoV-2 variants of concern emerged, including the Omicron variant, which has enhanced infectivity and immune invasion. Many antibodies and aptamers that bind the spike (S) of previous strains of SARS-CoV-2 either do not bind or bind with low affinity to Omicron S. In this study, we report a high-affinity SARS-CoV-2 Omicron RBD-binding aptamer (SCORe) that binds Omicron BA.1 and BA.2 RBD with nanomolar KD1. We employ aptamers SCORe.50 and SNAP4.74 in a multiplexed lateral flow assay (LFA) to distinguish between Omicron and wild-type S at concentrations as low as 100 pM. Finally, we show that SCORe.50 and its dimerized form SCOReD can neutralize Omicron S-pseudotyped virus infection of ACE2-overexpressing cells by >70%. SCORe therefore has potential applications in COVID-19 rapid diagnostics as well as in viral neutralization.

Aptamer Sandwich Lateral Flow Assay (AptaFlow) for Antibody-Free SARS-CoV-2 Detection.

The COVID-19 pandemic is among the greatest health and socioeconomic crises in recent history. Although COVID-19 vaccines are being distributed, there remains a need for rapid testing to limit viral spread from infected individuals. We previously identified the SARS-CoV-2 spike protein N-terminal domain (NTD) binding DNA aptamer 1 (SNAP1) for detection of SARS-CoV-2 virus by aptamer-antibody sandwich enzyme-linked immunoassay (ELISA) and lateral flow assay (LFA). In this work, we identify a new aptamer that also binds at the NTD, named SARS-CoV-2 spike protein NTD-binding DNA aptamer 4 (SNAP4). SNAP4 binds with high affinity (<30 nM) for the SARS-CoV-2 spike protein, a 2-fold improvement over SNAP1. Furthermore, we utilized both SNAP1 and SNAP4 in an aptamer sandwich LFA (AptaFlow), which detected SARS-CoV-2 UV-inactivated virus at concentrations as low as 106 copies/mL. AptaFlow costs <$1 per test to produce, provides results in <1 h, and detects SARS-CoV-2 at concentrations that indicate higher viral loads and a high probability of contagious transmission. AptaFlow is a potential approach for a low-cost, convenient antigen test to aid the control of the COVID-19 pandemic.

Discovery and Characterization of Spike N-Terminal Domain-Binding Aptamers for Rapid SARS-CoV-2 Detection.

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.

Identification of a DNA Aptamer That Binds to Human Monocytes and Macrophages.

As cancer strategies shift toward immunotherapy, the need for new binding ligands to target and isolate specific immune cell populations has soared. Based on prior work identifying a peptide specific for murine M2-like macrophages, we sought to identify an aptamer that could bind human M2-like macrophages. Tumor-associated macrophages (TAMs) adopt an M2-like phenotype and support tumor progression and dissemination. Here, we employed cell-SELEX to identify an aptamer ligand that targets this cell population over tissue resident (M0-like) or tumoricidal (M1-like) macrophages. Instead, we identified an aptamer that binds both human M0- and M2-like macrophages and monocytes, with highest binding affinity to M2-like macrophage (Kd ∼ 20 nM) and monocytes (Kd ∼ 45 nM) and minimal binding to other leukocytes. The aptamer binds to CD14+ but not CD16+ monocytes, and is rapidly internalized by these cells. We also demonstrate that this aptamer is able to bind human monocytes when both are administered in vivo to mice. Thus, binding to these cell populations (monocytes, M0-like and M2-like macrophages), this aptamer lends itself toward monocyte-specific applications, such as monocyte-targeted drug delivery or column selection.

Efficient Identification of Murine M2 Macrophage Peptide Targeting Ligands by Phage Display and Next-Generation Sequencing.

Peptide ligands are used to increase the specificity of drug carriers to their target cells and to facilitate intracellular delivery. One method to identify such peptide ligands, phage display, enables high-throughput screening of peptide libraries for ligands binding to therapeutic targets of interest. However, conventional methods for identifying target binders in a library by Sanger sequencing are low-throughput, labor-intensive, and provide a limited perspective (<0.01%) of the complete sequence space. Moreover, the small sample space can be dominated by nonspecific, preferentially amplifying "parasitic sequences" and plastic-binding sequences, which may lead to the identification of false positives or exclude the identification of target-binding sequences. To overcome these challenges, we employed next-generation Illumina sequencing to couple high-throughput screening and high-throughput sequencing, enabling more comprehensive access to the phage display library sequence space. In this work, we define the hallmarks of binding sequences in next-generation sequencing data, and develop a method that identifies several target-binding phage clones for murine, alternatively activated M2 macrophages with a high (100%) success rate: sequences and binding motifs were reproducibly present across biological replicates; binding motifs were identified across multiple unique sequences; and an unselected, amplified library accurately filtered out parasitic sequences. In addition, we validate the Multiple Em for Motif Elicitation tool as an efficient and principled means of discovering binding sequences.

Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection.

Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (∼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications.

Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells.

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

Aptamers 101: aptamer discovery and in vitro applications in biosensors and separations.

Aptamers are single-stranded nucleic acids that bind and recognize targets much like antibodies. Recently, aptamers have garnered increased interest due to their unique properties, including inexpensive production, simple chemical modification, and long-term stability. At the same time, aptamers possess similar binding affinity and specificity as their protein counterpart. In this review, we discuss the aptamer discovery process as well as aptamer applications to biosensors and separations. In the discovery section, we describe the major steps of the library selection process for aptamers, called systematic evolution of ligands by exponential enrichment (SELEX). We highlight common approaches and emerging strategies in SELEX, from starting library selection to aptamer-target binding characterization. In the applications section, we first evaluate recently developed aptamer biosensors for SARS-CoV-2 virus detection, including electrochemical aptamer-based sensors and lateral flow assays. Then we discuss aptamer-based separations for partitioning different molecules or cell types, especially for purifying T cell subsets for therapeutic applications. Overall, aptamers are promising biomolecular tools and the aptamer field is primed for expansion in biosensing and cell separation.

Aptamer-Based Chromatographic Methods for Efficient and Economical Separation of Leukocyte Populations.

The manufacturing process of chimeric antigen receptor T cell therapies includes isolation systems that provide pure T cells. Current magnetic-activated cell sorting and immunoaffinity chromatography methods produce desired cells with high purity and yield but require expensive equipment and reagents and involve time-consuming incubation steps. Here, we demonstrate that aptamers can be employed in a continuous-flow resin platform for both depletion of monocytes and selection of CD8+ T cells from peripheral blood mononuclear cells at low cost with high purity and throughput. Aptamer-mediated cell selection could potentially enable fully synthetic, traceless isolations of leukocyte subsets from a single isolation system.

Aptamer-Based Traceless Multiplexed Cell Isolation Systems.

In both biomedical research and clinical cell therapy manufacturing, there is a need for cell isolation systems that recover purified cells in the absence of any selection agent. Reported traceless cell isolation methods using engineered antigen-binding fragments or aptamers have been limited to processing a single cell type at a time. There remains an unmet need for cell isolation processes that rapidly sort multiple target cell types. Here, we utilized two aptamers along with their designated complementary strands (reversal agents) to tracelessly isolate two cell types from a mixed cell population with one aptamer-labeling step and two sequential cell elution steps with reversal agents. We engineered a CD71-binding aptamer (rvCD71apt) and a reversal agent pair to be used simultaneously with our previously reported traceless purification approach using the CD8 aptamer (rvCD8apt) and its reversal agent. We verified the compatibility of the two aptamer displacement mechanisms by flow cytometry and the feasibility of incorporating rvCD71apt with a magnetic solid state. We then combined rvCD71apt with rvCD8apt to isolate activated CD4+ T cells and resting CD8+ cells by eluting these target cells into separate fractions with orthogonal strand displacements. This is the first demonstration of isolating different cell types using two aptamers and reversal agents at the same time. Potentially, different or more aptamers can be included in this traceless multiplexed isolation system for diverse applications with a shortened operation time and a lower production cost.

Snf1 controls the activity of adr1 through dephosphorylation of Ser230.

The transcription factors Adr1 and Cat8 act in concert to regulate the expression of numerous yeast genes after the diauxic shift. Their activities are regulated by Snf1, the yeast homolog of the AMP-activated protein kinase of higher eukaryotes. Cat8 is regulated directly by Snf1, but how Snf1 regulates Adr1 is unknown. Mutations in Adr1 that alleviate glucose repression are clustered between amino acids 227 and 239. This region contains a consensus sequence for protein kinase A, RRAS(230)F, and Ser230 is phosphorylated in vitro by both protein kinase A and Ca(++) calmodulin-dependent protein kinase. Using an antiphosphopeptide antibody, we found that the level of Adr1 phosphorylated on Ser230 was highest in glucose-grown cells and decreased in a Snf1-dependent manner when glucose was depleted. A nonphosphorylatable Ser230Ala mutant was no longer Snf1 dependent for activation of Adr1-dependent genes and could suppress Cat8 dependence at genes coregulated by Adr1 and Cat8. Contrary to expectation, neither protein kinase A (PKA) nor Ca(++) calmodulin-dependent protein kinase appeared to have an important role in Ser230 phosphorylation in vivo, and a screen of 102 viable kinase deletion strains failed to identify a candidate kinase. We conclude that either Ser230 is phosphorylated by multiple protein kinases or its kinase is encoded by an essential gene. Using the Ser230Ala mutant, we explain a long-standing observation of synergy between Adr1 constitutive mutants and Snf1 activation and conclude that dephosphorylation of Ser230 via a Snf1-dependent pathway appears to be a major component of Adr1 regulation.

Traceless aptamer-mediated isolation of CD8+ T cells for chimeric antigen receptor T-cell therapy.

Chimeric antigen receptor T-cell therapies using defined product compositions require high-purity T-cell isolation systems that, unlike immunomagnetic positive enrichment, are inexpensive and leave no trace on the final cell product. Here, we show that DNA aptamers (generated with a modified cell-SELEX procedure to display low-nanomolar affinity for the T-cell marker CD8) enable the traceless isolation of pure CD8+ T cells at low cost and high yield. Captured CD8+ T cells are released label-free by complementary oligonucleotides that undergo toehold-mediated strand displacement with the aptamer. We also show that chimeric antigen receptor T cells manufactured from these cells are comparable to antibody-isolated chimeric antigen receptor T cells in proliferation, phenotype, effector function and antitumour activity in a mouse model of B-cell lymphoma. By employing multiple aptamers and the corresponding complementary oligonucleotides, aptamer-mediated cell selection could enable the fully synthetic, sequential and traceless isolation of desired lymphocyte subsets from a single system.

Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8.

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.

Snf1-dependent and Snf1-independent pathways of constitutive ADH2 expression in Saccharomyces cerevisiae.

The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sensitive alleles are present among the new constitutive mutants, indicating that essential genes play a role in ADH2 repression. Among the genes we cloned is MOT1, encoding a repressor that inhibits TBP binding to the promoter, thus linking glucose repression with TBP access to chromatin. Two genes encoding proteins involved in vacuolar function, FAB1 and VPS35, and CDC10, encoding a nonessential septin, were also uncovered in the search, suggesting that vacuolar function and the cytoskeleton have previously unknown roles in regulating gene expression. Constitutive activation of ADH2 expression by Adr1 is SNF1-dependent in a strain with a defective MOT1 gene, whereas deletion of SNF1 did not affect constitutive ADH2 expression in the mutants affecting vacuolar or septin function. Thus, the mutant search revealed previously unknown Snf1-dependent and -independent pathways of ADH2 expression.

Serum Stability and Affinity Optimization of an M2 Macrophage-Targeting Peptide (M2pep).

Tumor associated macrophages (TAMs) are a major stromal component of the tumor microenvironment in several cancers. TAMs are a potential target for adjuvant cancer therapies due to their established roles in promoting proliferation of cancer cells, angiogenesis, and metastasis. We previously discovered an M2 macrophage-targeting peptide (M2pep) which was successfully used to target and deliver a pro-apoptotic KLA peptide to M2-like TAMs in a CT-26 colon carcinoma model. However, the effectiveness of in vivo TAM-targeting using M2pep is limited by its poor serum stability and low binding affinity. In this study, we synthesized M2pep derivatives with the goals of increasing serum stability and binding affinity. Serum stability evaluation of M2pepBiotin confirmed its rapid degradation attributed to exolytic cleavage from the N-terminus and endolytic cleavages at the W10/W11 and S16/K17 sites. N-terminal acetylation of M2pepBiotin protected the peptide against the exolytic degradation while W10w and K(17,18,19)k substitutions were able to effectively protect endolytic degradation at their respective cleavage sites. However, no tested amino acid changes at the W10 position resulted in both protease resistance at that site and retention of binding activity. Therefore, cyclization of M2pep was investigated. Cyclized M2pep better resisted serum degradation without compromising binding activity to M2 macrophages. During the serum stability optimization process, we also discovered that K9R and W10Y substitutions significantly enhanced binding affinity of M2pep. In an in vitro binding study of different M2pep analogs pre-incubated in mouse serum, cyclic M2pep with K9R and W10Y modifications (cyclic M2pep(RY)) retained the highest binding activity to M2 macrophages over time due to its improved serum stability. Finally, we evaluated the in vivo accumulation of sulfo-Cy5-labeled M2pep and cyclic M2pep(RY) in both the CT-26 and 4T1 breast carcinoma models. Cyclic M2pep(RY) outperformed M2pep in both tumor localization and selective accumulation in M2-like TAMs. In conclusion, we report cyclic M2pep(RY) as our lead M2pep analog with improved serum stability and M2 macrophage-binding activity. Its enhanced utility as an in vivo M2-like-TAM-targeting agent was demonstrated in two tumor models, and is expected to be applicable for other tumor models or in models of M2 macrophage-related diseases.

Promoter binding by the Adr1 transcriptional activator may be regulated by phosphorylation in the DNA-binding region.

BACKGROUND: Post-translational modification regulates promoter-binding by Adr1, a Zn-finger transcriptional activator of glucose-regulated genes. Support for this model includes the activation of an Adr1-dependent gene in the absence of Adr1 protein synthesis, and a requirement for the kinase Snf1 for Adr1 DNA-binding. A fusion protein with the Adr1 DNA-binding domain and a heterologous activation domain is glucose-regulated, suggesting that the DNA binding region is the target of regulation. METHODOLOGY/PRINCIPAL FINDINGS: Peptide mapping identified serine 98 adjacent to the Zn-fingers as a phosphorylation site. An antibody specific for phosphorylated serine 98 on Adr1 showed that the level of phosphorylated Adr1 relative to the level of total Adr1 decreased with glucose derepression, in a Snf1-dependent manner. Relative phosphorylation decreased in a PHO85 mutant, and this mutant constitutively expressed an Adr1-dependent reporter. Pho85 did not phosphorylate Adr1 in vitro, suggesting that it affects Adr1 indirectly. Mutation of serine 98 to the phosphomimetic amino acid aspartate reduced in vitro DNA-binding of the recombinant Adr1 DNA-binding domain. Mutation to aspartate or alanine affected activation of a reporter by full-length Adr1, and in vivo promoter binding. CONCLUSIONS/SIGNIFICANCE: Mutation of Adr1 serine 98 affects in vitro and in vivo DNA binding, and phosphorylation of serine 98 in vivo correlates with glucose availability, suggesting that Adr1 promoter-binding is regulated in part by serine 98 phosphorylation.

The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

Snf1 protein kinase regulates Adr1 binding to chromatin but not transcription activation.

The yeast transcriptional activator Adr1 controls the expression of genes required for ethanol, glycerol, and fatty acid utilization. We show that Adr1 acts directly on the promoters of ADH2, ACS1, GUT1, CTA1, and POT1 using chromatin immunoprecipitation assays. The yeast homolog of the AMP-activated protein kinase, Snf1, promotes Adr1 chromatin binding in the absence of glucose, and the protein phosphatase complex, Glc7.Reg1, represses its binding in the presence of glucose. A post-translational process is implicated in the regulation of Adr1 binding activity. Chromatin binding by Adr1 is not the only step in ADH2 transcription that is regulated by glucose repression. Adr1 can bind to chromatin in repressed conditions in the presence of hyperacetylated histones. To study steps subsequent to promoter binding we utilized miniAdr1 transcription factors to characterize Adr1-dependent transcription in vitro. Yeast nuclear extracts prepared from glucose-repressed and glucose-derepressed cells are equally capable of supporting miniAdr1-dependent transcription and pre-initiation complex formation. Nuclear extracts prepared from a snf1 mutant support miniAdr1-dependent transcription but are partially defective in the formation of pre-initiation complexes with Mediator components being particularly depleted. We conclude that Snf1 regulates Adr1-dependent transcription primarily at the level of chromatin binding.

Hyperacetylation of chromatin at the ADH2 promoter allows Adr1 to bind in repressed conditions.

We report that in vivo increased acetylation of the repressed Saccharomyces cerevisiae ADH2 promoter chromatin, as obtained by disrupting the genes for the two deacetylases HDA1 and RPD3, destabilizes the structure of the TATA box-containing nucleosome. This acetylation-dependent chromatin remodeling is not sufficient to allow the binding of the TATA box-binding protein, but facilitates the recruitment of the transcriptional activator Adr1 and induces faster kinetics of mRNA accumulation when the cells are shifted to derepressing conditions.