Transcriptional Activation Domain Research Articles

Identification of plant transcriptional activation domains.

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs1. To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein-Protein Interactions.

Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium-chain, branched fatty acid to the N-terminus of one such heptameric lipopeptidomimetic (LPPM-8) increases the affinity for the coactivator Med25 >20-fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein-protein interactions (PPIs). The lipid structure, the peptide sequence, and the C-terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM-8 and its effectiveness as an inhibitor. LPPM-8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full-length protein in the cellular proteome. Further, genes regulated by Med25-activator PPIs are inhibited in a cell model of triple-negative breast cancer. Thus, LPPM-8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator-coactivator complexes.

A Lipopeptidomimetic of Transcriptional Activation Domains Selectively Disrupts the Coactivator Med25 Protein–Protein Interactions

AbstractShort amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium‐chain, branched fatty acid to the N‐terminus of one such heptameric lipopeptidomimetic (LPPM‐8) increases the affinity for the coactivator Med25 >20‐fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein–protein interactions (PPIs). The lipid structure, the peptide sequence, and the C‐terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM‐8 and its effectiveness as an inhibitor. LPPM‐8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full‐length protein in the cellular proteome. Further, genes regulated by Med25‐activator PPIs are inhibited in a cell model of triple‐negative breast cancer. Thus, LPPM‐8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator‐coactivator complexes.

Abstract 3101: Developing a drug repurposing screen for head and neck squamous cell carcinoma using a novel c-Rel bioassay

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Approximately 890,000 new cases are diagnosed each year, while incidence continues to rise and is anticipated to increase by 30% by 2030. HNSCC occurs most often in men in their 50-60s, although the incidence among younger individuals is increasing. Current standard of care practices include surgical resection, followed by adjuvant radiation or chemoradiation. HNSCC tumors exhibit frequent mutation of CDKN2A (22% of tumors) and TP53 (72% of tumors). Additionally, two members of the TP53 gene family, TP63 and TP73, are frequently altered in HNSCC. TP63 encodes two major isoforms, ΔNp63 which lacks the main transcription activation domain and acts as a dominant-negative inhibitor of transactivation (TA), and TAp63 (contains a p53-like TA domain). ΔNp63 is overexpressed in a majority of HNSCC tumors. Our lab has reported ΔNp63α interacts with activated c-Rel (a nuclear factor-KB family member) in HNSCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers. Consistent with a role in growth regulation, ΔNp63α:c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human HNSCCs and normal keratinocytes overexpressing ΔNp63α. The direct relationship between ΔNp63α and activated c-Rel is observed by strong nuclear co-localization in the proliferating compartment of primary head and neck SCC. Stimulation of HNSCC cells with TNFα results in the induction of the c-Rel oncoprotein that binds to ΔNp63, displacing and inactivating the tumor suppressor TAp73 from ΔNp63-TAp73 complexes. Using western blot analysis in human HNSCC cell lines, we confirmed that stimulation with TNFα enhances nuclear c-Rel localization. We then set out to design a high throughput screening bioassay for the purpose of evaluating compounds that can inhibit c-Rel accumulation in the nucleus under TNFα stimulation. By preventing activated c-Rel from interacting with ΔNp63α in the nucleus we aim to decrease uncontrolled proliferation and restore the tumor-suppressive functionality of the ΔNp63-TAp73 complex. A 384-well immunocytochemistry assay was developed using a confocal microscopy readout and an automated image analysis algorithm to quantify nuclear translocation. The optimized assay will be used to screen the NCATS collection of approved drugs and investigational oncology agents. Top candidates will undergo further investigation in vitro, ex vivo, and in vivo to explore the possibility of repurposing the small molecules for HNSCC therapeutics. Citation Format: Kristin Ann Altwegg, Kathryn E. King, Dvir Blivis, Ty C. Voss, Natalia J. Martinez, Wendy C. Weinberg, Mark J. Henderson. Developing a drug repurposing screen for head and neck squamous cell carcinoma using a novel c-Rel bioassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3101.

Locking it in: Capturing transient interactions of NFκB’s intrinsically disordered transcription activation domain with an unnatural amino acid crosslinker

Locking it in: Capturing transient interactions of NFκB’s intrinsically disordered transcription activation domain with an unnatural amino acid crosslinker

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Prognostic impact of CEBPA mutational subgroups in adult AML.

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIPInDel), frameshift InDel or nonsense mutations inducing translational stop (bZIPSTOP) or single base-pair missense alterations (bZIPms), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIPInDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIPInDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIPInDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIPInDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIPms).

Male with an apparently normal phenotype carrying a BRCA1 exon 20 duplication in trans to a BRCA1 frameshift variant

BackgroundReports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype.MethodsWe identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells.ResultsGenome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability.ConclusionsThis is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants.

Optimizing Rice Genomics: Employing the Hypercompact Cas12j2 System for Targeted Transcriptional Regulation and Epigenome Modification.

In the burgeoning field of genome engineering, the CRISPR-Cas systems have emerged as pivotal tools for precise genetic modifications in various organisms, including humans, animals, and plants. One significant obstacle in this arena is the substantial size of Cas proteins, such as SpCas9, which is approximately 190kDa, complicating their delivery, particularly via viral vectors. To overcome this challenge, our research introduces the hypercompact Cas12j2 system, a groundbreaking development with a size of merely ~80kDa, originally identified in Biggiephage. We demonstrate its application in plant genome editing, with a particular focus on rice. In this context, we have successfully adapted Cas12j2 for gene activation, achieving significant increases in gene expression, specifically up to a tenfold activation for OsER1 and a fourfold activation for OsNRT1.1A in stable transgenic rice plants. Moreover, we have ventured beyond mere gene editing to develop a Cas12j2-based approach for targeted epigenome editing, particularly in the context of DNA methylation. This was demonstrated through the targeted methylation of the OsGBSS1 promoter, as verified by Next-Generation Sequencing of bisulfite sequencing PCR products. This chapter presents a detailed protocol about utilizing the hypercompact Cas12j2 system in conjunction with specific effectors, such as transcriptional activation or repression domains, or methylation domains, to achieve targeted gene transcriptional regulation and epigenome modification in rice.

Regulation of fungal raw-starch-degrading enzyme production depends on transcription factor phosphorylation and recruitment of the Mediator complex

Filamentous fungus can produce raw-starch-degrading enzyme (RSDE) that efficiently degrades raw starch below starch gelatinization temperature. Employment of RSDE in starch processing can save energy. A key putative transcription factor PoxRsrA (production of raw-starch-degrading enzyme regulation in Penicilliumoxalicum) was identified to regulate RSDE production in P. oxalicum; however, its regulatory mechanism remains unclear. Here we show that PoxRsrA1434–1730 was the transcriptional activation domain, with essential residues, D1508, W1509 and M1510. SANT (SWI3, ADA2, N-CoR and TFIIIB)-like domain 1 (SANT1) bound to DNA at the sequence 5′-RHCDDGGD-3′ in the promoter regions of genes encoding major amylases, with an essential residue, R866. SANT2 interacted with a putative 3-hydroxyisobutyryl-CoA hydrolase, which suppressed phosphorylation at tyrosines Y1127 and Y1170 of PoxRsrA901–1360, thereby inhibiting RSDE biosynthesis. PoxRsrA1135–1439 regulated mycelial sporulation by interacting with Mediator subunit Med6, whereas PoxRsrA1440–1794 regulated RSDE biosynthesis by binding to Med31. Overexpression of PoxRsrA increased sporulation and RSDE production. These findings provide insights into the regulatory mechanisms of fungal RSDE biosynthesis.

Design of hypoxia responsive CRISPR-Cas9 for target gene regulation

The CRISPR–Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR–Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics.

Genetically Engineered Mouse Models of CIC-DUX4 Sarcoma Reveal an Unexpected Sensitivity to Histone Deacetylase (HDAC) Inhibition

Undifferentiated small round cell sarcomas constitute a highly aggressive subtype of soft tissue sarcoma that affects adolescents and young adults. Frequently, these cancers are caused by a gene fusion between the DNA binding domain of capicua (CIC), a canonical tumor suppressor that normally acts as a transcriptional repressor, with the transcriptional activation domain of double homeobox 4 (DUX4). Given the rare nature of CIC-DUX4 sarcoma (CDS), obtaining patient tissues to study the mechanisms of disease is very challenging. Moreover, conducting clinical trials to identify radiosensitizers or other novel treatments is not possible underscoring the need for a primary animal model. Here, we report the development and characterization of the first genetically engineered mouse model of CDS and the use of this model to investigate novel therapies. Using Cre-loxP technology, three genetically engineered mouse models of CDS were developed. The resulting tumors were characterized by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and RNA-sequencing. Cell lines from multiple primary and metastatic tumors were derived and used for Chromatin Immunoprecipitation (ChiP) sequencing, Rapid Immunoprecipitation mass spectrometry of Endogenous proteins (RIME), and targeted drug screening. In all three models, spontaneous (Cre-independent) recombination occurred in chimeric animals resulting in the formation of soft tissue tumors, widespread metastasis to the lungs, liver, and brain, and 100% lethality within 4-8 weeks of life. The tumors histologically resembled human CDS and stained positive for an HA (hemagglutinin) epitope tag confirming transgene expression. Of 108 chimeric pups (generation 0, G0), only one male bred before succumbing to metastatic disease. G2 progeny now exhibit resistance to spontaneous recombination and tumor development enabling reliable propagation of one of the models for experimentation. Using tumor derived cell lines for integrative analyses of multi-omics datasets, we find evidence for a model where CIC-DUX4 functions as a neomorphic and oncogenic transcriptional activator, which cooperates with ETS family transcription factors to drive the CDS transcriptional program. Lastly, small molecule epigenetic drug screens in mouse and human CDS cell lines point to an unexpected sensitivity to HDAC inhibitors, which we hypothesize may be attributable to ETS factor hyperactivity. Genetically engineered mouse models of CIC-DUX4 sarcoma form tumors that recapitulate the human disease. Using mouse tumors and tumor-derived cell lines, we identify a mechanistic role and potential therapeutic vulnerability that converges on the ETS family transcription factors.

DUX4-r exerts a neomorphic activity that depends on GTF2I in acute lymphoblastic leukemia.

Translocations producing rearranged versions of the transcription factor double homeobox 4 (DUX4-r) are one of the most frequent causes of B cell acute lymphoblastic leukemia (B-ALL). DUX4-r retains the DNA binding domain of wild-type DUX4 but is truncated on the C-terminal transcription activation domain. The precise mechanism through which DUX4-r causes leukemia is unknown, and no targeted therapy is currently available. We found that the rearrangement leads to both a loss and a gain of function in DUX4-r. Loss of CBP/EP300 transcriptional coactivator interaction leads to an inability to bind and activate repressed chromatin. Concurrently, a gain of interaction with the general transcription factor 2 I (GTF2I) redirects DUX4-r toward leukemogenic targets. This neomorphic activity exposes an Achilles' heel whereby DUX4-r-positive leukemia cells are exquisitely sensitive to GTF2I targeting, which inhibits DUX4-r leukemogenic activity. Our work elucidates the molecular mechanism through which DUX4-r causes leukemia and suggests a possible therapeutic avenue tailored to this B-ALL subtype.

Abstract P1145: All-in-one AAV CRISPR/dCas9VPR Vectors Enable Endogenous Gene Activation In Cardiomyocytes

The transcriptional landscape of cardiomyocytes harbors the cellular adaptation upon stress, but remains out of scope for therapies preventing heart failure progression. CRISPR/Cas9 mediated endogenous gene activation (CRISPRa) using enzymatically inactive Cas9 (dCas9) fused to transcription activation domains (VPR) allows for precise and fine-tuned gene activity control when targeted to promoter or enhancer regions of interest via guide RNAs (gRNAs). Transgenic mouse models and human induced pluripotent stem cell (hiPSC) lines were established for proof-of-concept (PoC) gene activation studies in cardiomyocytes. However, the size of the commonly used Streptococcus pyogenes Cas9 exceeds the packaging limits of gene therapy relevant adeno-associated virus (AAV), hampering the therapeutic use of the technology. We aimed to engineer AAV vectors based on smaller, enzymatically inactive Cas9 variants fused to a minimalized VPR-transactivation domain co-expressing a single gRNA for all-in-one gene activation strategies. We rendered mini-Cas9s enzymatically inactive by mutating the evolutionary conserved RUVC and HNH regions and C-terminally attached a minimal VPR domain as well as a HA-tag. As PoC, we expressed mini-dCas9VPR under the CMV promoter in HEK293T cells and confirmed expression of the Cas9 variants via immunoblotting. When we targeted mini-dCas9VPR to the anti-hypertrophic transcription factor KLF15 promoter region or the PoC target POU5F1 promoter, we could confirm significant gene activation compared to non-transfected and non-targeted gRNA controls (KLF15: 3-fold change, POU5F1: 4-fold change, n ≤ 4). We next exchanged the CMV promoter by the TNNT2 promoter to allow cardiomyocyte-specific expression of mini-dCas9VPR. Upon targeting the KLF15 promoter with these vectors transfected in hiPSC-cardiomyocytes, we observed significantly elevated KLF15 mRNA levels compared to controls (1.5-fold change, n ≤ 4) and robust expression of the mini-CRISPRa system, respectively. In conclusion, we present novel vectors for AAV mediated cardiomyocyte-specific endogenous gene regulation which are currently tested in hiPSC-based models, patient-derived cardiac slices and for in vivo gene activation.

Rice DSP controls stigma, panicle and tiller primordium initiation.

Tiller and seed number are key determinants of rice (Oryza sativa) yield. These traits are mainly affected by tiller, panicle, spikelet and stigma formation, but to date, no single gene involved in the development of all these organs has been identified. Here, we found a rice mutant defective stigma and panicle (dsp) with greatly reduced numbers of tillers and panicle branches, and ovaries lacking stigmas, due to defects in primordium initiation. We cloned DSP using sequencing-based mapping and verified its function with the CRISPR/Cas9 system. DSP encodes a transcription factor containing an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain that recognizes the GCC motif and a transcription-activating domain at the site of 244-314 that contains an angiosperm-related (AR) motif. Mutating the AR motif resulted in the dsp mutant phenotypes, whereas mutating the AP2/ERF domain led to seedling death. DSP directly regulated PINOID (PID) expression to determine the emergence of rice stigmas, and PID overexpression partially rescued the stigma defect in the dsp cr2-8 and dsp mutants. Moreover, DSP indirectly affected LAX PANICLE1 (LAX1) expression to determine tiller primordium formation and synergistically regulated panicle primordium development. Our results indicated that DSP was a key regulator that modulated different genetic pathways to control the initiation of stigma primordia, the axillary meristem formation of tillers and panicle branches, which revealed their molecular mechanisms and cross-networks, laying the vital foundation for rice yield and trait improvement.

The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast.

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Clusters of acidic and hydrophobic residues can predict acidic transcriptional activation domains from protein sequence.

Transcription factors activate gene expression in development, homeostasis, and stress with DNA binding domains and activation domains. Although there exist excellent computational models for predicting DNA binding domains from protein sequence, models for predicting activation domains from protein sequence have lagged, particularly in metazoans. We recently developed a simple and accurate predictor of acidic activation domains on human transcription factors. Here, we show how the accuracy of this human predictor arises from the clustering of aromatic, leucine, and acidic residues, which together are necessary for acidic activation domain function. When we combine our predictor with the predictions of convolutional neural network (CNN) models trained in yeast, the intersection is more accurate than individual models, emphasizing that each approach carries orthogonal information. We synthesize these findings into a new set of activation domain predictions on human transcription factors.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

1462-P: ASCL1—A Potentially Novel Type 1 Diabetes Gene

Background: New insights into type 1 diabetes (T1D) have recently come from studies of rare monogenic forms of the disease. Uncovering the genes that drive these monogenic forms can reveal mechanisms and druggable pathways in affected families that may be extended to the general T1D population. We have identified one such candidate gene: Achaete-scute complex 1 (ASCL1). ASCL1 encodes a basic helix-loop-helix (bHLH) transcription factor and is well-known for its role in neurogenesis, however it is less understood how this gene may impact immune tolerance. Recent studies have demonstrated that ASCL1 and its targets are expressed in medullary thymic epithelial cells (mTECs), which are key players in the removal of autoreactive T cells from the thymus. Methods: Whole exome sequencing (WES) was performed on individuals from our monogenic T1D registry and functional studies were performed in a human cell line using lentiviral vectors and bulk RNAseq. Results: We identified a novel coding mutation in the bHLH transcription factor ASCL1 in siblings with suspected monogenic T1D. WES revealed a heterozygous coding variant in ASCL1 that was predicted to be both deleterious and rare. This caused an amino acid substitution within the transcriptional activation domain of the ASCL1 protein. RNAseq analysis showed a broad reduction in the activation of gene targets in cells expressing the ASCL1 patient mutation. Conclusion: ASCL1 may play a role in central tolerance and be a new candidate gene for T1D. Disclosure K.C.Lavelle: None. K.Yang: None. J.Wang: None. C.Chamberlain: None. M.German: Advisory Panel; Encellin, Inc., Stock/Shareholder; ViaCyte, Inc. Funding The Leona M. and Harry B. Helmsley Charitable Trust; Larry L. Hillblom Foundation; Nora Eccles Treadwell Foundation; National Institute of Diabetes and Digestive and Kidney Diseases