Alexander Stark

Research Institute of Molecular Pathology | IMP

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate t...

The systematic determination of protein function is a key goal of modern biology, but remains challenging with current approaches. Here we present ORFtag, a versatile, cost-effective and highly efficient method for the massively parallel tagging and functional interrogation of proteins at the proteome scale. ORFtag uses retroviral vectors bearing a...

Background The COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria. Ai...

Determining protein function in a systematic manner is a key goal of modern biology, but remains challenging with current approaches. Here, we present ORFtag, a versatile, cost-effective and highly efficient method for the massively-parallel tagging and functional interrogation of proteins at proteome scale. Using mouse embryonic stem cells, we sho...

Enhancers are short DNA sequences that activate their target promoter from a distance; however, increasing the genomic distance between the enhancer and the promoter decreases expression levels. Many genes are controlled by combinations of multiple enhancers, yet the interaction and cooperation of individual enhancer elements is not well understood...

Genomic enhancers are key transcriptional regulators which, upon the binding of sequence-specific transcription factors, activate their cognate target promoters. Although enhancers have been extensively studied in isolation, a substantial number of genes have more than one simultaneously active enhancer, and it remains unclear how these cooperate t...

Gene expression is controlled by the precise activation and repression of transcription. Repression is mediated by specialized transcription factors (TFs) that recruit co-repressors (CoRs) to silence transcription, even in the presence of activating cues. However, whether CoRs can dominantly silence all enhancers or display distinct specificities i...

Enhancers are important cis-regulatory elements controlling cell-type specific expression patterns of genes. Furthermore, combinations of enhancers and minimal promoters are utilized to construct small, artificial promoters for gene delivery vectors. Large-scale functional screening methodology to construct genomic maps of enhancer activities has b...

Recruitment of RNA polymerase II (Pol II) to promoters is essential for transcription. Despite conflicting evidence, the Pol II preinitiation complex (PIC) is often thought to have a uniform composition and to assemble at all promoters via an identical mechanism. Here, using Drosophila melanogaster S2 cells as a model, we demonstrate that different...

The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, whereas the enhancer syntax, that is, the flexibilit...

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains...

Animal development and homeostasis critically depend on the accurate regulation of gene transcription, which includes the silencing of genes that should not be expressed. Repression is mediated by a specific class of transcription factors (TFs) termed repressors that, via the recruitment of co-repressors (CoRs), can dominantly prevent transcription...

Recruitment of RNA polymerase II (Pol II) to promoter regions is essential for transcription. Despite conflicting evidence, the Pol II Pre-Initiation Complex (PIC) is often thought to be of uniform composition and assemble at all promoters via an identical mechanism. Here, we show using Drosophila melanogaster S2 cells as a model that promoter clas...

The information about when and where each gene is to be expressed is mainly encoded in the DNA sequence of enhancers, sequence elements that comprise binding sites (motifs) for different transcription factors (TFs). Most of the research on enhancer sequences has been focused on TF motif presence, while the enhancer syntax, i.e. the flexibility of i...

Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties a...

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied and their repressive domains (RDs) are typically unknown, a...

All multicellular organisms rely on differential gene transcription regulated by genomic enhancers, which function through cofactors that are recruited by transcription factors1,2. Emerging evidence suggests that not all cofactors are required at all enhancers3–5, yet whether these observations reflect more general principles or distinct types of e...

Enhancer sequences control gene expression and comprise binding sites (motifs) for different transcription factors (TFs). Despite extensive genetic and computational studies, the relationship between DNA sequence and regulatory activity is poorly understood, and de novo enhancer design has been challenging. Here, we built a deep-learning model, Dee...

The identification of transcriptional enhancers and the quantitative assessment of enhancer activities is essential to understanding how regulatory information for gene expression is encoded in animal and human genomes. Further, it is key to understanding how sequence variants affect enhancer function. STARR‐seq enables the direct and quantitative...

Transcriptional cofactors (COFs) communicate regulatory cues from enhancers to promoters and are central effectors of transcription activation and gene expression¹. Although some COFs have been shown to prefer certain promoter types2–5 over others (for example, see refs 6,7), the extent to which different COFs display intrinsic specificities for di...

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Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans-activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high-throughput approach tAD-seq to functionally test thousands of candidate...

RNA polymerase II (Pol II) core promoters are specialized DNA sequences at transcription start sites of protein-coding and non-coding genes that support the assembly of the transcription machinery and transcription initiation. They enable the highly regulated transcription of genes by selectively integrating regulatory cues from distal enhancers an...

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfecti...

Enhancers are important genomic regulatory elements directing cell type-specific transcription. They assume a key role during development and disease, and their identification and functional characterization have long been the focus of scientific interest. The advent of next-generation sequencing and clustered regularly interspaced short palindromi...

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliab...

The hallmark of canonical Wnt signaling is the transcriptional induction of Wnt target genes by the beta-catenin/TCF complex. Several studies have proposed alternative interaction partners for beta-catenin or TCF, but the relevance of potential bifurcations in the distal Wnt pathway remains unclear. Here we study on a genome-wide scale the requirem...

(A) Schematic representation of potential protein products of Arm in arm-/--AFII7/8 (arm-/-) cells with premature termination codons (stop), which result from introduced frameshift mutations. (B) Full Western blot analysis from Fig 2. As presented in the blot, no truncated versions of Arm could be detected. (TIF)

(A) Schematic representation of potential protein products of Pan in pan-/--AF1AD26 (pan-/-) cells with premature termination codons (stop) due to introduced frameshift mutations. (B) qRT-PCR analysis of pan mRNA level with primer targeting its N-term (see S1 Table) in wild-type (WT) and pan-/--AF1AD26 (pan-/-) cells. Cells were stimulated with WCM...

(A) Scatterplots of replicates of STARR-seq in pan-/--AF1AD26 (pan-/-) cells treated with DMSO or CHIR99021 (CHIR). (B) Validation of candidate STARR-seq enhancers. Candidate enhancer sequences were cloned into the STARR-seq library luciferase vector, see Material and Methods. Wild-type (WT) and pan-/--AF1AD26 (pan-/-) cells were transfected with t...

qRT-PCR analysis of (A) positive and (B) negative candidate Wnt/Wg target genes in wild-type (WT), arm-/--AFII7/8 (arm-/-) and pan-/--AF1AD26 (pan-/-) cells. Cells were stimulated with WCM or CM for 24 h. Analysis of expression profiles of several Wg target genes after treatment versus control confirmed their induction after WCM stimulation. Fold e...

Validation of (A) induced and (B) repressed candidate STARR-seq enhancers with WCM. Candidate enhancer sequences were cloned into the STARR-seq library luciferase vector, see Material and Methods. Wild-type (WT) and pan-/--AF1AD26 (pan-/-) cells were transfected with the candidate luciferase reporter expression vector and Renilla expression vector...

Primer sequences for qRT-PCR, cloning of gRNAs, PCR. (XLSX)

(A) Scatterplots of replicates of STARR-seq in wild-type (WT) cells treated with DMSO or CHIR99021 (CHIR). (B) UCSC browser screenshot of STARR-seq tracks in WT cells for pxb. (C) Validation of peaks from the constitutive, induced, and repressed enhancer classes by luciferase assays. Log2 fold induction (CHIR-treated versus control) of normalized l...

CHIR99021 activates reliably and efficiently Wnt/Wg target genes in Drosophila cells. (A) Titration of CHIR99021 in Drosophila S2R+ cells. S2R+ cells were transfected with wingful luciferase reporter vector and Renilla. Red bars: promoter activation with 25 μM CHIR is as efficient as with Wg ligand. In green is the wingful promoter activity after s...

de novo motif search using DREME. (XLSX)

Primer sequences for STARR-seq enhancer validations and Pan motif validation. (XLSX)

Differential gene expression gives rise to the many cell types of complex organisms. Enhancers regulate transcription by binding transcription factors (TFs), which in turn recruit cofactors to activate RNA Polymerase II at core promoters. Transcriptional regulation is typically mediated by distinct combinations of TFs, enabling a relatively small n...

Gene expression is controlled by enhancers that activate transcription from the core promoters of their target genes. Although a key function of core promoters is to convert enhancer activities into gene transcription, whether and how strongly they activate transcription in response to enhancers has not been systematically assessed on a genome-wide...

Gene expression is regulated by genomic enhancers that recruit transcription factors and cofactors to activate transcription from target core promoters. Over the past years, thousands of enhancers and core promoters in animal genomes have been annotated, and we have learned much about the domain structure in which regulatory genomes are organized i...

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax...

Association of Polycomb complex with binding sites for developmental transcription factors (TFs). The plots show the percentage of TF binding sites for which the ChIP signal for the indicated Polycomb group protein or the Polycomb-associated histone modification H3K27me3 [68,75,84] is greater than a given threshold value (X-axis; as in main Fig 5F)...

De novo motif analysis and clustered Ubx binding sites at the loci of important developmental genes. (A) De novo motif recovered in Ubx-bound regions. (B) The number of Ubx peaks per 100 kb on chromosomes 2R, 3L, X and 4 (as in main Fig 4C and 4D). Each 100 kb window starts at a Ubx peak, covering all possible windows that contain at least one Ubx...

Strong association of Pc and Pho with Ubx binding sites. The boxplots show the distributions of Pc and Pho normalized ChIP signal (ChIP/input ratio [log2]) at the binding sites of the indicated transcriptional factors (as in Fig 5E). The binding sites are from [14] and [24] and are restricted to TFs that had Pc and Pho signals significantly higher...

One of the most important questions in biology is how transcription factors (TFs) and cofactors control enhancer function and thus gene expression. Enhancer activation usually requires combinations of several TFs, indicating that TFs function synergistically and combinatorially. However, while TF binding has been extensively studied, little is know...

Gene transcription in animals involves the assembly of RNA polymerase II at core promoters and its cell-type-specific activation by enhancers that can be located more distally. However, how ubiquitous expression of housekeeping genes is achieved has been less clear. In particular, it is unknown whether ubiquitously active enhancers exist and how de...