Skip to main contentSkip to breadcrumbsSkip to sub navSkip to doormat

Alexander Stark

The regulation of gene expression in response to developmental or environmental stimuli is a crucial mechanism in all organisms. We are fascinated by the question how transcription is regulated at the level of enhancer and core-promoter DNA elements, and the transcription factor and cofactor proteins that mediate transcription activation. We use genome-wide functional assays, bioinformatics, and mass-spectrometry; and develop highly-controllable reporter assays that provide direct functional readouts. Our goal is to understand transcription and – ultimately – how transcriptional networks define cellular and developmental programs.

Understanding transcriptional regulation

Transcription – the copying of genomic DNA into RNA – is one of the most fascinating processes in biology, particularly in animals in which different cell types transcribe different genes to acquire different morphologies and functions. This differential gene transcription is a prerequisite of multi-cellularity, the main driver of animal development, and underlies most of the differences we observe between closely related species; failures in this tightly regulated process are causal to many diseases including cancer.

To us, the discrepancy between the seeming simplicity of transcriptional regulation and the apparent difficulty of obtaining mechanistic insights to a satisfying level is particularly fascinating: enhancer sequences, typically drive gene expression patterns reliably in both their endogenous genomic contexts and in simple reporter systems. However, despite this defined sequence-to-function relationship, the exact sequence requirements for enhancer activity remain unknown. Similarly, how combinations of transcription factor and cofactor proteins control enhancer function and mediate the transcriptional activation of target core-promoters has remained elusive.

Our aim is to understand how transcription is regulated at the level of the two key types of regulatory genomic elements – enhancers and core-promoters – and the transcription factor and cofactor proteins that mediate transcription activation. To reach this goal, we follow an interdisciplinary approach, using genome-wide functional assays, bioinformatics, biochemistry, and mass-spectrometry. We develop and employ highly-controllable reporter assays that provide reliable functional readouts for each of questions we ask, while circumventing the many confounding issues that exist in complex gene regulatory systems in vivo.

Genome-wide quantitative enhancer activity maps

We have developed STARR–seq (self-transcribing-active-regulatory-region-sequencing), a massively parallel enhancer activity assay based on next-generation sequencing. STARR-seq allows for the identification of transcriptional enhancers in a direct and quantitative manner in entire genomes, drawing genome-wide quantitative enhancer activity maps. We are applying STARR-seq in Drosophila and human cells to understand the sequence basis of cell-type specific enhancer activities.

Figure 1: STARR-seq fragment densities in the Eip75 gene locus before and after treatment of Drosophila S2 cells with ecdysone. Induced peaks are shaded red while repressed peaks are shaded blue (Shlyueva et al., Molecular Cell 2014).

Genome-scale enhancer characterization in Drosophila embryos

Figure 2: Enhancer screen in transgenic Drosophila embryos. We show here a sample of embryos that stain positively in our enhancer assay and display diverse activity patterns. The embryos are in different stages and are shown in lateral orientation, anterior to the left. The inset shows a magnification of a blastoderm embryo with an enhancer activity signal in the dorsal ectoderm (Kvon et al., Nature 2014).

We have determined the temporal and spatial enhancer activity in Drosophila embryos of 7793 transcriptional reporter constructs integrated at a single defined genomic position in transgenic lines (Vienna-Tiles [VT] library). This provides the first genome-scale view on how developmental gene regulation is organized in an animal’s genome and how regulatory DNA sequences encode different patterns of gene expression.

All the data is available from http://enhancers.starklab.org.

Genome-wide assessment of enhancer responsiveness

We have developed STAP-seq (Self-Transcribing Active core-Promoter-sequencing) to measure the enhancer responsiveness of core-promoter candidates genome-wide. While STARR-seq assesses enhancer function using a constant core-promoter, STAP-seq quantifies the ability of core-promoter candidates to initiate transcription provided activating input from a defined constant enhancer. STAP-seq thereby enables the identification of transcription initiation sites (TSSs) and the quantification of how strongly each TSS activates transcription in response to enhancers, i.e. the TSS’s enhancer responsiveness.  

Direct identification of regulatory activities of transcription factors and cofactors

We have developed an enhancer complementation assay that allows for the testing of regulatory activities of transcription factors and cofactors regardless of their endogenous DNA binding specificities and developmental roles. By tethering these factors to different sites in mutant enhancers, we are able to quantify their regulatory activities in different contexts. Our results on the function of 474 Drosophila transcription factors and 338 transcriptional cofactors are available from http://factors.starklab.org.

Cracking the transcriptional regulatory code

We use bioinformatics and machine-learning to dissect regulatory sequences and determine sequence features (e.g. TF motifs) that are predictive of regulatory function and required for enhancer activity. Our goal is to: achieve a systematic understanding of the structure and functions of enhancers, i.e. to “crack” the regulatory code; predict enhancer activity from the DNA sequence; and understand how transcriptional networks define cellular and developmental programs.

Figure 3: STARR-seq fragment densities for two enhancers, each of which is specifically active in one of three Drosophila cell lines (S2: embryonic, OSC: adult ovarian, BG3: larval neuronal; Yáñez-Cuna et al., Genome Research 2014).

Comparative genomics – tracing sequences through evolution

Functional elements in a genome are often under evolutionary selection to maintain their functions in related organisms. We study the conservation and divergence of TF binding and enhancer activity and the underlying regulatory DNA sequences across different Drosophila species. Conservation and divergence allow for powerful insights into how gene regulation is encoded in the DNA sequence and how it changes during evolution.

Determining and understanding tissue-specific TF binding

Determining and understanding tissue-specific TF binding
Transcription factors are employed at developmental stages and in different tissues and typically bind to and regulate different targets in each context. We use tissue-specific ChIP-Seq to determine context-specific TF binding and study its sequence determinants, focusing on embryonic mesoderm and muscle development, the circadian clock, and homeobox (Hox) transcription factors.

Selected Publications

  • Arnold, CD., Zabidi, MA., Pagani, M., Rath, M., Schernhuber, K., Kazmar, T., Stark, A. (2016). Genome-wide assessment of sequence-intrinsic enhancer responsiveness at single-base-pair resolution. Nat Biotechnol.
  • Stampfel, G., Kazmar, T., Frank, O., Wienerroither, S., Reiter, F., Stark, A. (2015). Transcriptional regulators form diverse groups with context-dependent regulatory functions. Nature. 528(7580):147-51 
  • Zabidi, MA., Arnold, CD., Schernhuber, K., Pagani, M., Rath, M., Frank, O., Stark, A. (2015). Enhancer-core-promoter specificity separates developmental and housekeeping gene regulation. Nature. 518(7540):556-9 
  • Kvon, EZ., Kazmar, T., Stampfel, G., Yáñez-Cuna, JO., Pagani, M., Schernhuber, K., Dickson, BJ., Stark, A. (2014). Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature. 512(7512):91-5
  • Arnold, CD., Gerlach, D., Stelzer, C., Boryń, ŁM., Rath, M., Stark, A. (2013). Genome-wide quantitative enhancer activity maps identified by STARR-seq. Science. 339(6123):1074-7

Grants