Molecular Studies of Transcription
and Regulation of Developmental Genes

The regulation of gene transcription is fundamental to nearly all biological processes, including the development and growth of an organism. A key component of transcriptional regulation is the core promoter—the DNA region that directs the accurate initiation of transcription by RNA polymerase II. Previously, the core promoter was thought to act as a generic element with a universal mechanism. However, recent discoveries reveal significant diversity in core promoter structure and function, underscoring its complexity.

Our lab’s research aims to unravel the unique contributions of the core promoter to the regulation of eukaryotic gene expression. 

Schematic representation of the major core promoter elements.
Full information is available here

To achieve these goals, we integrate techniques from molecular biology, biochemistry, cell biology, bioinformatics, and developmental biology.

We employ Drosophila melanogaster (the fruit fly) as our model organism due to its long-standing contribution/use in genetics and developmental biology, as well as its molecular similarities to humans. 

Remarkably, many regulatory mechanisms in flies have parallels in human biology, enabling insights that extend beyond Drosophila. By examining how transcriptional regulation guides development in the fly, we gain valuable knowledge relevant to human biology.

Our studies focus on the molecular mechanisms underlying the formation of diverse body structures during development and emphasize the broader implications of core promoter diversity. This work is critical not only for understanding development but also for illuminating processes linked to diseases such as cancer, where transcriptional regulation can become dysregulated.

Together, our analyses of core promoters and the proteins that bind and regulate them are essential for deciphering fundamental molecular and mechanistic aspects of transcription. These insights contribute to the broader understanding of fundamental biological processes, including development and differentiation.

The core promoter composition establishes an additional dimension
in the dorsal-ventral gene regulatory network

We have discovered that the dorsal-ventral developmental GRN is dependent on the presence of the DPE motif. We demonstrated that over two-thirds of Dorsal target genes contain DPE sequence motifs, which is significantly higher than the proportion of DPE-containing promoters in Drosophila genes. Furthermore, we showed that multiple Dorsal target genes are evolutionarily conserved and functionally dependent on the DPE.

We envision the core promoter composition as an additional component of the dorsal-ventral gene regulatory network, which contributes to the combinatorial transcriptional output.

Dorsal target genes are classified according to their embryonic tissues: the mesoderm, neurogenic ectoderm, and dorsal ectoderm.

The Dorsal nuclear gradient is represented by the depth of the blue color.

The upper side of the cube displays the color coding of the possible core promoter elements combinations.

The front depicts selected Dorsal target genes with the corresponding color-coded core promoter composition.

The relative frequency of each core promoter combination among all Dorsal target genes in each of the three tissue (using the same color code) is shown on the right.

See here for additional discussion.

From promoter motif to cardiac function:
A single DPE motif affects transcription regulation and organ function in vivo

 

 

We recently demonstrated the in vivo importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. 

Pioneering a novel approach utilizing both CRISPR and nascent transcriptomics, we showed the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7bp substitution mutation results in massive perturbation of the Tinman regulatory network orchestrating dorsal musculature, manifested as physiological and anatomical changes in the  cardiac system, impaired specific activity features and significantly compromised viability of adult flies.

Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart.

See here for the article, featured on the cover of Development, Volume 151, Issue 14 (July 2024) and in BPoD – Biomedical Picture of the Day (August 7, 2024)

For detailed information on the Efficient in vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR approach, see this paper.

Involvement of TRF2 (TATA-box-binding protein-related factor 2)
in the regulation of diverse biological processes and specialized transcription programs

It is clear today that there is no universal transcription machinery and the term general transcription machinery should be replaced by basal transcription machinery.

Our lab studies the TRF2 (TATA-box-binding protein-related factor 2) basal transcription factor. As opposed to its name, TRF2 is much more than a TBP family member that doesn’t bind the TATA box.

Since its discovery in 1999, it has been known that TRF2 (TATA-box-binding protein-related factor 2), despite its homology to the TBP (TATA-box-binding protein) core domain, does not bind TATA-containing promoters. Microfluidic affinity analysis has demonstrated DNA binding of TRF2-containing complexes to DPE-containing promoters. It is likely that there are TRF2-associated factors (like TBP-associated factors), which assist TRF2 in binding to its target promoters.

Recent findings have highlighted the involvement of TRF2 in the regulation of diverse biological processes and specialized transcription programs. Hence, specific core promoter composition serves to recruit a specialized basal transcription factor, demonstrating the diversity of transcriptional regulation.

For a detailed discussion see this paper