Module TSS2: Using Sequence Alignment to Identify a TSS

In Drosophila melanogaster, the gene Antennapedia (Antp) is required for development of the fly’s body plan. It encodes a transcription factor that is part of the Hox gene complex and specifies segment development in the thorax. Absence of Antp expression in the adult fly results in transformation of a leg into an antenna. Ectopic expression in the head results in transformation of an antenna to a leg (or eye to wing).

The Antp gene in D. melanogaster has been annotated, and three TSSs have been defined by FlyBase. We will use information about the TSS for the Antp-RM isoform in D. melanogaster to help us identify the most likely TSS for isoform M of the Antp gene in another Drosophila species, D. eugracilis.

Using the strategies above we were able to pinpoint the transcription start site of D. eugracilis by comparing its sequence with that of D. melanogaster (Exercise 2). We also investigated the sequence of Exon 1 in multiple other Drosophila species using a comparative genomics track (Exercise 3).

The blastn comparison of the D. melanogaster Antp-RM exon 1 sequence against the D. eugracilis scaffold conducted in Exercise 2 worked well because of the sequence similarity at the beginning of exon 1 in both species. Therefore, using this strategy to identify the first transcribed nucleotide — the transcription start site — will work for any gene in any Drosophila species whose sequence is highly conserved with exon 1 of the orthologous gene in D. melanogaster. When the sequence is not highly conserved between genes, alternative evidence would be necessary for identifying the TSS.

The multiple sequence alignment investigated in Exercise 3 indicates that exon 1 of Antp-RM in D. melanogaster and in D. eugracilis share similar sequences near the TSS. The sequence similarity further supported the identification of the Antp-RM TSS in D. eugracilis.

Let’s take a closer look at the region surrounding the TSS to identify DNA motifs that might play a role in regulating Antp gene expression in vivo.

Transcription initiation and recruitment of RNA Polymerase II (RNA Pol II) to the promoter region of a gene is achieved with the aid of accessory proteins known as basal transcription factors [Aoyagi et al., 2000]. These basal transcription factors preferentially bind to specific nucleotide sequences within the core promoter region. The nucleotide sequences that are bound by a specific transcription factor can be experimentally determined and used to determine a sequence or DNA motif [Burke and Kadonaga, 1996].

DNA motif is a general term used to describe a short DNA sequence that recurs multiple times throughout the genome and is associated with a particular biological function. Motifs often correspond to binding sites for DNA-binding proteins, and different positions within the motif can show different levels of sequence conservation. Hence, a motif is often represented by a consensus sequence, which captures the variability of the motif instances within a species or among multiple species.

In order to capture the variations that are found in transcription factor binding sites (TFBS), a consensus motif often includes degenerate nucleotide symbols that represent multiple nucleotides. The IUPAC (International Union of Pure and Applied Chemistry) has generated a code to represent degenerate bases [Nomenclature Committee of the International Union of Biochemistry, 1986]. For example, if a site within a sequence motif can be occupied by either of the two pyrimidine bases (C or T), the consensus sequence would be represented by a "Y" at that location. The IUPAC code for degenerate bases is shown in Table 1.

It is not unusual for sequence motifs to possess sites in which two or more bases appear at similar frequencies (e.g., K and Y in the TCAKTY motif in Table 2). In fact, these differences in the motif instances could help modulate gene expression [Spivakov et al., 2012].

The most common core promoter motifs for Drosophila are shown in Table 2. For each motif, the consensus sequence is shown as well as the position of the motif relative to the TSS if they are known. Motifs that are enriched in broad promoters (e.g., the Ohler motifs 1, 5, 6, 7, and 8) do not have a known position relative to the TSS because broad promoters contain multiple TSSs [Ohler et al., 2002]. Note that the first transcribed nucleotide is assigned the position of +1.

A core promoter region can contain zero or more core promoter motifs. Some core promoter motifs are associated with the same basal transcription factor. For example, the Inr, TATA box, MTE, and DPE motifs are all associated with Transcription Factor II D (TFIID) [Theisen et al., 2010], and the BRE^u and BRE^d motifs are associated with the Transcription Factor II B (TFIIB) [Deng and Roberts, 2006]. The combination of motifs in a given promoter region can be a critical determinant in regulating gene expression [Juven-Gershon et al., 2006].

It is important to realize that promoters can vary widely, possessing different combinations of motifs [Vo ngoc et al., 2017]. For example, different isoforms of a single gene can have different promoters, each with a different combination of motifs. Screening of promoters for core promoter motifs using computational tools has shown that many promoters do not have any of the known core promoter motifs shown in Table 2 [Sloutskin et al., 2015]. Nevertheless, it is important to probe a putative TSS site location for the presence of these motifs as it provides more supporting evidence for the TSS annotation.

Although promoter motifs are commonly found in the promoter regions of protein-coding genes, there are many genes that do not appear to use such motifs for initiating transcription. Finding motifs within the predicted promoter region of a gene is exciting and confirms our current understanding of transcription initiation and regulation. The lack of promoter motifs for some genes, however, tells us that we still have a lot to learn about how the cell turns gene expression on and off!