A Web-Based Workflow for Selecting Gene- and Tissue-Specific Enhancers

We are interested in decoding the non-coding genome to understand how genes are regulated, to be expressed at the right time and the right place. We develop user-friendly protocols using web-based genomics tools to make enhanced discovery accessible to all biologists. Wide availability of multimodal data that can help narrow down the location of enhancers and the easy access of this data through open source web interfaces.

This enables comprehensive enhancer identification without requiring programming expertise from researchers. This protocol provides a foundation for anyone without a background in enhancer biology to start navigating publicly available data sets to retrieve enhancers for the genes of interest. We use TBX5, a well-known player in heart biology, as a case study.

Our workflow identified 21 enhancers, which may shed light on heart development mechanisms and congenital heart disease. We'll expand our protocol with new data sources, including spatial genomics, while developing additional user-friendly tools for biologists. To begin, open the Ensembl Genome Browser.

Select the genome assembly that matches the species and version of interest. Enter the gene of interest in the search field and click Go.From the results, click on the appropriate Ensembl Gene ID.Then scroll to the Summary"section and click the Region in Detail"link to visualize the surrounding region of the gene of interest. Now, search for the two genes flanking the gene of interest using the Gene Legend track.

These genes appear as visual elements representing merged Ensembl and Havana annotations within the basic gene annotations from the GENCODE track. Determine the directionality of the gene of interest by observing the greater than or less than signs beside the gene names. Click and drag the cursor across the intergenic region between these genes, then click Jump to Region"in the popup box to view the selected region.

Customize the display by clicking Add"or Remove Tracks"at the top of the track viewer. Use the zoom and navigation controls to adjust the view for enhanced visualization of the region. In the Region in Detail"tab viewer, click Configure This Page"in the sidebar.

In the Configure Region Image"sidebar, under Regulation, select Activity by Cell or Tissue. Use the cell or tissue search bar to find and select the desired tissues, or use the alphabetic index below the bar. Click on the Experiments"tab adjacent to the Cell or Tissue"option.

Choose H3K4me1 and H3K27ac as a marker for enhancers and H3K4me3 as a marker for promoters and click Configure Track Display. Then, select View Tracks"to visualize regions marked by H3K4me1 within the enhancer detection region and regions marked by H3K4me3 upstream of the gene of interest. Click on the colored visual or box elements in the H3K4me1 track to retrieve the genomic coordinates of regions marked within the defined detection region.

This opens the Hists and Pols popup, which displays the element's genomic location in base pairs. Alternatively, manually define the regions of interest for each genomic feature by clicking and dragging on the track to enclose graph peaks under the H3K4me1 or H3K27ac tracks. Then copy the genomic location coordinates into a text file and save the file in BED format.

Access the 4DN Data Portal. On the homepage, ensure that Experiment Sets"is selected as the Y-axis of the main stacked bar, Experiment Type"is selected as the X-axis, and the plot is grouped by organism. Locate the In Situ Hi-C bar along the X-axis and click on the portion representing human experiment sets.

In the popup, click the Browse"button and select cardiac muscle myoblasts"and our gene of interest, TBX5, associated with heart organogenesis. Click on the link in the Title"column of the relevant biosample corresponding to the tissue of interest. Then, in the Processed Files"tab, click Explore Data"to examine the Hi-C dataset in more detail.

Enter the coordinates of the identified promoter in the tissue of interest and right click the heat map to mark the region horizontally. These lines allow the promoter region to be visually tracked across the heat map. If required, adjust the view by dragging it vertically until the upper and lower bounds in the Y coordinates of the search bar align with the boundaries of the region of interest.

To remove any unwanted lines, right click the line and select Horizontal"or Vertical Rule, then click Close Series. Enter the coordinates of all experimentally validated control enhancers to calculate the interaction threshold based on their minimum interaction values. Display the three control enhancers from the literature alongside the promoter region from Ensembl using the pre-prepared view.

Then define the promoter enhancer threshold using these control enhancers by selecting the lowest non-zero interaction score as indicated by the color key on the right side of the heat map matrix. Enter the genomic coordinates of all H3K4me1-associated enhancer regions and mark vertically on the Hi-C heat map. Filter out weakly interacting regions by comparing the interaction scores of H3K4me3-marked regions against the interaction threshold.

Select the genomic coordinates showing interaction frequencies above this threshold, which appear as more concentrated or darker signals on the heat map, and save them in BED format. Three of the four cardiac enhancers identified in the VISTA Cardiac Enhancer Browser, HS2329, mm1282, and mm370, overlapped with regions predicted by the web-based enhancer detection protocol. Enhancer Two overlapped with both a region predicted by the enhancer detection protocol and an experimentally validated enhancer.

Enhancer 16 showed overlap with both predicted enhancer regions and prior experimental data. Enhancer Nine did not overlap with any predicted enhancers from the pipeline but showed partial H3K4me1 signal enrichment.