Interrogating Transcriptional Regulatory Sequences in Tol2-Mediated Xenopus Transgenics

<div><p>Identifying gene regulatory elements and their target genes in vertebrates remains a significant challenge. It is now recognized that transcriptional regulatory sequences are critical in orchestrating dynamic controls of tissue-specific gene expression during vertebrate development and in adult tissues, and that these elements can be positioned at great distances in relation to the promoters of the genes they control. While significant progress has been made in mapping DNA binding regions by combining chromatin immunoprecipitation and next generation sequencing, functional validation remains a limiting step in improving our ability to correlate <i>in silico</i> predictions with biological function. We recently developed a computational method that synergistically combines genome-wide gene-expression profiling, vertebrate genome comparisons, and transcription factor binding-site analysis to predict tissue-specific enhancers in the human genome. We applied this method to 270 genes highly expressed in skeletal muscle and predicted 190 putative <i>cis</i>-regulatory modules. Furthermore, we optimized Tol2 transgenic constructs in <i>Xenopus laevis</i> to interrogate 20 of these elements for their ability to function as skeletal muscle-specific transcriptional enhancers during embryonic development. We found 45% of these elements expressed only in the fast muscle fibers that are oriented in highly organized chevrons in the <i>Xenopus laevis</i> tadpole. Transcription factor binding site analysis identified >2 Mef2/MyoD sites within ∼200 bp regions in 6 of the validated enhancers, and systematic mutagenesis of these sites revealed that they are critical for the enhancer function. The data described herein introduces a new reporter system suitable for interrogating tissue-specific <i>cis</i>-regulatory elements which allows monitoring of enhancer activity in real time, throughout early stages of embryonic development, in <i>Xenopus</i>.</p></div

Prevalence ratios comparing pooled HIV prevalence among men who engage in transactional sex to general population men aged 15 and older.

1<p>Among studies confirming HIV infection with a biological assay and among studies reporting ≥50 participants;</p>2<p>All prevalence ratios significant (<i>P</i><0.05) except for Bangladesh and Canada.</p><p>N = number of subjects; k = number of studies; I² = variation in pooled prevalence ratio due to heterogeneity; 95% CI = 95% Confidence Interval.</p

Flow diagram of included studies.

<p>Flow diagram of included studies.</p

TFBS analysis.

<p>Top 20 predicted SMEs were examined for the presence of Mef2 and MyoD clusters. We found clusters of >2 Mef2/MyoD sites over regions >200 bp in all 6 SMEs shown to drive muscle expression in combination with <i>γ-cry</i> promoter (red) and 1/3 of the additional 3 enhancers that were found to drive muscle expression in combination with <i>krt8</i> promoter only (orange). Two of the negative elements also displayed this cluster (SME6/SME20), but none of the enhancers found to express in tissues other than muscle (green).</p

Evaluating promoter and enhancer specificity to determine optimum transgenic construc configuration for enhancer characterization.

<p>Evaluating promoter and enhancer specificity to determine optimum transgenic construc configuration for enhancer characterization.</p

Proportion of studies reporting individual factors that affect vulnerability to HIV by region, overall and specifically among men who engage in transactional sex.

1<p>Including syphilis, gonorrhea, chlamydia, human papilloma virus (HPV), and/or herpes simplex virus (HSV);</p>2<p>Risk factor reported among any population in the study (i.e., men who have sex with men);</p>3<p>Risk factor reported specifically among men who engage in transactional sex;</p>4<p>Fisher’s exact test comparing frequency of reporting factors by geographic region of study, overall and among men who report transactional sex.</p

Mef2C/MyoD sites are essential for SME8/16 muscle specific expression.

<p>Tol2 constructs containing tandem kidney (KE) and muscle (SME) enhancers in front of <i>γ-cry</i> promoter (A) were systematically mutated to remove the predicted Mef2 and MyoD sites (B, H), and compared to the ‘wildtype’ construct in transgenic efficiency and tissue specificity (C, H), as well as expression intensity (G, M). Mutating either Mef2C or MyoD sites reduced the number of embryos expressing in muscle, as well as reduced the expression intensity (D-F; I-K; G, M). [*<i>p</i>-value <0.05; **<i>p</i>-value <0.001].</p

Validating computational predictions for muscle enhancers in <i>Xenopus</i> trangenics.

<p>Predicted SMEs corresponded to evolutionary conserved elements proximal or distal to genes known to function in skeletal muscle (A-F). Six SMEs consistently expressed in skeletal muscle, in addition to eye (A′–F′). Genomic regions are color coded as follows: exons (blue); UTRs (yellow); repeats (green); conserved noncoding sequences (red); predicted SMEs are shaded in purple.</p

Pooled HIV prevalence by HIV measurement and sample size.

<p>N = number of subjects; k = number of studies; 95% CI = 95% Confidence Interval.</p

The top 20<b> </b>SME computational predictions were examined in combination with the <i>γ-cry</i> and the <i>Krt8</i> promoter.

*<p>elements show enhancer activity in a tissue other than the predicted tissue.</p