Nascent Wars: How RARE enhancers regulate Hoxb gene transcripts

Abstract

During development of an embryo, the anterior-posterior (A-P) body axis has to be properly defined, in order for body segments and organs to form in their correct locations. A family of transcription factors, the Hox genes, play an important role in determining the A-P body axis. In mammals, there are four Hox gene cluster (A-P) that are arranged on four different chromosomes. For proper A-P axis formation, the spatial and temporal domains of Hox genes within the clusters have to be precisely initiated and strictly maintained. Therefore, it is critical to understand the regulatory inputs that dictate the expression of Hox genes. It has been identified that Hox genes are responsive to morphogen gradients, e.g., retinoic acid (RA), along with cis-regulatory elements and long non-coding RNAs that are interspersed within the clusters. In this study, I have looked at retinoic acid response elements (RAREs), which help to incorporate RA signaling to regulate the expression of Hox genes. I have optimized the single molecule fluorescent in situ hybridization technique (smFISH), to look at newly synthesized or nascent Hoxb transcripts in mouse tissue sections. I found that three RAREs – DE, B4U, and ENE while having individual inputs into regulating Hoxb genes, also appear to work together to ensure proper levels of nascent transcripts in the neural tube and adjacent somites. Furthermore, I see that these RARE have different inputs along the axial level of the embryo, such that DE plays a greater role anteriorly while B4U plays a greater role posteriorly. In the DE-B4U double mutants, I observe that antagonism from the individual mutants is neutralized as levels of nascent transcripts in the double mutant appear similar to wildtype. Results from the triple DE-B4U-ENE mutants highlight that these RAREs are critical for nascent transcription of Hox genes, as in the triple mutant we observe a very low number of nascent transcripts. These results demonstrate how multiple RAREs integrate response to RA to fine-tune the transcript levels of Hox genes. My study is a starting point towards understanding the in vivo transcriptional dynamics of Hox genes in mouse embryos. This study is a step towards better understanding the enhancer-promoter interactions required for regulating genes that ensure proper embryonic development.