A micro-evolutionary change in target binding sites as a key determinant of Ultrabithorax function in Drosophila

Abstract

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

Motifizer: a tool for parsing high-throughput sequencing datasets and quantitative comparative analyses of transcription factor-binding sites

“…Site directed mutagenesis approach was used to mutate the Mes2 binding motif in the CG13222 enhancer using the following primer: 5’- TTGTCCTTCTGTCAATGTTTTGGAAAAAACCAATAAAGCTG-3’. Luciferase assays were carried out using the method explained in Khan et al ., 2021.…”

“…In an attempt to identify such co-transcription factors involved in Ubx mediated modulation of gene regulatory pathways, we developed a methodology as described in Fig 2A . We first generated three replicates of ChIP-seq data for Ubx in third instar haltere imaginal discs and analyzed it using the ChIP-seq module. The details analysis of enriched motifs has been described in a separate communication (Khan et al, 2021). Next, we combined the three biological replicates to identify high confidence peaks and annotated them using the Homer software (detailed in Supplementary Materials and Methods).…”