Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus.

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor, but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.

Functional analysis of limb enhancers in the developing fin.

Despite diverging ∼365 million years ago, tetrapod limbs and pectoral fins express similar genes that could be regulated by shared regulatory elements. In this study, we set out to analyze the ability of enhancers to maintain tissue specificity in these two divergent structures. We tested 22 human sequences that were previously reported as mouse limb enhancers for their enhancer activity in zebrafish (Danio rerio). Using a zebrafish enhancer assay, we found that 10/22 (45 %) were positive for pectoral fin activity. Analysis of the various criteria that correlated with positive fin activity found that both spatial limb activity and evolutionary conservation are not good predictors of fin enhancer activity. These results suggest that zebrafish enhancer assays may be limited in detecting human limb enhancers, and this limitation does not improve by the use of limb spatial expression or evolutionary conservation.

DNA topology of highly transcribed operons in Salmonella enterica serovar Typhimurium.

Bacteria differ from eukaryotes by having the enzyme DNA gyrase, which catalyses the ATP-dependent negative supercoiling of DNA. Negative supercoils are essential for condensing chromosomes into an interwound (plectonemic) and branched structure known as the nucleoid. Topo-1 removes excess supercoiling in an ATP-independent reaction and works with gyrase to establish a topological equilibrium where supercoils move within 10 kb domains bounded by stochastic barriers along the sequence. However, transcription changes the stochastic pattern by generating supercoil diffusion barriers near the sites of gene expression. Using supercoil-dependent Tn3 and γδ resolution assays, we studied DNA topology upstream, downstream and across highly transcribed operons. Whenever two Res sites flanked efficiently transcribed genes, resolution was inhibited and the loss in recombination efficiency was proportional to transcription level. Ribosomal RNA operons have the highest transcription rates, and resolution assays at the rrnG and rrnH operons showed inhibitory levels 40-100 times those measured in low-transcription zones. Yet, immediately upstream and downstream of RNA polymerase (RNAP) initiation and termination sites, supercoiling characteristics were similar to poorly transcribed zones. We present a model that explains why RNAP blocks plectonemic supercoil movement in the transcribed track and suggests how gyrase and TopA control upstream and downstream transcription-driven supercoiling.

Transcriptomic and epigenomic characterization of the developing bat wing.

Bats are the only mammals capable of powered flight, but little is known about the genetic determinants that shape their wings. Here we generated a genome for Miniopterus natalensis and performed RNA-seq and ChIP-seq (H3K27ac and H3K27me3) analyses on its developing forelimb and hindlimb autopods at sequential embryonic stages to decipher the molecular events that underlie bat wing development. Over 7,000 genes and several long noncoding RNAs, including Tbx5-as1 and Hottip, were differentially expressed between forelimb and hindlimb, and across different stages. ChIP-seq analysis identified thousands of regions that are differentially modified in forelimb and hindlimb. Comparative genomics found 2,796 bat-accelerated regions within H3K27ac peaks, several of which cluster near limb-associated genes. Pathway analyses highlighted multiple ribosomal proteins and known limb patterning signaling pathways as differentially regulated and implicated increased forelimb mesenchymal condensation in differential growth. In combination, our work outlines multiple genetic components that likely contribute to bat wing formation, providing insights into this morphological innovation.