Low affinity binding sites in an activating CRM mediate negative autoregulation of the Drosophila Hox gene Ultrabithorax

Abstract

Specification of cell identity and the proper functioning of a mature cell depend on precise regulation of gene expression. Both binary ON/OFF regulation of transcription, as well as more fine-tuned control of transcription levels in the ON state, are required to define cell types. The Drosophila melanogaster Hox gene, Ultrabithorax (Ubx), exhibits both of these modes of control during development. While ON/OFF regulation is needed to specify the fate of the developing wing (Ubx OFF) and haltere (Ubx ON), the levels of Ubx within the haltere differ between compartments along the proximal-distal axis. Here, we identify and molecularly dissect the novel contribution of a previously identified Ubx cis-regulatory module (CRM), anterobithorax (abx), to a negative auto-regulatory loop that decreases Ubx expression in the proximal compartment of the haltere as compared to the distal compartment. We find that Ubx, in complex with the known Hox cofactors, Homothorax (Hth) and Extradenticle (Exd), acts through low-affinity Ubx-Exd binding sites to reduce the levels of Ubx transcription in the proximal compartment. Importantly, we also reveal that Ubx-Exd-binding site mutations sufficient to result in de-repression of abx activity in a transgenic context are not sufficient to de-repress Ubx expression when mutated at the endogenous locus, suggesting the presence of multiple mechanisms through which Ubx-mediated repression occurs. Our results underscore the complementary nature of CRM analysis through transgenic reporter assays and genome modification of the endogenous locus; but, they also highlight the increasing need to understand gene regulation within the native context to capture the potential input of multiple genomic elements on gene control.

Characterisation of the role and regulation ofUltrabithoraxin sculpting fine-scale leg morphology

“…This suggests that the enhancer we have identified does indeed recapitulate the expression of Ubx in T2 and to some extent T3. Interestingly, Delker et al (2019) carried out FAIRE-seq to assay the open chromatin in developing halteres and wings, and while this revealed the accessibility of the abx region there was no distinctive peak in the region of the NV enhancer we have discovered aided by T2 leg-specific ATAC-seq. This suggests that while the enhancer we have identified is accessible and active in legs it is not used in the developing halteres or wings.…”

“…Ectopic expression has been observed previously with reporter constructs for Hox genes such as Scr and Ubx. abx fragments drive ectopic expression in imaginal discs that do not normally express Ubx and this has been suggested to be a consequence of their exclusion of a nearby PRE (Delker et al, 2019), and potentially the variably occupied CTCF site in the third intron (Fig. 2) (Magbanua et al, 2015).…”

“…This list also include several binding sites for Ubx itself suggesting there may be Ubx autoregulation in legs as found in other tissues ( Fig. 5; Table S1) (Delker et al, 2019).…”

“…Classically, this Hox gene represses wing identity and promotes haltere formation on the third thoracic (T3) segment through the direct regulation of potentially hundreds of genes (Bender et al, 1983;Diaz-de-la-Loza et al, 2020;Pavlopoulos and Akam, 2011;Tomoyasu, 2017;Weatherbee et al, 1998;Wilcox, 1984, 1985;White and Akam, 1985). Ubx also distinguishes the size and morphology of halteres at a more finescale level, in part through the autoregulation of differences in the expression levels between proximal and distal cells (Delker et al, 2019;Roch and Akam, 2000). In T3 legs, Ubx regulates the size, as well as bristle and trichome patterns in a concentration dependent manner (Casanova et al, 1985;Davis et al, 2007;Kerridge and Morata, 1982;Morata and Kerridge, 1981;Stern, 2003;Struhl, 1982).…”

Scarless engineering of theDrosophilagenome near any site-specific integration site

“…The final alleles generated by SIRT still have an attR scar. RMCE (recombinase mediated cassette exchange) (Bateman et al, 2006) based techniques represent a different strategy (Delker et al, 2019). In these approaches, the wild type locus is first replaced by an inverted attP cassette, two attP sites in the opposite orientation flanking a dominant marker.…”