Lmx1b‐dependent Activity of an Associated Enhancer Suggests Lmx1b Autoregulation
Authors: Ivey Lauren A. , Haro Endika , Pira Charmaine U. , Oberg Kerby C. 
Published: November 2023

IntroductionLmx1b is a homeobox transcription factor vital for proper limb dorsalization, as well as development of structures in the kidney, brain and eye. Within the developing limb bud, expression of Lmx1b is limited to the dorsal mesoderm.In humans, LMX1B mutations that produce haploinsufficiency cause Nail Patella Syndrome (NPS), a disorder with underdeveloped nails, deficient or absent patellae, and potential renal complications. Genome‐wide sites of murine Lmx1b binding by ChIP‐seq analysis identified a conserved Lmx1b‐bound interval (LBI) in non‐coding DNA 60 kb upstream of the Lmx1b gene. The LBI was associated with positive chromatin regulatory marks consistent with regulatory activity. Furthermore, the LBI contained 4 potential Lmx1b binding sites that were conserved across divergent vertebrates. Thus, we hypothesized that Lmx1b binding to this potential regulatory sequence might be required to autoregulate its own expression.MethodsThe fragment containing the LBI was isolated from murine genomic DNA and inserted into a reporter construct using the Herpes simplex virus tk minimal promoter linked to a GFP reporter. We then electroporated this construct into the presumptive wing of HH14 chicken embryos, along with a β‐actin promoter driven RFP plasmid to ascertain transfection efficiency.To characterize the role of Lmx1b binding on regulatory activity, we disrupted the individual Lmx1b binding sites by site‐directed mutagenesis. The empty reporter construct was used as a negative control. After 48 hrs, the limb buds were harvested and analyzed by fluorescence microscopy. GFP levels were quantified by ImageJ, and statistical analysis was performed using “R” computing software.ResultsThe LBI displayed robust enhancer activity within the dorsal mesoderm. Three of the four binding sites were associated with enhancer activity, and disruption of any one site caused a reduction in GFP expression but did not alter dorsal restriction. Compared to the mean GFP expression in the negative control, the fluorescence levels in the unmutated construct was 7.3 fold greater, while the three mutant plasmids expressed a 1.9 to 2.3‐fold increase. One‐way ANOVA tests supported these differences in the control and experimental groups as statistically significant (p ≤ 0.05).All three mutants had a statistically significant reduction in GFP expression when individually compared to the wild type enhancer, post‐hoc testing revealed no significant difference among the 3 mutants themselves or between the mutants individually and the negative control.Discussion/ConclusionsMutation of Lmx1b's DNA binding domain is known to cause a 5.8‐fold reduction in Lmx1b expression indicating autoregulation. Collectively, our data provide compelling evidence that Lmx1b binding to the Lmx1b‐associated regulatory module (LARM) is necessary for its function as an autoregulatory enhancer. Since LMX1B haploinsufficiency causes NPS, it is not unreasonable to speculate that mutations in this autoregulatory enhancer may also be a cause of this disorder.Support or Funding InformationFunding in part by a grant from the National Organization for Rare Disorders (NORD).