Lmx1b-targeted cis-regulatory modules involved in limb dorsalization

Abstract

Lmx1b is a homeodomain transcription factor responsible for limb dorsalization. Despite striking double-ventral (loss-of-function) and double-dorsal (gain-of-function) limb phenotypes, no direct gene targets in the limb have been confirmed. To determine direct targets, we performed a chromatin immunoprecipitation against Lmx1b in mouse limbs at embryonic day 12.5 followed by next-generation sequencing (ChIP-seq). Nearly 84% (=617) of the Lmx1b-bound genomic intervals (LBIs) identified overlap with chromatin regulatory marks indicative of potential -regulatory modules (PCRMs). In addition, 73 LBIs mapped to CRMs that are known to be active during limb development. We compared Lmx1b-bound PCRMs with genes regulated by Lmx1b and found 292 PCRMs within 1 Mb of 254 Lmx1b-regulated genes. Gene ontological analysis suggests that Lmx1b targets extracellular matrix production, bone/joint formation, axonal guidance, vascular development, cell proliferation and cell movement. We validated the functional activity of a PCRM associated with joint-related that provides a mechanism for Lmx1b-mediated joint modification and a PCRM associated with that suggests a role in autoregulation. This is the first report to describe genome-wide Lmx1b binding during limb development, directly linking Lmx1b to targets that accomplish limb dorsalization.

Identification of limb-specificLmx1bauto-regulatory modules with Nail-Patella Syndrome pathogenicity

“…S1). Thus, the observation that both LARM1 and LARM2 are bound by Lmx1b 7 , display dorsal restricted activity in limb buds, require Lmx1b binding sites for activity and interact with the Lmx1b promoter, in addition to human LMX1B being sufficient to activate LARM2 in the ventral mesoderm, collectively support the concept that LARM1/2 are bona fide Lmx1b autoregulatory enhancers. Interestingly, one of the enhancers of Apterous (ap), the Drosophila homologue of Lmx1b, is maintained by an autoregulatory loop, albeit indirectly through the ap targets vestigial and scalloped (Vg/Sd) 13 .…”

“…In this report, we characterize two conserved control Input limb DNA 7 , p300 20 , histone 3 acetylation at lysine 27 (H3k27Ac) 21 , histone 3 dimethylation at lysine 4 (H3k4me2) 22 , RNA polymerase II 23 , Med 12 23 and histone 3 trimethylation at lysine 27 (H3Kk7me3) 22 . Note that LARM1 consists of two conserved peaks, both of which are recognized by Lmx1b-targeted ChIP-seq 7 . showed activity restricted to the dorsal limb (7/7).…”

“…We recently identified two Lmx1b-bound CRMs, LARM1 and LARM2, and 66 kb upstream of the mouse Lmx1b gene, respectively (Fig. 1A) 7 , that are associated with active chromatin marks (p300, H3K27ac, H3K4me2, RNAPOL2 and Med12) during limb development (Fig. 1B).…”

“…Conservation analysis using the VISTA browser ( Fig. 1B) subdivided LARM1 into two conserved regions, a 5' element containing one conserved potential Lmx1b binding site and a 3' element with three conserved binding sites (based on the reported TMATWA binding motif) 7 ( Fig. 2A).…”

“…In the absence of Lmx1b function, transcription of the Lmx1b mRNA is decreased nearly 6fold in developing mouse limbs suggesting that one function of Lmx1b is the auto-amplification of its own expression 6 . Lmx1b-targeted chromatin immunoprecipitation combined with highthroughput sequencing (ChIP-seq) during limb dorsalization identified two highly conserved Lmx1b-bound cis-regulatory modules (CRMs) 60 kb upstream of the Lmx1b gene 7 . CRMs are DNA sequences enriched in transcription factor binding sites that regulate associated genes in a time and tissue specific manner.…”