Interactions between LHX3- and ISL1-family LIM-homeodomain transcription factors are conserved in Caenorhabditis elegans

Mugdha Bhati,David A Jacques,Jacqueline M Matthews,Estelle Llamosas,Cy M Jeffries,Hannah R Nicholas,Siavoush Dastmalchi,Nina Ripin

doi:10.1038/s41598-017-04587-8

Abstract

LIM-Homeodomain (LIM-HD) transcription factors are highly conserved in animals where they are thought to act in a transcriptional ‘LIM code’ that specifies cell types, particularly in the central nervous system. In chick and mammals the interaction between two LIM-HD proteins, LHX3 and Islet1 (ISL1), is essential for the development of motor neurons. Using yeast two-hybrid analysis we showed that the Caenorhabditis elegans orthologs of LHX3 and ISL1, CEH-14 and LIM-7 can physically interact. Structural characterisation of a complex comprising the LIM domains from CEH-14 and a LIM-interaction domain from LIM-7 showed that these nematode proteins assemble to form a structure that closely resembles that of their vertebrate counterparts. However, mutagenic analysis across the interface indicates some differences in the mechanisms of binding. We also demonstrate, using fluorescent reporter constructs, that the two C. elegans proteins are co-expressed in a small subset of neurons. These data show that the propensity for LHX3 and Islet proteins to interact is conserved from C. elegans to mammals, raising the possibility that orthologous cell specific LIM-HD-containing transcription factor complexes play similar roles in the development of neuronal cells across diverse species.

Highlights

LIM-containing proteins are commonly found in eukaryotes of all types, but LIM-homeodomain (LIM-HD) transcription factors are unique to and highly conserved in animals
We identified an ISL1LID-like domain in LIM-7 and tested the ability of this domain to interact with CEH-14
A manual alignment of the C-terminal regions of ISL1 and LIM-7 that used the spacing of the HD and LID domains as well as the structurally conserved LIM-binding motifs in the LID domains[26] as a guide, revealed a putative LIM interaction domain encompassing residues 347–376 of LIM-7 (LIM-7LID; Fig. 2A)

Summary

Introduction

LIM-containing proteins are commonly found in eukaryotes of all types, but LIM-homeodomain (LIM-HD) transcription factors are unique to and highly conserved in animals. The best characterised example of the LIM code relates to the developing ventral spinal cord in vertebrates It involves LIM-HD factors LIM homeobox protein 3 (LHX3) and Islet 1 (ISL1). In developing V2 interneurons LHX3 binds to LIM-domain binding protein 1 (LDB1), which is an essential cofactor for LIM-HD proteins[8], and this binary complex binds to LHX3 recognition elements on DNA, including those in the promoter region of Chx[10], a V2 interneuron marker[9] (Fig. 1C). In developing motor neurons ISL1 binds directly to LDB1, while LHX3 binds instead to ISL1 This ternary complex binds to ISL1/LHX3 recognition sites, including those in the promoter region of Hb9, which is a marker of motor neurons (Fig. 1C). The structures of LHX3 in complex with each of ISL1 and LDB1 revealed that both proteins bind LHX3 in the same manner, despite considerable sequence variation in the interaction domains[11]

Methods

Results

Conclusion