Critical Roles of the LIM Domains of Lhx3 in Recruiting Coactivators to the Motor Neuron-Specifying Isl1-Lhx3 Complex

Abstract

During spinal cord development, the LIM domains of the LIM homeodomain factor Lhx3 bind to either the LIM cofactor nuclear LIM interactor (NLI) or another LIM homeodomain factor, Isl1, assembling the tetrameric V2 interneuron-specifying Lhx3 complex (2NLI:2Lhx3) or the hexameric motor neuron-specifying Isl1-Lhx3 complex (2NLI:2Isl1:2Lhx3). However, the detailed molecular basis by which the Lhx3-LIM domains contribute to motor neuron specification still remains poorly understood. Here, we show that the Lhx3-LIM domains are essential for recruiting transcriptional coactivators to the Isl1-Lhx3 complex. Using a yeast genetic screening system, we identify Lhx3 point mutants that bind to NLI but not Isl1. Accordingly, these mutants fail to assemble the Isl1-Lhx3 complex. However, their interaction with coactivators is relatively intact, and they are fully functional in the Lhx3 complex and V2 interneuron specification. Interestingly, when these Lhx3 mutants are directly fused to Isl1, their transcriptional activity in the Isl1-Lhx3 complex is restored. We further show that this restoration reflects an unexpected role of the Lhx3-LIM domains, likely together with Isl1, to form an interaction interface for coactivators. Our results suggest that the Lhx3-LIM domains play critical roles in transactivation of the Isl1-Lhx3 complex by not only directing the assembly of the Isl1-Lhx3 complex but also recruiting coactivators to the complex.

FIG 1

LIM-HD complexes during embryonic spinal V2-IN and MN development. (A) Schematic representation of V2-INs and MNs derived from p2 and pMN domains, respectively, as well as the Lhx3 complex and the Isl1-Lhx3 complex, on their response elements. (B) Schematic representation of LIM-HD factors and their fusions used in the study. (C) Comparison of the LIM domains of Lhx3 and Lhx1. Two LIM domains, LIM1 and LIM2, are denoted by arrows.

FIG 2

Isolation of Lhx3 mutants that interact with NLI but not with Isl1. (A) Schematic illustration for the yeast one- plus two-hybrid system to identify Lhx3 mutants that lose interactions with Isl1. (B) Sequences of mutants that bind NLI but not Isl1. The listed mutants, m1 to m12, lacked interactions with Isl1 but retained interactions with NLI. For wild-type sequences, two LIM domains are denoted as arrows, and the residues conforming to the consensus zinc finger motifs [CX₂CX_16–23HX₂CX₂CX₂CX_16–21CX₂(CHD)] are highlighted in green. (C) Isl1 interaction-null mutants isolated from the yeast one- plus two-hybrid screening were tested for their interactions with NLI. Yeast two-hybrid assays on LexA fused to full-length Isl1 or NLI and B42 fused to full-length wild-type Lhx3 or the Lhx3-Y116C or Lhx3-F136S mutant. +++ and − represent strong (bright blue) and no (white to very weak blue) interactions, as visualized in the X-Gal plate. (D) CoIP experiments with in vitro-translated Isl1, NLI, wild-type Lhx3, and the Lhx3-Y116C and Lhx3-F136S mutants. Immunoprecipitation was carried out by IgG or anti-Lhx3 antibody, followed by immunoblotting by anti-HA antibody for Isl1 and NLI as well as Lhx3 antibody.

FIG 3

Lhx3-Y116C and Lhx3-F136S mutants are specifically impaired for Isl1-Lhx3 complex assembly. (A) EMSA with MNe, TeRE^1/2, and Isl1-RE as probes. Arrows in MNe and TeRE^1/2 panels indicate binding by a complex of Isl1 plus Lhx3. The arrow in the Isl1-RE panel indicates binding by Isl1. The arrowhead indicates binding by Lhx3 alone. (B) ChIP assays for Hb9 MNe and Chx10e using P19 cells transfected with Isl1 and wild-type or mutant Lhx3. (C and D) Luciferase reporter assays with TeRE::LUC (C) and MNe::LUC (D) in P19 cells, shown as relative fold activation.

FIG 4

Failure to specify MNs by Lhx3-Y116C and Lhx3-F136S. (A and B) Ectopic GFP expression from TeRE::GFP (A) and MNe::GFP (B) in the dorsal spinal cord. (C and D) Electroporation to assess ectopic Chx10⁺ V2 differentiation (C) and Hb9⁺ MN differentiation (D) in the dorsal spinal cord. The efficiency of V2 and MN induction was determined by the number of ectopic Chx10⁺ V2-INs (red) and Hb9⁺ MNs (red) among all electroporated cells expressing Lhx3 (green). Quantification results are shown. *, P values less than 0.001.

FIG 5

Direct fusion of Lhx3-Y116C and Lhx3-F136S to Isl1 enables them to specify MNs. (A) Electroporation to assess formation of ectopic Hb9⁺ MN differentiation in the dorsal spinal cord by Isl1-Lhx3, Isl1-Lhx3-F136S, and Isl1-Lhx3-Y116C fusions. (B) Luciferase reporter assays with MNe::LUC in P19 cells. In both types of assays, it is evident that Lhx3-Y116C and Lhx3-F136S directly fused to Isl1 act as a fully functional Isl1-Lhx3 complex. RLU, relative luciferase units.

FIG 6

Lhx3-Y116C and Lhx3-F136S interact with coactivators. (A) CoIP assays between coactivator RbBP5 with Lhx1, Isl1, and Lhx3 in HEK293T cells. (B and C) Luciferase reporter assays with TeRE::LUC (B) and HxRE::LUC (C) in P19 cells expressing Isl1, Lhx1, and Lhx3, alone or in combination as indicated. (D) CoIP assays between RbBP5 and L1-Lhx3, Lhx3, Lhx3-F136S, and Lhx3-Y116C in HEK293T cells.

FIG 7

Recruitment of coactivators to motor neuron enhancers via Isl1-Lhx3 complex. (A) CoIP assays between coactivators RbBP5 and p300 with Isl1-Lhx3, Isl1-Lhx3-F136S, Isl1-Lhx3-Y116C, and Isl1-L1-Lhx3 in HEK293T cells. (B) ChIP assays with anti-p300 and anti-RbBP5 antibodies to show the recruitment of coactivators to the motor neuron enhancers in P19 cells transfected with the indicated Isl1-Lhx3 fusion constructs. (C) ChIP assays with anti-p300 and anti-RbBP5 antibodies to show the recruitment of coactivators to the motor neuron enhancers in developing E12.5 mouse spinal motor neurons.

FIG 8

Lhx3-Y116C and Lhx3-F136S do not interact with NLI within the Isl1-Lhx3 complex. (A) GST-pulldown assays to examine the interaction between NLI and Lhx3, Lhx3-F136S, Lhx3-Y116C, or L1-Lhx3 in HEK293T cells. (B) GST pulldown assays to examine the interaction between NLI and Isl1^HD fused to Lhx3, Lhx3-F136S, Lhx3-Y116C, or L1-Lhx3 in HEK293T cells.