Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice

Abstract

How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. Here, we show that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, we test a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.

Developmental Origins of Human Cortical Oligodendrocytes and Astrocytes

“…1 F, I). We first examined GSX2 expression in the GW23 cortex; the GSX2 homeodomain is at the top of the hierarchical gene regulatory network that governs OBiN development in the mouse cortex and dorsal lateral ganglionic eminence (LGE) [ 39 , 58 – 61 ]. As expected, GSX2 + cells were observed in the GW23 cortex; these cells were also mainly located in the cortical IFL (Fig.…”

Gsx2 but not Gsx1 is necessary for early forebrain patterning and long-term survival in zebrafish

“…gsx1 and gsx2 are the vertebrate homologs of Drosophila melanogaster intermediate neuroblasts defective (ind). ind and the gsx genes similarly regulate dorsoventral (DV) patterning [20][21][22] , and Ind and murine GSX2 elicit similar regulatory outcomes based on monomer versus homodimer DNA binding 23 . Interestingly, ind and the gsx genes are expressed in similar patterns in the fly neuroectoderm 20 , mouse neural tube 24 , and Xenopus neural plate 25 , supporting models for conserved neuroaxis domain specification across species.…”

Mechanisms Underlying Hox-Mediated Transcriptional Outcomes

“…Moreover, the use of higher resolution binding assays such as Cleavage Under Targets & Release Using Nuclease (CUT&RUN) or ChIP-Exonuclease can provide near bp resolution binding that reveals if adjacent sites are also occupied near the Hox TF binding site. Such an approach was recently utilized for the Gsx2 homeodomain TF to reveal distinct monomer versus dimer binding events using CUT&RUN assays and nucleotide footprinting analysis ( Salomone et al, 2021 ). By combining high-resolution genomic binding data with transcriptomic studies using wild type and specific mutant cells (i.e., Hox mutant, Pbx/Exd mutant, etc), we will be better positioned to define which binding events are associated with gene expression changes.…”