Dynamic changes in cis-regulatory occupancy by Six1 and its cooperative interactions with distinct cofactors drive lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

Jinshu Xu,Aarthi Ramakrishnan,Elaine Y M Wong,Ting Zhang,Jianqiang Ding,Li Shen,Bernd Fritzsch,Yong-Hwee Eddie Loh,Pin-Xian Xu,Jun Li

doi:10.1093/nar/gkaa012

Abstract

The transcription factor Six1 is essential for induction of sensory cell fate and formation of auditory sensory epithelium, but how it activates gene expression programs to generate distinct cell-types remains unknown. Here, we perform genome-wide characterization of Six1 binding at different stages of auditory sensory epithelium development and find that Six1-binding to cis-regulatory elements changes dramatically at cell-state transitions. Intriguingly, Six1 pre-occupies enhancers of cell-type-specific regulators and effectors before their expression. We demonstrate in-vivo cell-type-specific activity of Six1-bound novel enhancers of Pbx1, Fgf8, Dusp6, Vangl2, the hair-cell master regulator Atoh1 and a cascade of Atoh1’s downstream factors, including Pou4f3 and Gfi1. A subset of Six1-bound sites carry consensus-sequences for its downstream factors, including Atoh1, Gfi1, Pou4f3, Gata3 and Pbx1, all of which physically interact with Six1. Motif analysis identifies RFX/X-box as one of the most significantly enriched motifs in Six1-bound sites, and we demonstrate that Six1-RFX proteins cooperatively regulate gene expression through binding to SIX:RFX-motifs. Six1 targets a wide range of hair-bundle regulators and late Six1 deletion disrupts hair-bundle polarity. This study provides a mechanistic understanding of how Six1 cooperates with distinct cofactors in feedforward loops to control lineage-specific gene expression programs during progressive differentiation of the auditory sensory epithelium.

Highlights

The transcription factor (TF) Six1 belongs to the sine oculis (So/Six) homeobox protein family that plays important roles in the development of multiple organs, including ear, urinary system and kidney [1,2,3,4]
To better characterize the chromatin structure associated with Six1, we used antibody-mediated ChIP on E13.5 cochleae to pull-down chromatin associated with the histone H3 Lys 27 acetylation (H3K27ac)––an epigenetic marker associated with active enhancers [26] and the histone H3 Lys 27 trimethylation mark (H3K27me3)––an epigenetic marker associated with transcriptional repression [27]
We identified a total of 14 967 Six1bound regions and observed clusters with varying levels of enrichment (Figure 1B and Supplementary Figure S1A). 5270 regions showed loss of or reduced Six1-occupancy at E16.5 with very weak or no H3K27ac-deposition, while 6616 regions showed new or increased binding at E16.5 with weaker H3K27ac-deposition at E13.5. 2981 Six1-bound sites retained occupancy upon differentiation and had strong H3K27ac-deposition, indicating that these regions are enhancers from E13.5. ∼66% of E16.5 and 37% of E13.5 peaks were marked by H3K27ac (Figure 1C and Supplementary Figure S1D, E), suggesting an increase in the proportion of Six1bound enhancers as differentiation proceeds

Summary

Introduction

The transcription factor (TF) Six belongs to the sine oculis (So/Six) homeobox protein family that plays important roles in the development of multiple organs, including ear, urinary system and kidney [1,2,3,4]. Overexpression of SIX1 is associated with many human cancers [5], while mutations in the human SIX1 cause Branchio-Oto-Renal (BOR) or Branchio-Oto (BO) syndrome [6]. The mammalian inner ear sensory organ for hearing––the organ of Corti––in the cochlea houses two types of hair cells: one row of inner and three rows of outer hair cells interdigitated with several subtypes of supporting cells––one inner border, one inner phalangeal, inner and outer pillar, and three rows of Deiters’ cells aligned in a medial-to-lateral direction, all of which differentiate from common precursors [9,10,11]. Failure to generate or maintain these epithelial cells in the organ of Corti causes irreversible deafness due to lack of regenerative capacity of the cochlea. Developmental programs that generate these distinct subtypes are not understood, presenting a major challenge for clin-

Methods

Results

Conclusion