Interplay between Chromatin and Trans-acting Factors Regulating the Hoxd4 Promoter during Neural Differentiation

Mark Featherstone,Mojgan Rastegar,Laila Kobrossy

doi:10.1074/jbc.m602555200

Abstract

Correct patterning of the antero-posterior axis of the embryonic trunk is dependent on spatiotemporally restricted Hox gene expression. In this study, we identified components of the Hoxd4 P1 promoter directing expression in neurally differentiating retinoic acid-treated P19 cells. We mapped three nucleosomes that are subsequently remodeled into an open chromatin state upon retinoic acid-induced Hoxd4 transcription. These nucleosomes spanned the Hoxd4 transcriptional start site in addition to a GC-rich positive regulatory element located 3' to the initiation site. We further identified two major cis-acting regulatory elements. An autoregulatory element was shown to recruit HOXD4 and its cofactor PBX1 and to positively regulate Hoxd4 expression in differentiating P19 cells. Conversely, the Polycomb group (PcG) protein Ying-Yang 1 (YY1) binds to an internucleosomal linker and represses Hoxd4 transcription before and during transcriptional activation. Sequential chromatin immunoprecipitation studies revealed that the PcG protein MEL18 was co-recruited with YY1 only in undifferentiated P19 cells, suggesting a role for MEL18 in silencing Hoxd4 transcription in undifferentiated P19 cells. This study links for the first time local chromatin remodeling events that take place during transcriptional activation with the dynamics of transcription factor association and DNA accessibility at a Hox regulatory region.

Highlights

Kingdom, and their expression is tightly regulated [3]
Modifications characteristic of transcriptionally active chromatin were correlated with transcriptional activation, starting at the 3Ј end of the gene and concluding more 5Ј at P1. This conversion was accompanied by recruitment of RNA polymerase II to the Hoxd4 locus, a process that did not occur in the absence of retinoic acid (RA) and not before those chromatin modifications took place. These results suggested that the Hoxd4 P1 promoter is not a nucleosome-free region and raised the question of whether nucleosome positioning plays an active role in mediating the repression and/or activation of Hoxd4 transcription
We showed that nucleosomes are positioned at the Hoxd4 P1 promoter and are remodeled following RA treatment of P19 cells

Summary

Introduction

Kingdom, and their expression is tightly regulated [3]. In mammals, 39 Hox genes are organized into four clusters, each located on a different chromosome [1]. Similar to Hoxb4 [32], interactions between the Hoxd4 3Ј neural enhancer and its proximal promoter P1 are important in initiation of Hoxd gene expression in the hindbrain of transgenic embryos [25] and in neurally differentiating P19 embryonal carcinoma cells [33]. We have correlated this enhancerpromoter interaction with chromatin changes occurring upon Hoxd gene activation in response to RA in neurally differentiating P19 cells and in the central nervous system of developing mouse embryos. These studies established P19 cells as a valid system for studying Hoxd enhancer-promoter function

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Results

Conclusion