The Forkhead Transcription Factor FoxI1 Remains Bound to Condensed Mitotic Chromosomes and Stably Remodels Chromatin Structure

Abstract

All forkhead (Fox) proteins contain a highly conserved DNA binding domain whose structure is remarkably similar to the winged-helix structures of histones H1 and H5. Little is known about Fox protein binding in the context of higher-order chromatin structure in living cells. We created a stable cell line expressing FoxI1-green fluorescent protein (GFP) or FoxI1-V5 fusion proteins under control of the reverse tetracycline-controlled transactivator doxycycline inducible system and found that unlike most transcription factors, FoxI1 remains bound to the condensed chromosomes during mitosis. To isolate DNA fragments directly bound by the FoxI1 protein within living cells, we performed chromatin immunoprecipitation assays (ChIPs) with antibodies to either enhanced GFP or the V5 epitope and subcloned the FoxI1-enriched DNA fragments. Sequence analyses indicated that 88% (106/121) of ChIP sequences contain the consensus binding sites for all Fox proteins. Testing ChIP sequences with a quantitative DNase I hypersensitivity assay showed that FoxI1 created stable DNase I sensitivity changes in condensed chromosomes. The majority of ChIP targets and random targets increased in resistance to DNase I in FoxI1-expressing cells, but a small number of targets became more accessible to DNase I. Consistently, the accessibility of micrococcal nuclease to chromatin was generally inhibited. Micrococcal nuclease partial digestion generated a ladder in which all oligonucleosomes were slightly longer than those observed with the controls. On the basis of these findings, we propose that FoxI1 is capable of remodeling chromatin higher-order structure and can stably create site-specific changes in chromatin to either stably create or remove DNase I hypersensitive sites.

Changes in retinoic acid signaling alter otic patterning

“…By promoting otic fate in response to Fgf signaling, Foxi1 in the preotic region of wild-type embryos and expanded Foxi1 expression in RAtreated embryos might be involved in lens fate repression. Consistent with this interpretation, microarray analysis covering approximately 20,000 zebrafish genes showed that only seven genes, including pax6b, are significantly repressed after overexpression of Foxi1 (Yan et al, 2006). However, it is also likely that the reduction or loss of lens tissue is not due to misexpression of foxi1 or to a posteriorization of the preplacodal domain, but rather due to the ectopic expression of pax8.…”

Epigenetic regulations in hematopoietic Hox code

“…Although many components of both PcG and TrxG proteins have been characterized, yet their relationship with other chromatin-remodeling factors remains to be elucidated. Forkhead transcription factor, Foxi1, stably binds to chromatin through the cell cycle and generally inhibits the accessibility of nucleases to chromatin (Yan et al, 2006b). Given that a cascade of Fox factors have been found to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals (Zaret et al, 2008), an attractive possibility is that the pioneer Fox proteins may be involved in PcG/TrxG complex-mediated chromatin modifications.…”

TGF-β and IL-6 signals modulate chromatin binding and promoter occupancy by acetylated FOXP3

“…Linker histone H1 can serve as a marker of excised chromatin (29,32). Linker histone H1 proteins released after MNase digestion were readily identified in the chromatin released from FOXP3-transfected Jurkat T cells (Fig.…”