Loss of WSTF results in spontaneous fluctuations of heterochromatin formation and resolution, combined with substantial changes to gene expression

Ashley E Culver-Cochran,Brian P Chadwick

doi:10.1186/1471-2164-14-740

Ashley E Culver-Cochran, Brian P Chadwick

Open Access

https://doi.org/10.1186/1471-2164-14-740

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Journal: BMC Genomics	Publication Date: Jan 1, 2013
Citations: 92	License type: cc-by

Affiliation: Florida State University

Abstract

BackgroundWilliams syndrome transcription factor (WSTF) is a multifaceted protein that is involved in several nuclear processes, including replication, transcription, and the DNA damage response. WSTF participates in a chromatin-remodeling complex with the ISWI ATPase, SNF2H, and is thought to contribute to the maintenance of heterochromatin, including at the human inactive X chromosome (Xi). WSTF is encoded by BAZ1B, and is one of twenty-eight genes that are hemizygously deleted in the genetic disorder Williams-Beuren syndrome (WBS).ResultsTo explore the function of WSTF, we performed zinc finger nuclease-assisted targeting of the BAZ1B gene and isolated several independent knockout clones in human cells. Our results show that, while heterochromatin at the Xi is unaltered, new inappropriate areas of heterochromatin spontaneously form and resolve throughout the nucleus, appearing as large DAPI-dense staining blocks, defined by histone H3 lysine-9 trimethylation and association of the proteins heterochromatin protein 1 and structural maintenance of chromosomes flexible hinge domain containing 1. In three independent mutants, the expression of a large number of genes were impacted, both up and down, by WSTF loss.ConclusionsGiven the inappropriate appearance of regions of heterochromatin in BAZ1B knockout cells, it is evident that WSTF performs a critical role in maintaining chromatin and transcriptional states, a property that is likely compromised by WSTF haploinsufficiency in WBS patients.

Highlights

Introduction of exogenous full length Williams syndrome transcription factor (WSTF)open reading frame (ORF) is sufficient to remove the appearance of DAPI-dense blocks We wondered if the DAPI-dense blocks in Bromodomain adjacent to zinc finger domain 1B (BAZ1B) knockout cells could be removed by reintroducing wild type WSTF protein
The human Inactive X chromosome (Xi) is primarily composed of two spatially distinct types of heterochromatin [3]; one is characterized by histone H3 trimethylated at lysine 9 (H3K9me3) [4,5] and association of heterochromatin protein 1 (HP1) [6], whereas the other is defined by histone H3
The repair template consisted of a central promoterless neomycin open reading frame (ORF) that was preceded by a splice acceptor and internal ribosome entry site, and followed by a polyadenylation signal [39]

Summary

Introduction

Introduction of exogenous full length WSTFORF is sufficient to remove the appearance of DAPI-dense blocks We wondered if the DAPI-dense blocks in BAZ1B knockout cells could be removed by reintroducing wild type WSTF protein. We have previously shown that the WSTF-ISWI chromatin remodeling complex (WICH) transiently associates with the human Xi as the chromosome is undergoing DNA replication [15] and, is a candidate for maintaining this chromatin organization. Current models suggest that Williams syndrome transcription factor (WSTF), one of two subunits in the WICH chromatin remodeling complex [16], assists in reforming chromatin states of the parental cell post DNA replication [17]. Consistent with this model, depletion of WSTF protein levels by RNA-interference (RNAi) resulted in aberrant heterochromatin formation [18]

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