SAF-A Forms a Complex with BRG1 and Both Components Are Required for RNA Polymerase II Mediated Transcription

Dzeneta Vizlin-Hodzic,Tomas Simonsson,Rikard Runnberg,Stina Simonsson,Jessica Ryme,Mary Bryk

doi:10.1371/journal.pone.0028049

Dzeneta Vizlin-Hodzic, Tomas Simonsson + Show 4 more

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https://doi.org/10.1371/journal.pone.0028049

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Abstract

BackgroundScaffold attachment factor A (SAF-A) participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II.MethodologyHere we use co-localization, co-immunoprecipitation (co-IP) and in situ proximity ligation assay (PLA) to identify Brahma Related Gene 1 (BRG1), the ATP-driven motor of the human SWI-SNF chromatin remodeling complex, as another SAF-A interaction partner in mouse embryonic stem (mES) cells. We also employ RNA interference to investigate functional aspects of the SAF-A/BRG1 interaction.Principal FindingsWe find that endogenous SAF-A protein interacts with endogenous BRG1 protein in mES cells, and that the interaction does not solely depend on the presence of mRNA. Moreover the interaction remains intact when cells are induced to differentiate. Functional analyses reveal that dual depletion of SAF-A and BRG1 abolishes global transcription by RNA polymerase II, while the nucleolar RNA polymerase I transcription machinery remains unaffected.ConclusionsWe demonstrate that SAF-A interacts with BRG1 and that both components are required for RNA Polymerase II Mediated Transcription.

Highlights

Transcription of nuclear genes is mediated by RNA polymerase II (Pol II) and proceeds through three main phases referred to as transcription initiation, transcription elongation and transcription termination [1]
We demonstrate that Scaffold attachment factor A (SAF-A) interacts with Brahma Related Gene 1 (BRG1) and that both components are required for RNA Polymerase II Mediated Transcription
SAF-A interacts with BRG1 in mouse embryonic stem (mES) cells The spatial distributions of endogenous SAF-A and endogenous BRG1 were first examined in mES cells by immunofluorescence followed by confocal microscopy with sequential scanning of two channels, each corresponding to one protein

Summary

Introduction

Transcription of nuclear genes is mediated by RNA polymerase II (Pol II) and proceeds through three main phases referred to as transcription initiation, transcription elongation and transcription termination [1]. SAF-A, known as heterogeneous nuclear ribonucleoprotein U (hnRNP U), is an abundant nuclear protein that binds singleand double-stranded DNA [3,4] and RNA [5], and organizes chromatin into functional gene domains [6]. It is important during early embryonic development and previous reports have indicated that SAF-A is involved in transcriptional regulation of specific genes due to its association with elements within the promoter regions of apo-D, bmal and developmentally regulated genes shh, klf and oct4 [7,8,9,10,11] as well as with transcription factors such as the glucocorticoid hormone receptor, WT1, BRN4, SOX2 and OCT4 [10,12,13,14]. Scaffold attachment factor A (SAF-A) participates in the regulation of gene expression by organizing chromatin into transcriptionally active domains and by interacting directly with RNA polymerase II

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