Coactivators and general transcription factors have two distinct dynamic populations dependent on transcription

Abstract

SAGA and ATAC are two distinct chromatin modifying co‐activator complexes with distinct enzymatic activities involved in RNA polymerase II (Pol II) transcription regulation. To investigate the mobility of co‐activator complexes and general transcription factors in live‐cell nuclei, we performed imaging experiments based on photobleaching. SAGA and ATAC, but also two general transcription factors (TFIID and TFIIB), were highly dynamic, exhibiting mainly transient associations with chromatin, contrary to Pol II, which formed more stable chromatin interactions. Fluorescence correlation spectroscopy analyses revealed that the mobile pool of the two co‐activators, as well as that of TFIID and TFIIB, can be subdivided into “fast” (free) and “slow” (chromatin‐interacting) populations. Inhibiting transcription elongation decreased H3K4 trimethylation and reduced the “slow” population of SAGA, ATAC, TFIIB and TFIID. In addition, inhibiting histone H3K4 trimethylation also reduced the “slow” populations of SAGA and ATAC. Thus, our results demonstrate that in the nuclei of live cells the equilibrium between fast and slow population of SAGA or ATAC complexes is regulated by active transcription via changes in the abundance of H3K4me3 on chromatin.

SAGA Is a General Cofactor for RNA Polymerase II Transcription

“…This discrepancy could be explained by an inherent difficulty to ChIP the SAGA complex (HTM. Timmers, unpublished observations), which can be due to its very dynamic interaction with chromatin ( Vosnakis et al, 2017 ).…”

Chromosome-wide co-fluctuation of stochastic gene expression in mammalian cells

“…The above three groups of genes (evolved linked protein complex genes, linked protein complex genes, and unlinked protein complex genes) were constructed using stratified sampling so that their mean expression levels across tissues are not significantly different (see Methods). For comparison, we performed the same analysis but replaced H1 histones with TFIIB, a general transcription factor that is involved in the formation of the RNA polymerase II preinitiation complex and has a high diffusion rate [53]. The trends shown in Fig.…”

Imaging of native transcription factors and histone phosphorylation at high resolution in live cells

“…5, D–F ), indicating that the observed Pol II cluster size shift reflected in vivo Pol II behavior changes after transcription inhibition. Using photobleaching techniques, it has been shown that, when transcription elongation is inhibited, total bound Pol II is released from the chromatin in general and becomes mobile ( Kimura et al, 2002 ; Hieda et al, 2005 ; Vosnakis et al, 2017 ). Thus, our results show that when transcription elongation is inhibited by Flavo the larger Pol II foci dissociate, because Pol II molecules are released from these sites and become mobile.…”

“…It has been well established that the function of transcription factors and coactivator complexes involved in chromatin-dependent processes are tightly linked to their mobility and interactions with diverse posttranslational modifications (PTMs) in the nuclear environment ( Snapp et al, 2003 ; Kimura, 2005 ; Hager et al, 2009 ; Cisse et al, 2013 ; Vosnakis et al, 2017 ). Our current understanding of transcription regulation dynamics is often based on approaches, called fluorescence recovery after photobleaching and florescence loss in photobleaching, in which fluorescently tagged factors in the nucleus, or a whole cellular compartment, are bleached and the fluorescence redistribution is followed over time in live cells ( Kimura et al, 1999 , 2002 ; Dundr et al, 2002 ; Kimura, 2005 ; Gorski et al, 2008 ; van Royen et al, 2011 ).…”