Nuclear proteins: finding and binding target sites in chromatin

Royen, Martin E. van; Zotter, Angelika; Ibrahim, Shehu; Geverts, Bart; Houtsmuller, Adriaan B.

doi:10.1007/s10577-010-9172-5

Cited by 45 publications

(39 citation statements)

References 106 publications

(122 reference statements)

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“…Our FRAP data show that ELL-GFP mobility is much higher than the mobility of XPB-GFP and similar to that of Cdk7-GFP (Fig.1E). Although the nucleoplasm is not a homogenous medium, a clear relationship exists between molecular weight and diffusion speed (14,15). The fact that Cdk7 (∼40 kDa alone, ∼110 kDa within the CAK) diffuses much faster than XPB indicates that these proteins diffuse in the nucleus separately, but at the same time does not exclude that a fraction of these proteins are part of the same, much larger, complex, that is, TFIIH (>500 kDa) (15).…”

Section: Resultsmentioning

confidence: 99%

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair

Mourgues

Gautier

Lagarou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major actor of both nucleotide excision repair subpathways of which transcription-coupled repair highlights the interplay between DNA repair and transcription. Using an unbiased proteomic approach, we have identified the protein eleven-nineteen lysine-rich leukemia (ELL) as a TFIIH partner. Here we show that ELL is recruited to UV-damaged chromatin in a Cdk7-dependent manner (a component of the cyclin-dependent activating kinase subcomplex of TFIIH). We demonstrate that depletion of ELL strongly hinders RNA polymerase II (RNA Pol II) transcription resumption after lesion removal and DNA gap filling. Lack of ELL was also observed to increase RNA Pol II retention to the chromatin during this process. Identifying ELL as an essential player for RNA Pol II restart during cellular DNA damage response opens the way to obtaining a mechanistic description of transcription resumption after DNA repair.elongation factor | CAK | LEC | Cockayne syndrome | TCR D amage to DNA induced by UV irradiation is repaired by the nucleotide excision repair (NER) system. Three DNA repair-deficient disorders emphasize the importance of NER in genome stability (1). In eukaryotic cells, NER can be divided into two pathways: global genome repair (GGR), repairing lesions throughout the genome, and transcription-coupled repair (TCR), which repairs lesions on the transcribed strand of active genes (2). After damage detection, the basal transcription/repair factor II H (TFIIH), containing the XPB and XPD ATPase/helicases is needed to locally unwind the DNA double helix around the lesion. Furthermore, in vitro UV irradiation elicits a change in the composition of TFIIH. Indeed the majority of TFIIH present on UV-damaged chromatin does not contain the ternary cyclindependent activating kinase (CAK) complex (3). More specifically, the CAK complex is found to be only implicated in TCR but not during GGR, where it seems to be released from the remaining TFIIH components, concomitantly with the recruitment of subsequent NER factors (3). To better our understanding of TFIIH functions in vivo, we have used a combination of improved immunoprecipitation assays and proteomic analysis to identify and characterize TFIIH-interacting partners.Our quantitative proteomic approach has identified several putative TFIIH partners, of which the four most enriched constitute the Little Elongation Complex (LEC): eleven-nineteen lysinerich leukemia (ELL), EAF1 (ELL-associated factor 1), KIAA0947, and NARG2 (4). The proposed role of the LEC is to regulate the transcription of small nuclear RNA genes (5). Although ELL and EAF1 are also part of the Super Elongation Complex (SEC), known to regulate the transcriptional elongation c...

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Section: Resultsmentioning

confidence: 99%

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair

Mourgues

Gautier

Lagarou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…1 This could be a characteristic feature of the search mechanism employed by a number of chromatin interacting proteins. [32][33][34] However, while both PCNA and ISWI remodelers fit into this scheme some specific features of chromatin remodelers can be identified from the comparative mobility study conducted here. In contrast to PCNA, ISWIs thoroughly probe a large number of nucleosomes by a continuous sampling mechanism, thus ensuring that potential targets are reliably identified.…”

Section: Methodsmentioning

confidence: 90%

Binding kinetics of human ISWI chromatin-remodelers to DNA repair sites elucidate their target location mechanism

Erdel

Rippe

2011

Nucleus

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Chromatin remodelers translocate nucleosomes along the DNA chain in an ATP-dependent manner. This catalytic activity is particularly important for DNA replication and repair since both processes require a significant amount of nucleosome translocations and assembly during DNA synthesis. Recently, we have studied the mobility and interactions of the human ISWI family chromatin remodelers Snf2H and Snf2L as well as Acf1, one of the non-catalytic subunits present in the ACF and CHRAC complexes of Snf2H. We proposed that these protein complexes identify their nucleosomal substrates via a continuous sampling mechanism. It rationalizes the relatively high nuclear mobility and abundance observed for all ISWI proteins in terms of fast target location. According to our model a certain type of ISWI complex visits a given nucleosome in the human genome on the timescale of several seconds to a few minutes. Here, we show that the ISWI proteins Snf2H, Snf2L as well as Acf1 accumulate at UV-induced DNA damage sites within tens of seconds and reach a plateau after a few minutes. These findings corroborate the predictions of the continuous sampling mechanism as an efficient way for targeting chromatin remodelers to sites in the genome that require their activity. In comparison to the mobility of PCNA (proliferating cell nuclear antigen) that also accumulates at DNA repair sites the specifics of substrate location by chromatin remodelers are further characterized.

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“…Numerous studies have investigated gene‐specific functions of the complexes and genome‐wide approaches have been applied to define their binding sites. However, it has been shown that transcription‐associated proteins exhibit a broad range of mobility in the nuclei of living cells, with the stability of transcription factor‐chromatin interactions linked to function (van Royen et al , 2011), and this information was missing for SAGA and ATAC complexes. Our FRAP and FLIP analyses classified the two coactivators as highly mobile factors, with no detectable immobile fractions, suggesting transient chromatin associations for these complexes.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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.

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Nuclear proteins: finding and binding target sites in chromatin

Cited by 45 publications

References 106 publications

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair

ELL, a novel TFIIH partner, is involved in transcription restart after DNA repair

Binding kinetics of human ISWI chromatin-remodelers to DNA repair sites elucidate their target location mechanism

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

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