Detection and Identification of Transcription Factors as Interaction Partners of Alien in vivo

Kob, Robert; Baniahmad, Aria; Escher, Niko; Eggeling, Ferdinand von; Melle, Christian

doi:10.4161/cc.6.8.4108

Cited by 5 publications

(11 citation statements)

References 15 publications

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“…For this reason, we first performed a protein-protein complex detection assay to identify interacting proteins of endogenously expressed S100A11 in crude extracts of HaCaT cells, an immortalized human keratinocyte line (Boukamp et al, 1988). This assay was used previously for the identification of protein complexes involved in cell cycle regulation as well as of the transcription machinery Kob et al, 2007). S100A11-containing protein complexes were captured by a specific antibody against S100A11 coupled to IDM beads followed by elution of the captured proteins and analysis of the complex composition using SELDI-MS ( Figure 1A).…”

Section: Identification Of Rad54b As An Interactingmentioning

confidence: 99%

Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

Murzik

Hemmerich

Weidtkamp‐Peters

et al. 2008

MBoC

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S100A11 is involved in a variety of intracellular activities such as growth regulation and differentiation. To gain more insight into the physiological role of endogenously expressed S100A11, we used a proteomic approach to detect and identify interacting proteins in vivo. Hereby, we were able to detect a specific interaction between S100A11 and Rad54B, which could be confirmed under in vivo conditions. Rad54B, a DNA-dependent ATPase, is described to be involved in recombinational repair of DNA damage, including DNA double-strand breaks (DSBs). Treatment with bleomycin, which induces DSBs, revealed an increase in the degree of colocalization between S100A11 and Rad54B. Furthermore, S100A11/ Rad54B foci are spatially associated with sites of DNA DSB repair. Furthermore, while the expression of p21 WAF1/CIP1 was increased in parallel with DNA damage, its protein level was drastically down-regulated in damaged cells after S100A11 knockdown. Down-regulation of S100A11 by RNA interference also abolished Rad54B targeting to DSBs. Additionally, S100A11 down-regulated HaCaT cells showed a restricted proliferation capacity and an increase of the apoptotic cell fraction. These observations suggest that S100A11 targets Rad54B to sites of DNA DSB repair sites and identify a novel function for S100A11 in p21-based regulation of cell cycle. INTRODUCTIONProteins may exist in several complexes in a spatial and temporal manner to accomplish distinct functions. Analyses of the interacting partners will provide a strong insight into the physiological role of a particular factor. Therefore, it is essential to identify ideally all interacting partners of proteins in vivo to precisely be able to define their biological function. The investigation of protein complexes of solely endogenously expressed proteins avoid the tendency to detect false positive protein-protein interactions of examinations performed in vitro. A multitude of proteins are involved in both the detection and the repair of DNA damages. It is conceivable that some protein complexes involved in these processes are not yet discovered. A severe form of DNA damage that threaten the integrity of the genome are DNA double-strand breaks (DSBs). DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements that can contribute to cell dysfunction, cell death, or carcinogenesis (Hoeijmakers, 2001). Homologous recombination is a major DNA repair pathway by which DSBs are repaired (Lettier et al., 2006).For the identification of specific interacting proteins of S100A11 (S100C, calgizzarin) we used in the present study a proteomic approach comprising mass spectrometry and immunological techniques. S100A11 belongs to the group of S100 proteins that are considered as multitasking proteins involved in several biological processes such as the Ca 2ϩ signaling network, cell growth and motility, cell cycle progression, transcription, and cell differentiation (Schafer and Heizmann, 1996;Donato, 2001;Eckert et al., 2004). It has been proposed that the S100 proteins are involved in...

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Section: Identification Of Rad54b As An Interactingmentioning

confidence: 99%

Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

Murzik

Hemmerich

Weidtkamp‐Peters

et al. 2008

MBoC

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“…Further insights of Alien-mediated corepressor function were gained by using an in vivo proteomic approach to identify novel Alien interacting partners (28)(29)(30). Interestingly, conserved cell cycle factors were among proteins that interact with Alien.…”

Section: Alien Interacts With Evolutionarily Conserved Factors Regulamentioning

confidence: 99%

“…Interestingly, other factors such as two transcription factor IIH (TFIIH) subunits, XBP and XPD/p44, were identified by in vivo proteomic approaches (28)(29)(30). TFIIH is an eukaryotic multiprotein complex exhibiting exceptional evolutionary conservation of its structure.…”

Section: Alien Interacts With Evolutionarily Conserved Factors Regulamentioning

confidence: 99%

The corepressor Alien as a novel tumor suppressor?

Baniahmad

2011

Hormone Molecular Biology and Clinical Investigation

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Alien has been characterized as a corepressor for nuclear hormone receptors that harbor a silencing domain such as the thyroid hormone receptor (TR), the vitamin D3 receptor (VDR) and DAX-1. In addition, the androgen receptor (AR), a steroid hormone receptor, interacts with Alien. Alien enhances gene silencing mediated by TR, VDR and DAX-1, whereas Alien inhibits AR-mediated transactivation. The inhibition of AR by Alien seems to be restricted to cases where AR is bound to AR antagonists. In line with this, Alien inhibits AR target gene expression and human prostate cancer cell proliferation in an antagonist-specific manner indicating that Alien has an inhibitory role for cell cycle progression. Alien mediates gene silencing by recruitment of histone deacetylase activity and interestingly through nucleo-some assembly activity. Hereby, Alien enhances nucleosome positioning mediated by nucleosome assembly protein 1, which suggests a novel molecular mechanism of corepressor function. Using a proteomic approach to identify Alien interacting partners, we detected the cell cycle factor E2F1 to bind to Alien in vivo. The E2F1-mediated transactivation and E2F target gene expression is inhibited by Alien, and in line with this Alien is observed to repress cell cycle progression.

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“…Protein-protein interaction techniques are a powerful method to detect protein complexes in a cell. An in vivo proteomic approach was assessed to identify Alien-interacting partners, combining detection of protein interactions of solely endogenously-expressed proteins with surface-enhanced laser desorption/ionization - mass spectrometry (SELDI-MS) and immunological techniques [ Escher et al, 2007 ; Kob et al, 2007 ; Tenbaum et al, 2007 ]. Using this in vivo approach in addition to HDACs, several members of the E2F transcription factor family were identified as interacting partners of Alien ( Figure 1 and [ Escher et al, 2007 ]).…”

Section: Alien Interacts With Transcription Factors Involved In Cell mentioning

confidence: 99%

The coregulator Alien

2007

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Alien has characteristics of a corepressor for selected members of the nuclear hormone receptor (NHR) superfamily and also for transcription factors involved in cell cycle regulation and DNA repair. Alien mediates gene silencing and represses the transactivation of specific NHRs and other transcription factors to modulate hormone response and cell proliferation. Alien is a highly conserved protein and is expressed in a wide variety of tissues. Knockout of the gene encoding Alien in mice is embryonic lethal at a very early stage, indicating an important evolutionary role in multicellular organisms. From a mechanistic perspective, the corepressor function of Alien is in part mediated by histone deacetylase (HDAC) activity. In addition, Alien seems to modulate nucleosome assembly activity. This suggests that Alien is acting on chromatin not only through recruitment of histone-modifying activities, but also through enhancing nucleosome assembly.

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Detection and Identification of Transcription Factors as Interaction Partners of Alien in vivo

Cited by 5 publications

References 15 publications

Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

Rad54B Targeting to DNA Double-Strand Break Repair Sites Requires Complex Formation with S100A11

The corepressor Alien as a novel tumor suppressor?

The coregulator Alien

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