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SAMPLES (32)

mace:id
Technology # Array version
Species
Organ
Tissue
Physiopathology
Type
Name

Attached file download project data file ('.map')
Attached file (see: ruid website) download project data file ('.map' RUID converted)
Attached file download raw data files ('.zip')
Attached file download annotation files ('.zip')

User name	Arndt Benecke
Email	arndt@ihes.fr
Phone / Fax number	+33160926665 / +33160926609
Location	Institut des Hautes Etudes Scientifiques (Integrative Biology) - 35, route de Chartres - 91440 Bures sur Yvette, France

Scientific description

TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process.

Technical description

RNA interference. Double-stranded siRNAs were produced with a Silencer siRNA kit (Ambion) according to the manufacturer's instructions. Target sequences were obtained with the Dharmacon siDesign engine using the less-stringent parameters. A total of 1.5 x 105 HeLa cells were transfected by using Lipofectamine 2000 (Invitrogen) in 35-mm plates. Sequences of the specific siRNAs are available upon request.

Microarray analysis. A 25,000-gene human oligonucleotide microarray covering most of the known human transcripts has been used for transcriptome analysis. This oligonucleotide collection is a public resource (RÃ©seau National des GÃ©nopoles, France, and MRC, United Kingdom). Information about these oligonucleotides is available at http://medcal.ipmc.cnrs.fr:8080/mediante. Oligonucleotides (synthesized by Proligo) were diluted to 50 ÂµM in 50% dimethyl sulfoxide and 100 mM phosphate buffer and spotted using a ÂµGridII arrayer (BioRobotics) onto amino silane-coated slides (UltraGAPS TM; Corning).

Total RNA from HeLa cells treated with siRNA targeting TAF9, TAF9b, or luciferase (Luc) was extracted (Trizol, Invitrogen). Total RNA (220 ng) was amplified by linear PCR, and the amplification products were labeled by Cy3 and Cy5 and purified using Nucleospin Extract II columns (Macherey Nagel). Labeled cDNAs were hybridized in a Discovery station using ChipHybe 80 hybridization buffer at 42Â°C for 8 h without any final stringency washes (Ventana Medical System hybridization automate, reagents, and microarray hybridization procedure). All the protocols used are available at http://www-microarrays.u-strasbg.fr/Chips/index.html. After slides scanning (ScanArray4000; Perkin-Elmer), the obtained TIF images were quantified using Imagene 5 (BioDiscovery), and the raw data were normalized using the Median. For each different RNA sample, a flip-flop experiment was performed using the RNA obtained from luciferase RNA interference-treated HeLa cells as a reference. The normalized ratio of RNA sample to reference RNA (log2) was calculated, and this ratio was directly used for the comparison of different samples.

Reference

TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9.

Frontini M, Soutoglou E, Argentini M, Bole-Feysot C, Jost B, Scheer E, Tora L.

Mol Cell Biol. 2005 Jun;25(11):4638-49

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/15899866