Subunits of ADA-two-A-containing (ATAC) or Spt-Ada-Gcn₅-acetyltrasferase (SAGA) coactivator complexes enhance the acetyltransferase activity of GCN₅

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a. To better understand how the activity of human (h) hGCN5 is regulated in the two related, but different, HAT complexes we carried out in vitro HAT assays. We compared the activity of hGCN5 alone with its activity when it was part of purified recombinant hATAC or hSAGA HAT modules or endogenous hATAC or hSAGA complexes using histone tail peptides and full-length histones as substrates. We demonstrated that the subunit environment of the HAT complexes into which GCN5 incorporates determines the enhancement of GCN5 activity. On histone peptides we show that all the tested GCN5-containing complexes acetylate mainly histone H3K14. Our results suggest a stronger influence of ADA2b as compared with ADA2a on the activity of GCN5. However, the lysine acetylation specificity of GCN5 on histone tails or full-length histones was not changed when incorporated in the HAT modules of ATAC or SAGA complexes. Our results thus demonstrate that the catalytic activity of GCN5 is stimulated by subunits of the ADA2a- or ADA2b-containing HAT modules and is further increased by incorporation of the distinct HAT modules in the ATAC or SAGA holo-complexes

Phosphoproteome and Transcriptome of RA-Responsive and RA-Resistant Breast Cancer Cell Lines.

Retinoic acid (RA), the main active vitamin A metabolite, controls multiple biological processes such as cell proliferation and differentiation through genomic programs and kinase cascades activation. Due to these properties, RA has proven anti-cancer capacity. Several breast cancer cells respond to the antiproliferative effects of RA, while others are RA-resistant. However, the overall signaling and transcriptional pathways that are altered in such cells have not been elucidated. Here, in a large-scale analysis of the phosphoproteins and in a genome-wide analysis of the RA-regulated genes, we compared two human breast cancer cell lines, a RA-responsive one, the MCF7 cell line, and a RA-resistant one, the BT474 cell line, which depicts several alterations of the "kinome". Using high-resolution nano-LC-LTQ-Orbitrap mass spectrometry associated to phosphopeptide enrichment, we found that several proteins involved in signaling and in transcription, are differentially phosphorylated before and after RA addition. The paradigm of these proteins is the RA receptor α (RARα), which was phosphorylated in MCF7 cells but not in BT474 cells after RA addition. The panel of the RA-regulated genes was also different. Overall our results indicate that RA resistance might correlate with the deregulation of the phosphoproteome with consequences on gene expression

Workflow for the phosphoproteomics strategy.

<p>(A) Phosphoproteome. Nuclear and cytoplasmic extracts were prepared and divided in two: one half was digested with trypsin/Lys-C and the other half with chymotrypsin. A small fraction of the trypsin/Lys-C digests was analyzed directly without further purification. The remaining digests were subjected to phosphopeptide enrichment and MS analysis. (B) RARα phosphorylation. Whole cell extracts were prepared from MCF7 and BT474 cells with and without a 30 min RA treatment. RARα was immunoprecipitated and the eluates were thermolysin-digested. Phosphopeptides were enriched and analyzed by nano-LC-LTQ-Orbitrap MS.</p

Broussonetia kaempferi Sieb.

原著和名: ツルカウゾ科名: クワ科 = Moraceae採集地: 長崎県 多良岳麓 (肥前 多良岳麓)採集日: 1970/5/14採集者: 萩庭丈壽整理番号: JH045354国立科学博物館整理番号: TNS-VS-99535

ChIP-qPCR analysis of RARα recruitment at the <i>Cy26a1</i> gene promoter.

<p>(A) Kinetic ChIP experiments performed with RA-treated MCF7 and BT474 cells and determining the recruitment of RARα to the R1 and R2 response elements of the <i>Cyp26a1</i> gene. Values correspond to a representative experiment among 3. (B) ChIP experiments performed with MEFs expressing RARαWT or RARαS77A and determining the recruitment of RARα to the R1 and R2 elements of the <i>Cyp26a1</i> gene. Values are the mean ±SD of three experiments.</p

List of phosphoproteins, grouped per biological functions that were detected in the nuclear extracts of MCF7 and/or BT474 cells, in the two replicate experiments.

<p>For each protein, the phosphopeptides were analyzed manually. The site classes are assigned as acidic (A), basic (B) or proline-directed (P). Phosphoproteins detected in both cell lines (Black), in MCF7 cells only (Green) or in BT474 cells only (Blue). Indicated is whether phosphorylation occurs in the absence of RA only (-), in the presence of RA only (+) or both in the absence and presence of RA (±).</p

Overview of phosphorylation in MCF7 and BT474 cells.

<p>(A) Relative frequency of mono- (1P), bi-(2P) and tri- (3P) phosphorylated peptides in the cytosolic and nuclear extracts of MCF7 and BT474 cells with and without RA treatment. T: total number of phosphopeptides. C: cytosolic extracts, N: nuclear extracts. (B) Relative phosphorylation of Serine (S), Thr (T) and Tyr (Y) residues (Ag: ambiguous). The values are the average ±SD of two experiments.</p

Comparison of the RA-regulated genes in MCF7 and BT474 cells.

<p>(A) Venn diagram showing that 80% of the genes that are RA-regulated in MCF7 cells are not in BT474 cells. (B) and (C) 3D pie charts showing the categories of genes that are RA-regulated in MCF7 only and in BT474 cells only. The genes were selected using the Manteia GO statistical analysis on GO analysis with a P value <0,01. (D) Heatmaps showing the genes that are RA-regulated in both cell lines.</p

Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome

International audienceFragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5' UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS