Zita Nagy

Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation.

Transcription in eukaryotes is a tightly regulated, multistep process. Gene-specific transcriptional activators, several different co-activators and general transcription factors are necessary to access specific loci to allow precise initiation of RNA polymerase II transcription. As the dense chromatin folding of the genome does not allow the access of these sites by the huge multiprotein transcription machinery, remodelling is required to loosen up the chromatin structure for successful transcription initiation. In the present review, we summarize the recent evolution of our understanding of the function of two histone acetyl transferases (ATs) from metazoan organisms: GCN5 and PCAF. Their overall structure and the multiprotein complexes in which they are carrying out their activities are discussed. Metazoan GCN5 and PCAF are subunits of at least two types of multiprotein complexes, one having a molecular weight of 2 MDa (SPT3-TAF9-GCN5 acetyl transferase/TATA binding protein (TBP)–free–TAF complex/PCAF complexes) and a second type with about a size of 700 kDa (ATAC complex). These complexes possess global histone acetylation activity and locus-specific co-activator functions together with AT activity on non-histone substrates. Thus, their biological functions cover a wide range of tasks and render them indispensable for the normal function of cells. That deregulation of the global and/or specific AT activities of these complexes leads to the cancerous transformation of the cells highlights their importance in cellular processes. The possible effects of GCN5 and PCAF in tumorigenesis are also discussed.

The metazoan ATAC and SAGA coactivator HAT complexes regulate different sets of inducible target genes

Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus-specific transcription. The GCN5 HAT was identified as a subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) multiprotein complex. Vertebrate cells express a second HAT, PCAF, that is 73% identical to GCN5. Here, we report the characterization of the mammalian ATAC (Ada-Two-A-Containing) complexes containing either GCN5 or PCAF in a mutually exclusive manner. In vitro ATAC complexes acetylate lysine 14 of histone H3. Moreover, ATAC- or SAGA-specific knock-down experiments suggest that both ATAC and SAGA are involved in the acetylation of histone H3K9 and K14 residues. Despite their catalytic similarities, SAGA and ATAC execute their coactivator functions on distinct sets of inducible target genes. Interestingly, ATAC strongly influences the global phosphorylation level of histone H3S10, suggesting that in mammalian cells a cross-talk exists linking ATAC function to H3S10 phosphorylation.

The Transcriptional Histone Acetyltransferase Cofactor TRRAP Associates with the MRN Repair Complex and Plays a Role in DNA Double-Strand Break Repair

ABSTRACT Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates

All DNA‐related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5‐containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non‐histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and α‐tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5‐mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.

The human SPT20-containing SAGA complex plays a direct role in the regulation of endoplasmic reticulum stress-induced genes

ABSTRACT One of the central questions in eukaryotic transcription is how activators can transmit their signal to stimulate gene expression in the context of chromatin. The multisubunit SAGA coactivator complex has both histone acetyltransferase and deubiquitination activities and remodels chromatin to allow transcription. Whether and how SAGA is able to regulate transcription at specific loci is poorly understood. Using mass spectrometry, immunoprecipitation, and Western blot analysis, we have identified human SPT20 (hSPT20) as the human homologue of the yeast Spt20 and show that hSPT20 is a bona fide subunit of the human SAGA (hSAGA; previously called TFTC/STAGA/PCAF) complex and that hSPT20 is required for the integrity of the hSAGA complex. We demonstrate that hSPT20 and other hSAGA subunits, together with RNA polymerase II, are specifically recruited to genes induced by endoplasmic reticulum (ER) stress. In good agreement with the recruitment of hSAGA to the ER stress-regulated genes, knockdown of hSTP20 hampers ER stress response. Surprisingly, hSPT20 recruitment was not observed for genes induced by another type of stress. These results provide evidence for a direct and specific role of the hSPT20-containing SAGA complex in transcriptional induction of ER stress-responsive genes. Thus, hSAGA regulates the transcription of stress-responsive genes in a stress type-dependent manner.

DNA repair: easy to visualize, difficult to elucidate

Faithful repair of DNA damage is essential for the maintenance of genome integrity. Recent advances in the local induction of DNA damage and in cell biological imaging techniques have extended our understanding of DNA repair gained from biochemical and genetic approaches; these advances now reveal that the assembly of DNA repair complexes at sites of DNA damage is spatially and temporally regulated. Visualization of the dynamics of double strand breaks in living cells has also provided valuable insights into how chromosomal translocations form. Here we outline the most commonly used tools to induce and visualize the DNA repair process and we discuss the advantages and disadvantages inherent to each method.

Identification of a Small TAF Complex and Its Role in the Assembly of TAF-Containing Complexes

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation.

TATA binding protein associated factor 3 (TAF3) interacts with p53 and inhibits its function

BackgroundThe tumour suppressor protein p53 is a sequence specific DNA-binding transcription regulator, which exerts its versatile roles in genome protection and apoptosis by affecting the expression of a large number of genes. In an attempt to obtain a better understanding of the mechanisms by which p53 transcription function is regulated, we studied p53 interactions.ResultsWe identified BIP2 (Bric-à-brac interacting protein 2), the fly homolog of TAF3, a histone fold and a plant homeodomain containing subunit of TFIID, as an interacting partner of Drosophila melanogaster p53 (Dmp53). We detected physical interaction between the C terminus of Dmp53 and the central region of TAF3 both in yeast two hybrid assays and in vitro. Interestingly, DmTAF3 can also interact with human p53, and mammalian TAF3 can bind to both Dmp53 and human p53. This evolutionarily conserved interaction is functionally significant, since elevated TAF3 expression severely and selectively inhibits transcription activation by p53 in human cell lines, and it decreases the level of the p53 protein as well.ConclusionWe identified TAF3 as an evolutionarily conserved negative regulator of p53 transcription activation function.

Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin

The nucleotide excision repair preincision complex assembles on chromatin even in the absence of DNA damage through the sequential and ordered addition of its individual subunits.

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.