RNA polymerase II transcription with partially assembled TFIID complexes.

The recognition of core promoter sequences by the general transcription factor TFIID is the first step in the process of RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is composed of the TATA binding protein (TBP) and of 13 TBP associated factors (TAFs). Inducible Taf7 knock out (KO) results in the formation of a Taf7-less TFIID complex, while Taf10 KO leads to serious defects within the TFIID assembly pathway. Either TAF7 or TAF10 depletions correlate with the detected TAF occupancy changes at promoters, and with the distinct phenotype severities observed in mouse embryonic stem cells or mouse embryos. Surprisingly however, under either Taf7 or Taf10 deletion conditions, TBP is still associated to the chromatin, and no major changes are observed in nascent Pol II transcription. Thus, partially assembled TFIID complexes can sustain Pol II transcription initiation, but cannot replace holo-TFIID over several cell divisions and/or development.

The related coactivator complexes SAGA and ATAC control embryonic stem cell self-renewal through acetyltransferase-independent mechanisms.

SAGA (Spt-Ada-Gcn5 acetyltransferase) and ATAC (Ada-two-A-containing) are two related coactivator complexes, sharing the same histone acetyltransferase (HAT) subunit. The HAT activities of SAGA and ATAC are required for metazoan development, but the role of these complexes in RNA polymerase II transcription is less understood. To determine whether SAGA and ATAC have redundant or specific functions, we compare the effects of HAT inactivation in each complex with that of inactivation of either SAGA or ATAC core subunits in mouse embryonic stem cells (ESCs). We show that core subunits of SAGA or ATAC are required for complex assembly and mouse ESC growth and self-renewal. Surprisingly, depletion of HAT module subunits causes a global decrease in histone H3K9 acetylation, but does not result in significant phenotypic or transcriptional defects. Thus, our results indicate that SAGA and ATAC are differentially required for self-renewal of mouse ESCs by regulating transcription through different pathways in a HAT-independent manner.

Histone H2Bub1 deubiquitylation is essential for mouse development, but does not regulate global RNA polymerase II transcription.

Co-activator complexes dynamically deposit post-translational modifications (PTMs) on histones, or remove them, to regulate chromatin accessibility and/or to create/erase docking surfaces for proteins that recognize histone PTMs. SAGA (Spt-Ada-Gcn5 Acetyltransferase) is an evolutionary conserved multisubunit co-activator complex with modular organization. The deubiquitylation module (DUB) of mammalian SAGA complex is composed of the ubiquitin-specific protease 22 (USP22) and three adaptor proteins, ATXN7, ATXN7L3 and ENY2, which are all needed for the full activity of the USP22 enzyme to remove monoubiquitin (ub1) from histone H2B. Two additional USP22-related ubiquitin hydrolases (called USP27X or USP51) have been described to form alternative DUBs with ATXN7L3 and ENY2, which can also deubiquitylate H2Bub1. Here we report that USP22 and ATXN7L3 are essential for normal embryonic development of mice, however their requirements are not identical during this process, as Atxn7l3-/- embryos show developmental delay already at embryonic day (E) 7.5, while Usp22-/- embryos are normal at this stage, but die at E14.5. Global histone H2Bub1 levels were only slightly affected in Usp22 null embryos, in contrast H2Bub1 levels were strongly increased in Atxn7l3 null embryos and derived cell lines. Our transcriptomic analyses carried out from wild type and Atxn7l3-/- mouse embryonic stem cells (mESCs), or primary mouse embryonic fibroblasts (MEFs) suggest that the ATXN7L3-related DUB activity regulates only a subset of genes in both cell types. However, the gene sets and the extent of their deregulation were different in mESCs and MEFs. Interestingly, the strong increase of H2Bub1 levels observed in the Atxn7l3-/- mESCs, or Atxn7l3-/- MEFs, does not correlate with the modest changes in RNA Polymerase II (Pol II) occupancy and lack of changes in Pol II elongation observed in the two Atxn7l3-/- cellular systems. These observations together indicate that deubiquitylation of histone H2Bub1 does not directly regulate global Pol II transcription elongation.

Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair.

DNA repair is critical to maintaining genome integrity, and its dysfunction can cause accumulation of unresolved damage that leads to genomic instability. The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex and the nuclear pore-associated transcription and export complex 2 (TREX-2) couple transcription with mRNA export. In this study, we identify a novel interplay between human TREX-2 and the deubiquitination module (DUBm) of SAGA required for genome stability. We find that the scaffold subunit of TREX-2, GANP, positively regulates DNA repair through homologous recombination (HR). In contrast, DUBm adaptor subunits ENY2 and ATXNL3 are required to limit unscheduled HR. These opposite roles are achieved through monoubiquitinated histone H2B (H2Bub1). Interestingly, the activity of the DUBm of SAGA on H2Bub1 is dependent on the integrity of the TREX-2 complex. Thus, we describe the existence of a functional interaction between human TREX-2 and SAGA DUBm that is key to maintaining the H2B/HB2ub1 balance needed for efficient repair and HR.

Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription.

The human general transcription factor TFIID is composed of the TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). In eukaryotic cells, TFIID is thought to nucleate RNA polymerase II (Pol II) preinitiation complex formation on all protein coding gene promoters and thus, be crucial for Pol II transcription. In a child with intellectual disability, mild microcephaly, corpus callosum agenesis and poor growth, we identified a homozygous splice-site mutation in TAF8 (NM_138572.2: c.781-1G > A). Our data indicate that the patient's mutation generates a frame shift and an unstable TAF8 mutant protein with an unrelated C-terminus. The mutant TAF8 protein could not be detected in extracts from the patient's fibroblasts, indicating a loss of TAF8 function and that the mutation is most likely causative. Moreover, our immunoprecipitation and proteomic analyses show that in patient cells only partial TAF complexes exist and that the formation of the canonical TFIID is impaired. In contrast, loss of TAF8 in mouse embryonic stem cells and blastocysts leads to cell death and to a global decrease in Pol II transcription. Astonishingly however, in human TAF8 patient cells, we could not detect any cellular phenotype, significant changes in genome-wide Pol II occupancy and pre-mRNA transcription. Thus, the disorganization of the essential holo-TFIID complex did not affect global Pol II transcription in the patient's fibroblasts. Our observations further suggest that partial TAF complexes, and/or an altered TFIID containing a mutated TAF8, could support human development and thus, the absence of holo-TFIID is less deleterious for transcription than originally predicted.

Tip60 complex binds to active Pol II promoters and a subset of enhancers and co-regulates the c-Myc network in mouse embryonic stem cells.

BACKGROUND: Tip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood. RESULTS: Gel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of "free" Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50-60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions. CONCLUSIONS: Our study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs.

The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) coactivator complex contains distinct chromatin-modifying activities and is recruited by DNA-bound activators to regulate the expression of a subset of genes. Surprisingly, recent studies revealed little overlap between genome-wide SAGA-binding profiles and changes in gene expression upon depletion of subunits of the complex. As indicators of SAGA recruitment on chromatin, we monitored in yeast and human cells the genome-wide distribution of histone H3K9 acetylation and H2B ubiquitination, which are respectively deposited or removed by SAGA. Changes in these modifications after inactivation of the corresponding enzyme revealed that SAGA acetylates the promoters and deubiquitinates the transcribed region of all expressed genes. In agreement with this broad distribution, we show that SAGA plays a critical role for RNA polymerase II recruitment at all expressed genes. In addition, through quantification of newly synthesized RNA, we demonstrated that SAGA inactivation induced a strong decrease of mRNA synthesis at all tested genes. Analysis of the SAGA deubiquitination activity further revealed that SAGA acts on the whole transcribed genome in a very fast manner, indicating a highly dynamic association of the complex with chromatin. Thus, our study uncovers a new function for SAGA as a bone fide cofactor for all RNA polymerase II transcription.

Mof-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation.

The histone acetyltransferase (HAT) Mof is essential for mouse embryonic stem cell (mESC) pluripotency and early development. Mof is the enzymatic subunit of two different HAT complexes, MSL and NSL. The individual contribution of MSL and NSL to transcription regulation in mESCs is not well understood. Our genome-wide analysis show that i) MSL and NSL bind to specific and common sets of expressed genes, ii) NSL binds exclusively at promoters, iii) while MSL binds in gene bodies. Nsl1 regulates proliferation and cellular homeostasis of mESCs. MSL is the main HAT acetylating H4K16 in mESCs, is enriched at many mESC-specific and bivalent genes. MSL is important to keep a subset of bivalent genes silent in mESCs, while developmental genes require MSL for expression during differentiation. Thus, NSL and MSL HAT complexes differentially regulate specific sets of expressed genes in mESCs and during differentiation.

DNA binding by Sgf11 protein affects histone H2B deubiquitination by Spt-Ada-Gcn5-acetyltransferase (SAGA).

The yeast Spt-Ada-Gcn5-acetyltransferase (SAGA) complex is a transcription coactivator that contains a histone H2B deubiquitination activity mediated by its Ubp8 subunit. Full enzymatic activity requires the formation of a quaternary complex, the deubiquitination module (DUBm) of SAGA, which is composed of Ubp8, Sus1, Sgf11, and Sgf73. The crystal structures of the DUBm have shed light on the structure/function relationship of this complex. Specifically, both Sgf11 and Sgf73 contain zinc finger domains (ZnF) that appear essential for the DUBm activity. Whereas Sgf73 N-terminal ZnF is important for DUBm stability, Sgf11 C-terminal ZnF appears to be involved in DUBm function. To further characterize the role of these two zinc fingers, we have solved their structure by NMR. We show that, contrary to the previously reported structures, Sgf73 ZnF adopts a C2H2 coordination with unusual tautomeric forms for the coordinating histidines. We further report that the Sgf11 ZnF, but not the Sgf73 ZnF, binds to nucleosomal DNA with a binding interface composed of arginine residues located within the ZnF α-helix. Mutational analyses both in vitro and in vivo provide evidence for the functional relevance of our structural observations. The combined interpretation of our results leads to an uncommon ZnF-DNA interaction between the SAGA DUBm and nucleosomes, thus providing further functional insights into SAGA's epigenetic modulation of the chromatin structure.

The human TREX-2 complex is stably associated with the nuclear pore basket.

In eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC. Here, we characterize the dynamics of human TREX-2 and show that it stably associates with the NPC basket. Moreover, the association of TREX-2 with the NPC requires the basket nucleoporins NUP153 and TPR, but is independent of transcription. Differential profiles of mRNA nuclear accumulation reveal that TREX-2 functions similarly to basket nucleoporins, but differently from NXF1. Thus, our results show that TREX-2 is an NPC-associated complex in mammalian cells and suggest that it is involved in putative NPC basket-related functions.

The tightly controlled deubiquitination activity of the human SAGA complex differentially modifies distinct gene regulatory elements.

The multisubunit SAGA coactivator complex facilitates access of general transcription factors to DNA through histone acetylation mediated by GCN5. USP22 (ubiquitin-specific protease 22) was recently described as a subunit of the human SAGA complex that removes ubiquitin from monoubiquitinated histone H2B and H2A in vitro. Here we demonstrate an allosteric regulation of USP22 through multiple interactions with different domains of other subunits of the SAGA deubiquitination module (ATXN7, ATXN7L3, and ENY2). Downregulation of ATXN7L3 by short hairpin RNA (shRNA) specifically inactivated the SAGA deubiquitination activity, leading to a strong increase of global H2B ubiquitination and a moderate increase of H2A ubiquitination. Thus, SAGA is the major H2Bub deubiquitinase in human cells, and this activity cannot be fully compensated by other deubiquitinases. Here we show that the deubiquitination activity of SAGA is required for full activation of SAGA-dependent inducible genes. Interestingly, the reduction of the SAGA deubiquitination activity and the parallel increase in H2B ubiquitation at inducible target genes before activation do not induce aberrant gene expression. Our data together indicate that different dynamic equilibriums of H2B ubiquitination/deubiquitination are established at different gene regulatory elements and that H2B ubiquitination changes are necessary but not sufficient to trigger parallel activation of gene expression.

Automated recombinant protein expression screening in Escherichia coli.

To fit the requirements of structural genomics programs, new as well as classical methods have been adapted to automation. This chapter describes the automated procedure developed within the Structural Biology and Genomics Platform, Strasbourg for performing recombinant protein expression screening in Escherichia coli. The procedure consists of parallel competent cells transformation, cell plating, and liquid culture inoculation, implemented for up to 96 samples at a time.

A comparison of inoculation methods to simplify recombinant protein expression screening in Escherichia coli.

In the past five years, Structural Genomics (SG) initiatives have established an automated pipeline for protein production in Escherichia coli to rapidly screen various conditions, resulting in soluble expression of recombinant proteins to aid in carrying out structural studies. However, some steps of the procedure are still extensive and require manual handling. Here, we present a comparative study of one step of the process, E. coli cultivation, using a set of 12 expression vectors encoding for fusion proteins of seven independent target proteins. First, we show that performing E. coli growth in auto-inducible medium (ZYM-5052) results in a comparable protein expression/solubility profile to that obtained when growing cells in classical Luria-Bertani (LB) medium. Second, we show that the transformation mix can be used directly to inoculate a culture, saving time and circumventing the error-prone step of colony picking, without impairing cell growth and the protein expression/solubility profile. Thus, we show that a basic, but nevertheless essential, step of a protein production pipeline, E. coli cultivation, can be simplified to a single event that is fully compatible with complete automation.