Petros Papadopoulos

The architecture of human general transcription factor TFIID core complex

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6u2009Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8–TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.

Isolation and Characterization of Hematopoietic Transcription Factor Complexes by in Vivo Biotinylation Tagging and Mass Spectrometry

Abstract: We have described the application of a simple biotinylation tagging approach for the direct purification of tagged transcription factor complexes, based on the use of artificial short peptide tags that are specifically and efficiently biotinylated by the bacterial BirA biotin ligase, which is co‐expressed in cells with the tagged factor. We used this approach to initially characterize complexes formed by the hematopoietic transcription factor GATA‐1 in erythroid cells. GATA‐1 is essential for the erythroid differentiation, its functions encompassing upregulation of erythroid genes, repression of alternative transcription programs, and suppression of cell proliferation. However, it was not clear how all of these GATA‐1 functions are mediated. Our work describes, for the first time, distinct GATA‐1 interactions with the essential hematopoietic factor Gfi‐1b, the repressive MeCP1 complex, and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA‐1/FOG‐1 and GATA‐1/TAL‐1 complexes. We also provide evidence that distinct GATA‐1 complexes are associated with specific GATA‐1 functions in erythroid differentiation, for example, GATA‐1/Gfi‐1b with the suppression of cell proliferation and GATA‐1/FOG‐1/MeCP1 with the repression of other hematopoietic transcription programs. We next applied the biotinylation tag to Ldb‐1, a known partner of GATA‐1, and characterized a number of novel interaction partners that are essential in erythroid development, in particular, Eto‐2, Lmo4, and CdK9. Last, we are in the process of applying the same technology to characterize the factors that are bound to the suppressed γ‐globin promoter in vivo.

Analysis of phenotype and outcome in essential thrombocythemia with CALR or JAK2 mutations

The JAK2 V617F mutation, the thrombopoietin receptor MPL W515K/L mutation and calreticulin (CALR) mutations are mutually exclusive in essential thrombocythemia and support a novel molecular categorization of essential thrombocythemia. CALR mutations account for approximately 30% of cases of essential thrombocythemia. In a retrospective study, we examined the frequency of MPL and CALR mutations in JAK2 V617F-negative cases of essential thrombocythemia (n=103). In addition, we compared the clinical phenotype and outcome of CALR mutant cases of essential thrombocythemia with a cohort of JAK2 V617F-positive essential thrombocythemia (n=57). CALR-positive cases represented 63.7% of double-negative cases of essential thrombocythemia, and most carried CALR type 1 or type 2 indels. However, we also identified one patient who was positive for both the JAK2 V617F and the CALR mutations. This study revealed that CALR mutant essential thrombocythemia is associated with younger age, higher platelet counts, lower erythrocyte counts, leukocyte counts, hemoglobin, and hematocrit, and increased risk of progression to myelofibrosis in comparison with JAK2 V617F-positive essential thrombocythemia. Analysis of the CALR mutant group according to indel type showed that CALR type 1 deletion is strongly associated with male gender. CALR mutant patients had a better overall survival than JAK2 V617F-positive patients, in particular patients of age 60 years or younger. In conclusion, this study in a Belgian cohort of patients supports and extends the growing body of evidence that CALR mutant cases of essential thrombocythemia are phenotypically distinct from JAK2 V617F-positive cases, with regards to clinical and hematologic presentation as well as overall survival.

TAF10 Interacts with the GATA1 Transcription Factor and Controls Mouse Erythropoiesis.

ABSTRACT The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs), is a prerequisite for the transcription of protein-coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA, are also necessary for tightly regulated transcription initiation. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. By ablating TAF10 specifically in erythroid cells in vivo, we observed a differentiation block accompanied by deregulated GATA1 target genes, including Gata1 itself, suggesting functional cross talk between GATA1 and TAF10. Additionally, we analyzed by mass spectrometry the composition of TFIID and SAGA complexes in mouse and human cells and found that their global integrity is maintained, with minor changes, during erythroid cell differentiation and development. In agreement with our functional data, we show that TAF10 interacts directly with GATA1 and that TAF10 is enriched on the GATA1 locus in human fetal erythroid cells. Thus, our findings demonstrate a cross talk between canonical TFIID and SAGA complexes and cell-specific transcription activators during development and differentiation.

Screening of JAK2 V617F and MPL W515 K/L negative essential thrombocythaemia patients for mutations in SESN2, DNAJC17, ST13, TOP1MT, and NTRK1.

Essential thrombocythaemia (ET) is a myeloproliferative neoplasm characterized by a sustained elevation of the platelet count and a tendency for thrombosis and haemorrhage. Cytogenetic abnormalities are rare in ET, accounting for <5% of cases. Molecular abnormalities include JAK2 V617F [Mendelian Inheritance in Man (MIM) reference 614521], a mutation found in 50% of ET patients (Kralovics etxa0al, 2005). In addition, a small fraction of JAK2 V617F-negative ET patients (about 10%) have activating point mutations in the thrombopoietin receptor gene (MPL, MIM 159530) (Kilpivaara & Levine, 2008). Other genes were also found to be mutated in ET, such as TET2, LNK and ASXL1, but at a low frequency. As such, the underlying molecular cause remains to be discovered in a substantial fraction of ET cases. n nRecently, Hou etxa0al (2012) developed a high-throughput single-cell sequencing method for sequencing the cancer genome with high accuracy at the nucleotide level to facilitate the analysis of tumour evolution in cancers. They applied their method on single cells derived from an ET JAK2 V617F-negative patient and identified several mutated genes. Based on the type, frequency, in silico analysis and score of the detected mutations, eight genes were identified as potential candidate drivers: SESN2, DNAJC17, ST13, TOP1MT, NTRK1, ABCB5, FRG1, and ASNS. However, their recurrence rate in ET was not determined. As none of these genes have been linked to ET before, our aim was to screen a large cohort of ET patients to verify the recurrence rate of these mutations. n nPeripheral blood (nxa0=xa031) or bone marrow (nxa0=xa033) was collected from a cohort of 64 patients diagnosed with ET according to the World Health Organization criteria (Vardiman etxa0al, 2009) and verified to be JAK2 V617F-negative and MPL wild-type by allele-specific polymerase chain reaction (PCR) and Sanger sequencing, respectively. We checked the presence and recurrence of the mutations in SESN2, DNAJC17, ST13 and TOP1MT, which exhibited the highest scores in the study reported by Hou etxa0al (2012), and NTRK1, which encodes a tyrosine kinase, in a large cohort of 64 JAK2 V617F- and MPL W515L/K-negative ET patients. PCR primers were designed to amplify the full exonic region encompassing the reported mutations: SESN2 p.P87S, TOP1MT p.S479L, ST13 p.Q349*, NTRK1 p.N323S, DNAJC17 p.A292P (Table u200b(Table1).1). The generated amplicons were analysed by Sanger sequence analysis. n n n nTable 1 n nOverview of the investigated genes, exons, and mutations. n n n nOur sequencing experiments did not reveal any of the mutations reported by Hou etxa0al (2012). However, our sequencing covered the exon involved in each gene, detecting two other novel heterozygous mutations. The first mutation was in exon 11 of DNAJC17 (c.877C>T, p.R293W), and was predicted to be benign by polyphen 2 (http://genetics.bwh.harvard.edu/pph2/) (Adzhubei etxa0al, 2010). This mutation was also detected in the buccal swab of the patient indicating that it is germline. The second mutation was in exon 11 of TOP1MT (c.1400A>G, p.N467S). It was predicted to be damaging by polyphen 2 and SIFT (http://sift.bii.a-star.edu.sg/) and was not present in the buccal swab corroborating it as an acquired mutation. TOP1MT is localized on human chromosome 8q24.3. It is highly homologous to the nuclear TOP1 gene and consists of 14 exons. TOP1MT is a mitochondrial topoisomerase encoded by the genomic DNA. It is a type IB enzyme, which sustains the befitting conformation of DNA during replication, transcription, recombination and repair. In addition to the main form of TOP1MT mRNA, two alternatively spliced forms were found, which appeared more frequent in renal cell carcinoma compared to normal adjacent tissue (Zhang etxa0al, 2007). TOP1MT was found to be overexpressed in haematological cancer cell lines in parallel with MYC overexpression leading to the assumption that MYC expression induces TOP1MT expression (Zoppoli etxa0al, 2011). n nWe assessed the effect of the mutation p.N467S on the structure of the protein by implementing in silico analysis. Amino acid sequences of wild-type and mutated TOP1MT sequences (Refseq: {type:entrez-protein,attrs:{text:NP_443195,term_id:16418461,term_text:NP_443195}}NP_443195; Uniprot: {type:entrez-protein,attrs:{text:Q969P6,term_id:20140694,term_text:Q969P6}}Q969P6) were supplied to the automated I-TASSER server (http://zhanglab.ccmb.med.umich.edu/) (Roy etxa0al, 2010). The obtained 3D structures were viewed on DeepView–Swiss-PdbViewer (http://www.expasy.org/spdbv/) (Guex & Peitsch, 1997). Models were energy minimized by performing two cycles of steepest descent with 50 steps each and one cycle of conjugate gradient of 200 steps with a minimum ΔE of 0·01xa0kJ/mol together with a harmonic constraint of 100xa0kJ/mol. The resulting models were further refined by ModRefiner (http://zhanglab.ccmb.med.umich.edu/ModRefiner) in order to obtain the best possible conformation of TOP1MT (Xu & Zhang, 2011). The final images were rendered on POV-Ray v3.6 (http://www.povray.org). By comparison, the major changes caused by p.N467S mutation were the gain of an α helix and the loss of a β strand (Figu200b(Fig1A)1A) which are in close proximity to the bound DNA molecule. This suggests that this mutation might affect the interaction of TOP1MT with the DNA molecule (Figu200b(Fig11B). n n n nFigure 1 n nTOP1MT p.N467S leads to a structural change that might affect the interaction of TOP1MT with DNA. In comparison to wild-type (Wt) TOP1MT, the mutated (Mt) TOP1MT gained an α helix (red arrows) and lost a β strand (blue arrows) in addition ... n n n nMoreover, TOP1MT is a highly conserved protein; by aligning TOP1MT from different species on Jalview we could confirm that Asparagine at position 467 is conserved (Figu200b(Fig1C).1C). Moreover, although this mutation has not been reported before in the catalogue of somatic mutations in cancer (COSMIC), two other mutations were reported in close proximity, c.1384G>A p.V462M and c.1390A>G p.I464V in adenocarcinoma and endometrioid carcinoma, respectively (Figu200b(Fig1D).1D). To check the recurrence of the p.N467S in ET, we screened exon 11 of TOP1MT gene in 38 additional JAK2 V617F-negative MPL wild-type ET cases, but did not detect any additional mutant cases. n nIn summary, in this series of JAK2 V617F and MPL W515K/L negative ET cases, we did not detect any of the mutations that were previously proposed as potential candidate drivers (Hou etxa0al, 2012). However, a novel mutation in exon 11 of TOP1MT was found. This establishes this gene as recurrently mutated in JAK2 V617F and MPL W515K/L negative ET, and suggests that TOP1MT mutations are involved, at low frequency, in the pathogenesis of ET. However, its functional consequences remain to be investigated. Given that the candidate mutations, as proposed by Hou etxa0al (2012) were predicted to be protein damaging, the possibility remains that other exons of these genes might harbour loss of function mutations. The quest for the full complement of driver mutations in ET therefore remains open.