Sequential Digestion with Trypsin and Elastase in Cross-Linking Mass Spectrometry.

Cross-linking mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid compositions of some protein regions impede the detection of cross-linked residues, although it would yield invaluable information for protein modeling. Here, we report on a sequential-digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin-cleavage sites. We exploited intrinsic substrate-recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

Measuring dynamics of eukaryotic transcription initiation: Challenges, insights and opportunities.

Transcription of protein-encoding genes in eukaryotic cells is a dynamically coordinated process. Many of the key transcription regulators contain functionally essential intrinsically disordered regions (IDRs), the dynamic nature of which creates extra challenges to traditional biochemical analyses. Recent advances in single-molecule fluorescence imaging technology have enabled direct visualization of these rapid, complex and dynamic molecular interactions in real time.

High levels of γ-glutamyl hydrolase (GGH) are associated with poor prognosis and unfavorable clinical outcomes in invasive breast cancer.

BACKGROUND: Previously, we performed analysis of gene expression in 46 axillary lymph node negative tumors and identified molecular gene signatures that resulted in different clinical outcomes. The aim of this study was to determine the correlation of γ-glutamyl hydrolase (GGH), fatty acid amide hydrolase (FAAH), Pirin (PIR) and TAF5-like RNA polymerase II, p300/CBP-associated factor (PCAF)-associated factor, 65 kDa (TAF5L), selected from identified gene signatures, with clinical outcomes as well as classical clinicopathological characteristics in primary invasive breast cancer patients. METHODS: The protein levels of GGH, FAAH, PIR and TAF5L were assessed by immunohistochemistry (IHC) on a panel of 80 primary invasive breast tumors. Quantitative real-time PCR (qRT-PCR) and western blot analysis were performed to verify the expression levels of the candidate biomarkers. Patient disease-specific survival (DSS) and recurrence-free survival (RFS) were evaluated using the Kaplan-Meier method. The prognostic biomarkers were identified by univariate analysis with a log-rank test and by multivariate analysis with Cox proportional hazards regression models. RESULTS: The GGH and FAAH protein levels were significantly up-regulated in invasive breast cancer tumors compared with adjacent non-cancerous tissues. Furthermore, the protein levels of GGH and FAAH were significantly correlated in tumor tissues. Tumoral GGH protein expression was significantly correlated with shorter DSS and RFS. Furthermore, the protein expression of GGH was positively correlated with undifferentiated tumors (BRE grade III) and ER/PR expressing tumors. Multivariate regression analysis showed that only GGH protein expression independently predicts DSS. No such correlations were found for FAAH, PIR and TAF5L protein expression. However, elevated protein levels of FAAH were positively associated with high number of lymph node involvement and upregulated levels of PIR were positively related with lymph node metastasis. The TAF5L was pronouncedly down-regulated in primary invasive breast cancer tissues compared to matched adjacent non-cancerous tissues. CONCLUSION: These data show for the first time that cytoplasmic GGH might play a relevant role in the development and progression of invasive breast cancer, warranting further investigations. Our findings suggest that GGH serve as a potential biomarker of unfavorable clinical outcomes over short-term follow-up in breast cancer. The GGH may be a very attractive targeted therapy for selected patients.

Expression analysis of four testis-specific genes AURKC, OIP5, PIWIL2 and TAF7L in acute myeloid leukemia: a gender-dependent expression pattern.

Cancer testis antigens (CTAs) have normal expression restricted in the testis and also inappropriate expression in various tumor types. Special and favorable characteristics of these genes, as being immunogenic and therefore having the potential to be used as tumor vaccine, have made them as one of the star attractions of the cancer research. Acute myeloid leukemia (AML) is a highly heterogeneous hematological disorder with various reported changes in gene expression. To find new CTA genes in AML, we analyzed the expression pattern of four testis-specific genes AURKC, OIP5, PIWIL2 and TAF7L using real-time quantitative PCR in 51 AMLs and 6 myelodysplastic syndrome cases in comparison with 33 healthy controls. The expression of the studied genes, noticeably OIP5 and TAF7L, differed between studied groups in a gender-dependent manner. Upregulation of OIP5 was observed only in ~41 % of the female AML patients as compared to the healthy females. The remaining ~59 % of the male AML patients, when compared to the healthy males, displayed downregulation of TAF7L. This finding may affect many aspects of AML such as diagnosis, prognosis assessment and treatment choice.

Genome-wide localization analysis of a complete set of Tafs reveals a specific effect of the taf1 mutation on Taf2 occupancy and provides indirect evidence for different TFIID conformations at different promoters.

In Saccharomyces cerevisiae, TFIID and SAGA principally mediate transcription of constitutive housekeeping genes and stress-inducible genes, respectively, by delivering TBP to the core promoter. Both are multi-protein complexes composed of 15 and 20 subunits, respectively, five of which are common and which may constitute a core sub-module in each complex. Although genome-wide gene expression studies have been conducted extensively in several TFIID and/or SAGA mutants, there are only a limited number of studies investigating genome-wide localization of the components of these two complexes. Specifically, there are no previous reports on localization of a complete set of Tafs and the effects of taf mutations on localization. Here, we examine the localization profiles of a complete set of Tafs, Gcn5, Bur6/Ncb2, Sua7, Tfa2, Tfg1, Tfb3 and Rpb1, on chromosomes III, IV and V by chromatin immunoprecipitation (ChIP)-chip analysis in wild-type and taf1-T657K mutant strains. In addition, we conducted conventional and sequential ChIP analysis of several ribosomal protein genes (RPGs) and non-RPGs. Intriguingly, the results revealed a novel relationship between TFIIB and NC2, simultaneous co-localization of SAGA and TFIID on RPG promoters, specific effects of taf1 mutation on Taf2 occupancy, and an indirect evidence for the existence of different TFIID conformations.

The homologous Drosophila transcriptional adaptors ADA2a and ADA2b are both required for normal development but have different functions.

In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.

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

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.

Role of TAFII-17, a VDR binding protein, in the increased osteoclast formation in Paget's Disease.

UNLABELLED: In contrast to normal OCL precursors, pagetic OCL precursors express MVNP and form OCL at physiologic concentrations of 1,25(OH)2D3, as do normal OCL precursors transfected with the MVNP gene. Using a GST-VDR chimeric protein, we identified TAFII-17 as VDR binding protein expressed by pagetic OCL precursors and MVNP transduced normal OCL precursors. TAF(II)-17 was in part responsible for the increased 1,25(OH)2D3 responsivity of pagetic OCL precursors. INTRODUCTION: Pagetic osteoclasts (OCLs) and their precursors express measles virus nucleocapsid protein (MVNP) and form large numbers of OCLs at low concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Similarly, normal OCL precursors transfected with MVNP also form OCLs at low concentrations of 1,25(OH)2D3. These results suggest that expression of MVNP in OCL precursors enhances vitamin D receptor (VDR)-mediated gene transcription. MATERIALS AND METHODS: To determine the mechanism for the increased OCL formation capacity of pagetic OCL precursors in response to 1,25(OH)2D3, lysates from pagetic and MVNP-transduced normal OCL precursors were incubated with a GST-VDR chimeric protein. RESULTS: A 17-kDa peptide that bound VDR was detected in MVNP-transduced cells and pagetic OCL precursors treated with 1,25(OH)2D3. This peptide was identified as TAFII-17, a component of the TFIID transcription complex. Expression of increased levels of TAFII-17 in cells allowed TAFII-17 to bind to VDR at low concentrations of 1,25(OH)2D3. An antisense oligonucelotide (AS-ODN) to TAFII-17 significantly decreased OCL formation in response to 1,25(OH)2D3 in pagetic but not normal marrow cultures by approximately 40%. Transfection of TAFII-17 or MVNP into NIH3T3 cells increased VDR transcriptional activity as measured by DR-3 reporter assays. CONCLUSION: These data show that expression of the MVNP gene in OCL precursors results in increased levels of TAFII-17. TAFII-17 can bind VDR at low concentrations of 1,25(OH)2D3. These results suggest that MVNP expression in Paget's OCL precursors increases expression of a component(s) of the VDR transcription complex that can increase OCL formation.