Results of a collaborative study on DNA identification of aged bone samples

AimA collaborative exercise with several institutes was organized by the Forensic DNA Service (FDNAS) and the Institute of the Legal Medicine, 2nd Faculty of Medicine, Charles University in Prague, Czech Republic, with the aim to test performance of different laboratories carrying out DNA analysis of relatively old bone samples. MethodsEighteen laboratories participating in the collaborative exercise were asked to perform DNA typing of two samples of bone powder. Two bone samples provided by the National Museum and the Institute of Archaelogy in Prague, Czech Republic, came from archeological excavations and were estimated to be approximately 150 and 400 years old. The methods of genetic characterization including autosomal, gonosomal, and mitochondrial markers was selected solely at the discretion of the participating laboratory. ResultsAlthough the participating laboratories used different extraction and amplification strategies, concordant results were obtained from the relatively intact 150 years old bone sample. Typing was more problematic with the analysis of the 400 years old bone sample due to poorer quality. ConclusionThe laboratories performing identification DNA analysis of bone and teeth samples should regularly test their ability to correctly perform DNA-based identification on bone samples containing degraded DNA and potential inhibitors and demonstrate that risk of contamination is minimized

N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly

The interleukin-1α precursor suppresses hypersensitivity of <i>snf1</i>Δ strain to 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of the His3 imidazoleglycerol-phosphate dehydratase.

<p>This suppressive property of pre-IL-1alpha (pre) is more profound on SD media containing glycerol/ethanol as a carbon source (SDgly) in case of the <i>snf1-108</i> strain carrying incomplete <i>SNF1</i> deletion. Mature interleukin-1alpha (Mat) is not able to rescue the 3-AT hypersensitivity of both <i>snf1</i>Δ and <i>snf1-108</i> strains and was used as a control. We did not observe any differences in growth between strains producing pre-IL-1alpha and mature IL-1alpha on SD agar plates which did not contain and/or contain only a minute amount of 3-AT. This is an example of 3 independent biological experiments.</p

A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.

<p>Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,<i>ahc1</i>Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.</p

The SAGA and ADA complex subunits co-purify as a part of the IL-1α precursor-binding complex.

<p>Co-immunoprecipitation experiments with an anti-Flag antibody using yeast strains from a TAP tag library transformed with IL-1α expression vectors revealed that both of the HAT complexes bound pre-IL-1α (pre) but not mature IL-1α (Mat). Control cells (ctrl) carry the empty plasmid pYX212. Western blotting was performed using an anti-CBP antibody that recognizes the TAP tag at the C-terminus of the respective HAT complex subunits. For each line of the IP experiment, 1.4 mL of the cell lysate prepared from 5.10⁸ yeast cells in average was used. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Disruption of the SAGA and ADA complexes confirmed binding of the IL-1α precursor to the HAT/Core module and suggested the mutually exclusive role of Spt7 and Ahc1 in SAGA complex assembly.

<p>Co-immunoprecipitation was performed using yeast lysates with an anti-Flag antibody that recognizes the Flag tag at the N-terminus of the IL-1α precursor. Western blotting was performed with an anti-CBP antibody which identifies the TAP tag at the C-terminus of the respective HAT complex subunits. (A) Gcn5 does not bind to pre-IL-1α, and because it is not required for SAGA or ADA complex integrity, its deletion has no effect on Ahc1 or Spt8 co-immunoprecipitation with the IL-1α precursor (pre). Deletion of the AHC2 gene doesn’t impair co-IP of pre-IL-1α with Gcn5, Spt8 and Spt7. (B) The disruption of the ADA HAT complex did not affect the co-immunoprecipitation of Gcn5 and Spt8 with IL-1α. However, the interaction between Spt7 and the IL-1α precursor was significantly weakened. In experiments with TAP/Spt7,<i>ahc1</i>Δ strain, we received either no or very low signal (the latter is depicted) of TAP-tagged Spt7, with a success rate 3∶1, respectively. (C) The disruption of the SAGA complex abolished the interaction between Spt8 and the IL-1α precursor but had no effect on Ahc1 binding to the IL-1α precursor. Control cells (ctrl) carry the empty plasmid pYX212. For each line of the IP experiment, 3.5 mL of cell lysate prepared from 20.10⁸ yeast cells in average was used, except TAP/Spt8,<i>gcn5</i>Δ, TAP/Spt8,<i>ahc1</i>Δ, TAP/Spt8,<i>spt7</i>Δ and TAP/Gcn5,<i>ahc1</i>Δ strains, where 1.3 mL of cell lysates from 9.10⁸ yeast cells each were applied. Inputs contain 16.7 µL of the corresponding lysates taken before the lysates were used for immunoprecipitation.</p

Subcellular localization of pre-IL-1α and IL-1αMat in <i>Saccharomyces cerevisiae.</i>

<p>The IL-1α precursor (pre-IL-1α) is exclusively localized in the nucleus of yeast cells, which is in contrast to the observed cytoplasmic localization of mature IL-1α (IL-1αMat). Control cells (ctrl) carry the empty pUG36 vector. The cell nuclei are stained with DAPI.</p