s2fieldsofstudy: Biology Chemistry

text:
CK2 Is the Regulator of SIRT1 Substrate-Binding Affinity, Deacetylase Activity and Cellular Response to DNA-Damage

SIRT1, an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase, protects cells from stress-induced apoptosis, and its orthologues delay aging in lower eukaryotes. SIRT1 increases survival in response to stress such as DNA damage by deacetylating a number of substrates including pro-apoptotic protein p53. The molecular mechanism by which DNA-damage activates SIRT1 is not known. By screening a kinase inhibitor library, we identified CK2 as a SIRT1 kinase. CK2 is a pleiotropic kinase with more than 300 substrates and well-known anti-apoptotic and pro-growth activities. We find that CK2 is recruited to SIRT1 after ionizing radiation (IR) and phosphorylates conserved residues Ser 154, 649, 651 and 683 in the N- and C-terminal domains of mouse SIRT1. Phosphorylation of SIRT1 increases its deacetylation rate but not if the four Ser residues are mutated. In addition, phosphorylation of SIRT1 increases its substrate-binding affinity. CK2-mediated phosphorylation increases the ability of SIRT1 to deacetylate p53 and protect cells from apoptosis after DNA damage. Based on these findings, we propose that CK2 protects against IR-induced apoptosis partly by phosphorylating and activating SIRT1. Thus, this work suggests that SIRT1 is a component of the expansive anti-apoptotic network controlled by CK2. Since expression of both CK2 and SIRT1 is upregulated with tumorigenesis and downregulated with senescence, the CK2-SIRT1 link sheds new light on how CK2 may regulate cancer development and aging.

Introduction
Sirtuins [1], which are composed of seven members (SIRT1-SIRT7), are class III histone/protein deacetylases (HDAC). Unlike the other classes of HDACs, they require the coenzyme NAD+ (nicotinamide adenine dinucleotide) [2,3]. Sirtuins are orthologs of yeast Sir2 (silencing information regulator) [2,3], which mediates chromatin silencing [4,5] and slows the aging rate by suppressing the production of extrachromosomal rDNA circles [6]. Within the Sirtuin family, SIRT1 [7] is most closely related to yeast Sir2. Calorie restriction, the only known intervention that extends life span in mammals, increases SIRT1 expression in some tissues [8,9]. There is no evidence that SIRT1 regulates aging in mammals, but it does increase resistance to various forms of stress [8,10,11,12,13,14]. One very well characterized pathway regulated by SIRT1 is DNA-damage induced apoptosis. SIRT1 deacetylates and decreases the transcriptional activity of pro-apoptotic p53 and increases cell survival after DNA damage [12,15,16,17]. Consistent with the pro-survival function of SIRT1, p53 is hyperacetylated in mice lacking SIRT1, and thymocytes derived from these mice have increased sensitivity to IR [15]. However, the biochemical pathway that activates SIRT1 in response to DNA damage has not been discovered.
CK2, a tetrameric enzyme composed of two catalytic subunits (aa, aa' or a'a') and two regulatory b subunits, is a ubiquitously expressed and evolutionarily conserved serine/threonine protein kinase [18]. CK2a and CK2a' have approximately 90% identity in their catalytic domains and have similar enzymatic properties including substrate specificity. CK2 is a pleiotropic kinase that has more than 300 putative targets [19] and can be found in the nucleus, cytoplasm and specific structures and organelles such as the plasma membrane, Golgi, ribosomes and endoplasmic reticulum [20]. CK2 is essential for viability [21] and plays a critical role in tumor development [22]. Phosphorylation by CK2 prevents caspase-mediated cleavage of a number of proteins involved in the regulation of cell survival [23], and suppression of CK2 decreases cell proliferation and viability [24,25]. Although CK2 has a high basal activity, it can be stimulated with growth factors [26], serum [27] and stresses such as DNA damage [28,29]. CK2 function is also modulated by translocation to specific sites within the cell. For example, stresses such as ionizing radiation IR [25] and hypoxia [30] cause nuclear accumulation of CK2.
Here, we report that IR leads to CK2-SIRT1 interaction and CK2-mediated phosphorylation at four Ser residues. SIRT1 phosphorylation increases its substrate-binding affinity and its deacetylase activity. As a result, SIRT1 phosphorylation increases p53 deacetylation and survival after DNA-damage.

SIRT1 phosphorylation is CK2-dependent
Phosphorylation is one of the most common mechanisms by which protein function is regulated. To identify a potential kinase that phosphoryates SIRT1, we screened a library of 81 kinase-inhibitors (10 mM) (Table S1) for their ability to inhibit 32 P incorporation into SIRT1. We incubated HEK 293T cells stably expressing WT Flag-tagged mouse SIRT1 in 100 mCi/ml [ 32 P]orthophosphoric acid for one hr and isolated SIRT1 by immunoprecipitating with anti-FLAG antibody (Fig. 1A). From this screen, we identified four kinase-inhibitors that suppressed SIRT1 phosphorylation by more than 50%: Staurosporin (panspecific), by 86.6%; Tyrphostin 9 (PDGF receptor kinase inhibitor), by 79.8%; GW 5074 (c-Raf inhibitor), by 74.5%; TBCA (the most potent CK2 inhibitor) [31], by 72.5%. Two other CK2 inhibitors used in this screen, DRB and Apigenin, did not significantly inhibit 32 P incorporation because of weak inhibitory activity at the concentration that we used here. We also tested whether inhibitors of other growth or stress-related kinases that were not included in the screen affected 32 P incorporation into SIRT1. As shown in Fig. 1B, only TBCA inhibited 32 P incorporation into SIRT1.
The localization of PDGF receptor and c-Raf are limited to the plasma membrane and cytoplasm, respectively, whereas CK2 can be found in the nucleus, cytoplasm and specific structures and organelles such as the plasma membrane, Golgi, ribosomes and endoplasmic reticulum [20]. Although SIRT1 has been detected in both cytoplasm and nucleus in some cell types [32,33], SIRT1 is a nuclear protein in most cell lines [34,35], including the HEK 293T cell line that we used for this kinase inhibitor screen (data not shown). Based on common nuclear localization, we chose to investigate the possibility that CK2 is a SIRT1 kinase.

Identification of CK2 phosphorylation sites in SIRT1
To investigate whether CK2 phosphorylates SIRT1 directly, we produced fragments of mouse SIRT1 proteins in E. coli and performed in vitro kinase reactions using CK2 a( Fig. 2A). We found that CK2 phosphorylation sites were located in N-terminal (a.a. 1-235) and C-terminal (a.a. 611-737) domains but not in the Sirtuin (deacetylase) domain (Fig. 2B). The consensus sequence for CK2 phosphorylation is S/TXXD/E, where X can be any amino acid residue. There are four evolutionarily-conserved CK2 consensus sequences (Ser 154,649,651,683) within the CK2 phosphorylated regions of SIRT1 (*, Fig. 2C). To identify the CK2 phosphorylation sites, we mutated the four CK2 consensus sites to Ala either individually or in groups ( Fig. 2D-I). Mutating Ser 154 to Ala (S154A) completely abolished CK2 phosphorylation of the Nterminal fragment indicating that Ser 154 is the only CK2 phosphorylation site in the N-terminal fragment (Fig. 2D). Mutating Ser 649 to Ala (S649A) in the C-terminal fragment significantly reduced SIRT1 phosphorylation but did not abolish it (Fig. 2E). Mutating Ser 651 to Ala (S651A) in fragment CT-2 consistently reduced SIRT1 phosphorylation, albeit slightly (Fig. 2F). Mutating Ser 649 and 651 to Ala decreased phosphorylation of full-length SIRT1 more than mutating Ser 649 alone, indicating that both Ser 649 and 651 are phosphorylated by CK2 (Fig. 2G). Mutating Ser 649, 651 and 683 to Ala in the C-terminal fragment completely abolished phosphorylation of the C-terminal fragment but mutating Ser 649 and 651 did not, indicating that Ser 683 is also phosphorylated by CK2 (Fig. 2H). Mutating Ser 154, 649, 651 and 683 (4A) completely abolished CK2 phosphorylation of full-length SIRT1 (Fig. 2I), indicating that CK2 phosphorylates all four conserved Ser residues in vitro.

CK2-mediated phosphorylation of SIRT1 in vivo
To confirm that the four Ser residues were phosphorylated in vivo, we performed the [ 32 P]-orthophosphoric acid incorporation assay after transiently expressing wild-type (WT) and 2A (S649/ 651A) and 4A (S154/649/651/683A) mutant SIRT1 in HEK  Table S1). Each bar represents the intensity of phosphorylated SIRT1 normalized to the total SIRT1 level after immunoprecipitation (IP) with M2 Flag antibody. The value of vehicle-treated sample was set to 100%. (B) In vivo 32 P labeling of SIRT1 in the presence of inhibitors of stress-activated kinases that are indicated. doi:10.1371/journal.pone.0006611.g001 293T cells (Fig. 3A). Compared with WT SIRT1, the 2A and 4A mutants showed progressively decreasing phosphorylation intensity. Even in 4A mutant, there was some residual phosphorylation, indicating that other kinase(s) also phosphorylate SIRT1 in vivo. To directly visualize phosphorylation at these four Ser residues, we attempted to make phospho-specific antibodies recognizing each of the four phosphorylated Ser residues. However, due to the acidic nature of some of these sites, we were able to make a phospho-specific antibody only for Ser 649 (Ser 659 in human SIRT1). To test the specificity of the phospho-S649 (P-S649) antibody, we performed CK2 kinase reactions using either WT or 4A SIRT1 as substrates with or without TBCA in the reaction mixture. As shown in Fig. 3B, P-S649 antibody recognized SIRT1 phosphorylated by CK2 in vitro but not 4A SIRT1. P-S649 antibody also recognized WT but not 4A SIRT1 in transiently transfected cells (Fig. 3C). Ser 649 phosphorylation of endogenous SIRT1 was poorly visible in untreated cells but was induced 90 min after IR (Fig. 3D). The interaction between SIRT1 and the two catalytic subunits of CK2 also increased significantly 90 min after IR (Fig. 3D). To demonstrate that SIRT1 phosphorylation is mediated by CK2, we transiently expressed SIRT1 along with siRNA for either a scrambled sequence or for both catalytic subunits of CK2 (a and a'). Two hours before they were harvested, cells were exposed to IR to induce Ser 649 phosphorylation. Knock-down of CK2 dramatically reduced Ser 649 phosphorylation, indicating that CK2 phosphorylates Ser 649 in vivo (Fig. 3E). In agreement with this, treatment of cells with CK2-inhibitor TBCA (Fig. 3F) or coexpression of kinase-dead (KD) CK2 a' (Fig. 3G) significantly inhibited Ser 649 phosphorylation. Our inability to detect Ser 649 phosphorylation in endogenous SIRT1 without IR (Fig. 3D) was unexpected because CK2 has high basal activity and also because we did not use IR when we discovered through our kinase inhibitor screen ( Fig. 1) that TBCA inhibited SIRT1 phosphorylation. We believe that this apparent contradiction is due to the low sensitivity of P-S649 antibody. Ser 649 phosphorylation is visible in transiently expressed SIRT1 because there is more SIRT1 protein in the cell and the vector DNA triggers the DNA-break signal in the way IR does (data not shown).
To determine how phosphorylation by CK2 affects SIRT1, we measured the deacetylase activity of SIRT1 after CK2 phosphorylation in vitro. Deacetylation of Lys 382 in p53, which is known to be mediated by SIRT1 [16], was used as the indicator for SIRT1 activity. We monitored p53 deacetylation after 20, 40 or 60 min reaction time (Fig. 4A). For all three reaction times, deacetylation of p53 by WT SIRT1 was increased after CK2 phosphorylation, but not if TBCA was present during the phosphorylation reaction. However, deacetylation of p53 by 4A SIRT1 was not affected by CK2. It should be noted that in the absence of CK2 phosphorylation, the deacetylase activity of 4A SIRT1 is comparable to that of WT SIRT1, indicating that the four Ser residues are not essential for SIRT1 activity in vitro.

Phosphorylation by CK2 activates SIRT1
In response to DNA damage, Lys 382 in p53 is acetylated by histone acetyltransferase p300 [36,37]. Acetylation of p53 increases its affinity to the target DNA sequence and thus its transcriptional activity. We investigated the effect of CK2 on SIRT1 activity in U2OS cells using p53 (K382) acetylation as the endogenous marker for the deacetylase activity of SIRT1. The acetylation level of K382 at any given moment is determined by the rate of acetylation and deacetylation at that moment. To determine the time course of CK2-mediated activation of SIRT1, we visualized acetylation of K382 at different time points after IR in cells transfected with siRNA for either a scrambled sequence or for both catalytic subunits of CK2 (Fig. 4B). Consistent with the observation that acetylation of K382 by p300 occurs 2 hr after IR [37], K382 acetylation peaked 2 hr after IR. However, K382 acetylation at 2 hr was higher when CK2 was knocked-down and did not return to baseline even at 7 hr, indicating that CK2 increases the deacetylase activity of SIRT1. Although CK2 enhanced SIRT1 activity at 2 hr point, the acetylation level of K382 at this time is still higher than before IR, suggesting that p300 activity exceeds SIRT1 activity at 2 hr point. Although this experiment suggests that CK2-activated SIRT1 deacetylated p53, we cannot rule out the possibility that activated SIRT1 decreased p53 acetylation by an indirect mechanism, such as improved DNA repair leading to reduced p300-mediated acetylation of p53. However, as of now, there is no evidence that SIRT1 improves DNA repair.
In order to confirm that CK2 phosphorylation increases SIRT1 activity, we transiently expressed p53 along with WT SIRT1 or 4A SIRT1 in H1299 cells and monitored p53 acetylation. The deacetylase activity of WT SIRT1 was inhibited in the presence of  (2) or with TBCA (20 mM). Immunoblotting was performed with S649-specific phospho-antibody (P-S649). Total SIRT1 levels are shown below. (C) Transiently expressed V5-WT or 4A SIRT1 was immunoprecipitated with V5 antibody and immunoblotted with P-S649 antibody. Immunoprecipitation with HA antibody was used as a control. (D) HeLa cells were harvested 60 and 90 min after IR (20 Gy) and endogenous SIRT1 was immnoprecipitated with either SIRT1 antibody or a control antibody, IgG. Immunoblotting was performed with P-S649 antibody to visualize SIRT1 phosphorylation and with SIRT1 antibody to visualize total SIRT1 levels. SIRT1-CK2 interaction was visualized by immunoblotting with antibody specific for CK2 a or CK2 a'. (E) Flag-tagged WT SIRT1 transiently expressed in HeLa cells with siRNA for either a scrambled sequence (Scr) or both catalytic subunits of CK2. Two hr after IR (20 Gy), SIRT1 was immunoprecipitated and then immunoblotted with P-S649 to visualize CK2 phosphorylation. (F) Lysates from HeLa cells co-expressing Flag-tagged WT SIRT1 treated with DMSO (2) or with 20 mM TBCA (+) starting 1 hr before IR (20 Gy). Two hr after IR, SIRT1 was immunoprecipitated with Flag antibody and then immunoblotted with P-S649 antibody. (G) Lysates from HeLa cells co-expressing Flag-tagged WT SIRT1 and either WT or KD CK2 were harvested 2 hr after IR. SIRT1 was immunoprecipitated with a Flag antibody and then immunoblotted with P-S649 antibody. doi:10.1371/journal.pone.0006611.g003 TBCA but the deacetylase activity of 4A SIRT1, which was lower than that of WT SIRT1, was insensitive to TBCA (Fig. 4C). Since there is a possibility that the effect of TBCA on SIRT1 activity was due to an off-target hit of TBCA, we overexpressed kinase-dead (KD) CK2 a' to inhibit the CK2 activity in a dominant-negative fashion (Fig. 4D). The deacetylase activity of WT SIRT1 as measured by p53 deacetylation was suppressed in the presence of KD CK2 a', but the deacetylase activity of 4A SIRT1 was not affected by KD CK2 a'. To determine if CK2 regulates deacetylation of other SIRT1 substrates, we repeated the experiment using RelA/p65 subunit of NF-kB, another known SIRT1 substrate (Fig. 4E) [38]. The deacetylase activity of WT SIRT1, as measured by RelA/p65 deacetylation, was suppressed in the presence of KD CK2 a', but the deacetylase activity of 4A SIRT1 was not affected by KD CK2 a'. We also tried to knockdown CK2 using siRNA specific for the catalytic subunits of CK2 in cells transiently expressing either WT or 4A SIRT1. However, we noticed that the combination of CK2 siRNA and 4A SIRT1 expression caused cell death, and therefore did not continue this experiment. Thus, CK2-dependent phosphorylation of SIRT1 stimulates the deacetylase activity of SIRT1 against at least two known substrates, p53 and NF-kB.

Phosphorylation by CK2 increases SIRT1 substratebinding
Since the CK2 phosphorylation sites are distant from the Sirtuin domain of SIRT1, it is curious that deacetylation of its substrate is increased by CK2 phosphorylation. One possibility is that SIRT1 phosphorylation changes its substrate binding affinity. To investigate whether phosphorylation by CK2 affects SIRT1-substrate interaction, we performed a GST-pull down assay using acetylated GST-p53 fusion protein and WT or 4A mutant SIRT1 that has been preincubated with CK2. As shown in Fig. 5, preincubation of WT SIRT1 with CK2 increased WT SIRT1-p53 interaction in a TBCAsensitive manner. In contrast, preincubation of 4A SIRT1 with CK2 did not affect SIRT1-p53 interaction. To determine if acetylation of The deacetylase activity of the endogenous SIRT1 in the presence of siRNA for either a scrambled sequence or for both catalytic subunits of CK2. P53 K382 acetylation was visualized 2 and 7 hr after IR (20Gy) in U2OS cells. Total levels of p53, SIRT1, CK2a, CK2a' and actin are shown below. (C) H1299 cells co-expressing V5-tagged WT or 4A SIRT1 and p53 in the presence or absence of TBCA were harvested 6 hr after IR (20 Gy). Acetylation of p53 was visualized by immunoblotting with acetylated p53 (K382) antibody. (D) V5-tagged WT or 4A SIRT1 was transiently co-expressed with p53 and either HA-tagged WT or KD CK2a' in H1299 cells. Cells were harvested 6 hr after IR and deacetylation of p53 was visualized by immunoblotting with acetylated p53 (K382) antibody. (E) V5-tagged WT or 4A SIRT1 was transiently co-expressed with FLAG-RelA/p65 (a component of NF-kB) and either HA-tagged WT or KD CK2a' in H1299 cells. FLAG-RelA/p65 was immunoprecipitated with FLAG antibody and immunoblotted with either FLAG antibody to visualize the total level of FLAG-RelA/p65 or Ac-Lys antibody to visualize the acetylation level. doi:10.1371/journal.pone.0006611.g004 the substrate is required for the increased SIRT1-p53 interaction, we also performed the GST-pull down assay using unacetylated GST-p53. CK2-mediated phosphorylation increased SIRT1 interaction with unacetylated GST-p53 (Fig. 5, bottom), indicating that the acetyl-group on the substrate is not a feature that is required for the enhanced affinity. Taken together, these findings indicate that one mechanism by which CK2-mediated phosphorylation increases SIRT1 deacetylase activity is increasing interaction with its substrate. We cannot rule out the possibility that CK2-mediated phosphorylation also increases SIRT1 activity by other mechanisms.
Anti-apoptotic function of SIRT1 is enhanced by CK2mediated phosphorylation SIRT1 protects cells from apoptosis induced by DNA damage [11,12,14,16]. The most commonly studied role of SIRT1 in stress resistance is p53 deacetylation and suppression of p53-induced apoptosis. To measure the effect of CK2-mediated phosphorylation of SIRT1 on p53 mediated apoptosis, H1299 cells were cotransfected with p53 and either WT or 4A SIRT1 and treated with the genotoxin etoposide for 24 hr. Quantification of apoptosis indicated that WT SIRT1 suppressed etoposide-induced apoptosis by 42% and 4A SIRT1, by 16%, compared to vector (pcDNA)alone control (Fig. 6A). Thus, the CK2 phosphorylation sites are important for the anti-apoptotic effects of SIRT1. Since the 4A mutation does not significantly affect the intrinsic SIRT1 activity (see Fig. 4A), the low magnitude of the anti-apoptotic effect of 4A SIRT1 is most likely due to the baseline protection from endogenous SIRT1 in all the samples. Deacetylation of p53 was significantly stronger with WT SIRT1 than with 4A SIRT1 (Fig. 6B) in these cells. Collectively, these results suggest that the anti-apoptotic function of SIRT1 is enhanced by CK2-mediated phosphorylation.
Despite the prominent role of SIRT1 in DNA damageresponse, the signaling pathway that activates SIRT1 after DNA-damage was unknown. Our results indicate that CK2 activates SIRT1 by phosphorylating four Ser residues in N-and Cterminal domains. Although very little is known about the function of the N-and C-terminal domains of SIRT1, our finding that CK2-mediated phosphorylation increases SIRT1-p53 interaction suggests that the N-and C-terminal domains regulate substrate recruitment. A full understanding of how these terminal domains and their phosphorylation regulate SIRT1 function is hampered by the lack of 3D structure information on the full-length SIRT1 or on the terminal domains. It remains to be determined whether CK2-mediated phosphorylation affects SIRT1 activity by simply increasing substrate recruitment or whether other mechanisms are also involved.
To our knowledge, this work represents the first identification of a signal pathway that activates the catalytic function of SIRT1 in response to DNA damage. While this work was in progress, Zschoernig et. al. [39] reported that CK2 phosphorylates Ser 649/ 651 but did not determine whether phosphorylation affected SIRT1 in any way or under what conditions CK2 phosphorylates SIRT1. Also, Sasaki et. al. [40] reported that SIRT1 is activated by phosphorylation at multiple sites and two of these sites (Thr 530 and Ser 540 in human SIRT1) are phosphorylated by cyclinB/ Cdk1, and mutation of these residues disturbed cell cycle  progression. However, phosphorylation by cyclinB/Cdk1 in vitro did not affect the catalytic activity of SIRT1. Thus, it is not clear how phosphorylation by cyclinB/Cdk1 activates SIRT1 activity. Interestingly, SIRT1 activity is activated by sumoylation of Lys 734 [13]; and DNA-damage causes SENP1-mediated desumoylation and inhibition of SIRT1 activity. SIRT1 activity is also regulated by proteins that bind SIRT1 directly. AROS, which binds to the N-terminal domain of SIRT1, activates SIRT1 [41]. It is not known if AROS-SIRT1 interaction is regulated by DNA damage or other forms of stress. In contrast, DBC1, which binds to the Sirtuin domain of SIRT1, inhibits SIRT1 activity in response to DNA-damage [11,14]. Thus, these previous reports and our findings indicate that DNA-damage has both inhibitory and stimulatory effects on SIRT1 activity.
Previous studies have shown that CK2 can phosphorylate Ser 392 [42] of p53 and enhance its activity by increasing tetramerization and DNA binding [43]. Therefore, CK2 may regulate p53 activity directly and indirectly through SIRT1 as we have demonstrated here. However, Ser 392 is phosphorylated after UV irradiation but not after IR or etoposide [28,44], suggesting that CK2 does not phosphorylate p53 in response to double-stranded breaks. Since we utilized IR and etoposide, not UV, in this work, it is unlikely that direct p53 phosphorylation played a significant role in CK2-dependent regulation of p53 that we describe here.
Our findings show that SIRT1 is a component of an expansive anti-apoptotic network that is controlled by CK2. Since expression of both CK2 and SIRT1 is upregulated with tumorigenesis [22,45,46] and downregulated with senescence [47,48], the CK2-SIRT1 pathway is likely to play an important role in regulating cellular survival and death associated with cancer and aging. Although SIRT1 has anti-apoptotic functions, it may also have anti-tumorigenesis functions as well. SIRT1 is important for genetic stability [49], and when it is overexpressed in colonic epithelial cells, it deacetylates b-catenin and suppresses colon cancer cell growth [50]. Thus, SIRT1 appears to mediate a delicate balance between its anti-growth and pro-growth properties.

Protein kinase inhibitor library screening
The kinase inhibitors were derived from the Protein kinase inhibitor library (BIOMOL international) and other commercial sources. HEK 293T cells stably expressing WT Flag-tagged mouse SIRT1 were seeded onto 6 well plates at 5610 6 cells per well. One day after plating, cells were pre-incubated with phosphate free DMEM media (Gibco) containing 10 mM protein kinase inhibitor for 1 hr. After pre-incubation, 100 mCi/ml [ 32 P]-orthophosphoric acid was added into each well directly and incubated for another 2 hr. Labeled Flag-tagged SIRT1 proteins were immunoprecipitated with Flag M2-Agarose (Sigma), separated by 4-12 % SDS-PAGE and transferred to nitrocellulose. The amounts of [ 32 P]labeled and loaded SIRT1 proteins were detected by autoradiography and immunoblotting with SIRT1 antibody, respectively.

In vitro deacetylase assay
For in vitro deacetylase assays, 2 mg purified His-tagged WT and 4A mutant full-length SIRT1 proteins were incubated with CK2a enzyme with or without 20 mM TBCA in a kinase buffer described above except radioactive ATP was not included. After the kinase reaction, the samples were loaded onto CENTRISEP spin columns (Princeton separations) pre-equilibrated with 16PBS to remove the excess ATP and free additives in CK2 a enzyme solution. 1/10 volume of above phosphorylated or mockphosphorylated SIRT1 proteins were added to a deacetylase buffer (50 mM HEPES at pH 7.0, 1 mM DTT, 150 mM NaCl, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche)) containing 0.2 mg acetylated GST-p53, and 0.5 mM NAD + in a 20 ml final reaction volume. The reactions were performed at 37uC for the indicated durations and stopped by adding SDS sample buffer. Heated samples were subjected to 4-12% SDS PAGE, transferred to nitrocellulose, and immunoblotted with Ac-p53 (K382) antibody (Cell Signaling Technologies). The loading amounts of SIRT1 and GST-p53 proteins were visualized with Coomassie stain and Ponceau S stain.

Acetylation of GST-p53 protein
Glutathione S-transferase (GST) fused to human p53 fragment (amino acids 373-385) was expressed in E. coli and purified on glutathione Sepharose beads according to the vendor's manual. Purified GST-p53 was acetylated by His-tagged active core p300/ CBP associated factor (PCAF, amino acids 352-832) in HAT buffer (Millipore) containing 5 mM acetyl CoA. As shown in Fig.  S1, K382 was strongly acetylated by this method. After incubating for 3 hrs, the reaction mixture was incubated with glutathione Sepharose and acetylated GST-p53 proteins were purified and dialyzed against 20 mM HEPES buffer (pH 7.0) containing 2 % glycerol, 1 mM DTT, and 50 mM NaCl.

GST-Pull down assay
To measure the interaction between mouse SIRT1 and p53 peptide, 0.2 mg GST-p53 and 0.2 mg mouse SIRT1 (preincubated with CK2 with or without TBCA) was incubated with glutathioneagarose beads (GE) in lysis buffer containing 150 mM NaCl for 2 hr at room temperature. The beads were washed once with lysis buffer and twice with RIPA buffer containing 300 mM NaCl. Proteins retained on the beads were eluted with 2X SDS sample buffer and subjected to SDS-PAGE. Bound SIRT1 was visualized by immunoblotting and GST-p53 was visualized by Ponceau staining.

Proteins, Cell lines and Expression constructs
Wild-type and alanine mutant His-tagged mouse SIRT1 were constructed in pET-15b prokaryotic expression vector using NdeI and XhoI restriction sites, expressed in E.coli, and purified on Ni 2+ -NTA beads (Qiagen) following the manufacturer's protocol. GST fusion constructs of full-length and truncated mouse SIRT1 were generated by PCR amplification and subcloned into pGEX 4T-3 vector using SalI and XhoI sites. All GST-fusion constructs were transformed into BL21 competent cells and the recombinant proteins were purified and dialyzed as described above. To produce GST-PC, optimized CK2 substrate (Fig. 2B), nucleotides corresponding to RRRDDDSDDD were ligated into pGEX 4T-3 vector using Bam H1 and XhoI sites. To produce the mammalian SIRT1 expression vectors, WT and mutant mouse SIRT1 coding sequences were subcloned into pcDNA6.0 (Invitrogen) and FLAG-CMV expression vector (Sigma) using KpnI and XhoI sites. Wildtype and kinase-dead dominant negative CK2 a' expression vectors were obtained from Dr. Litchfield (the University of Ontario, Canada). H1299 non-small cell lung cancer cells, Hela cervical cancer cells, HEK 293T cells, Hepa 1-6 mouse hepatoma cell, and U2OS human osteosarcoma cells were maintained in DMEM medium supplemented with 10% FBS. The expression vector for FLAG-RelA/65 (1-313) was previously published [38].

Apoptosis assay
To measure p53-dependent apoptosis, we co-transfected H1299 cells with expression vectors for p53 and pcDNA (empty), WT or 4A mutant SIRT1 and K K plasmid. Transfected cells were selected using autoMax system (Miltenyi Biotec Inc.) 36 hr after transfection. After a further 24 hr, the cells were treated with etoposide (20 mM) for 24 hr and apoptosis was measured by the cell death detection ELISA Plus (Roche) according to the instruction manual.

Immunoblotting and immunoprecipitation assays
Whole cell lysates were prepared by adding a lysis buffer containing phosphate buffered saline supplemented with 1 % Triton X-100, protease and phosphatase inhibitor cocktails (Roche). Extracts were separated by 4-12 % SDS-PAGE gels, transferred to nitrocellose mambrane and probed with primary antibodies as indicated. To immunoprecipitate, cells treated as indicated were extracted with lysis buffer and incubated with recommended amount of primary antibodies by manufacturer for overnight at 4uC and then further incubated with protein A/G agarose beads (Pierce) for 2 hrs. These beads were loaded onto Micro Bio-Spin chromatography columns (Bio-Rad), washed four times with lysis buffer, which was then removed by centrifugation. Immunoprecipitated proteins were denatured with SDS-sample buffer and released from the column by centrifugation. Antibodies for SIRT1 (Upstate Biotechnology), Acetyl-Lys (Cell Signaling Technology), CK2a and a' (Santa Cruz Biotechnology) were purchased from commercial sources.

P-S649 antibody generation
The phosphospecific antibody was manufactured by 21st Century Biochemicals (Marlborough, MA). Briefly, a peptide corresponding to the sequence C-Ahx-IFHGAEVY[pS]D-amide was manufactured by Fmoc chemistry, HPLC purified to.90%, and the mass and sequence verified by nanospray MS and CID MS/MS, respectively. This peptide was conjugated to a carrier protein and was used to immunize two New Zealand rabbits. After multiple boosts and bleeds, serum that detected the target via western blots was pooled, immunodepleted by passing the serum over cross-linked agarose conjugated with unmodified peptide, and subsequently affinity purified using phosphopeptide-cross-linked agarose.


s2fieldsofstudy: Biology Medicine

text:
Passage and concentration-dependent effects of Indomethacin on tendon derived cells

Background Non-steroidal anti-inflammatory drugs (NSAID) are commonly used in the treatment of tendinopathies such as tendonitis and tendinosis. Despite this, little is known of their direct actions on tendon-derived cells. As NSAIDs have been shown to delay healing in a number of mesenchymal tissues we have investigated the direct effects of indomethacin on the proliferation of tendon-derived cells. Results and Discussion The results obtained were dependent on both the type of cells used and the method of measurement. When measured using the Alamar blue assay, a common method for the measurement of cell proliferation and viability, no effect of indomethacin was seen regardless of cell source. It is likely that this lack of effect was due to a paucity of mitochondrial enzymes in tendon cells. However, when cell number was assessed using the methylene blue assay, which is a simple nuclear staining technique, an Indomethacin-induced inhibition of proliferation was seen in primary cells but not in secondary subcultures. Conclusion These results suggest that firstly, care must be taken when deciding on methodology used to investigate tendon-derived cells as these cells have a quite different metabolism to other mesenchymal derive cells. Secondly, Indomethacin can inhibit the proliferation of primary tendon derived cells and that secondary subculture selects for a population of cells that is unresponsive to this drug.

Introduction
Non-steroidal inflammatory drugs (NSAIDs) are commonly used for the treatment of a number of musculoskeletal sports injuries including the inflammation of tendons and ligaments. A number of studies have however, suggested that NSAIDs may delay soft tissue healing although the exact mechanism of action for this is unknown [1][2][3][4]. Some in vitro investigations on the effects of NSAIDs on tenocytes have been performed [5][6][7][8][9]. However, they have used limited dose-ranges of NSAIDs and subcultures of tenocytes. We have previously argued that sub-culturing tenocytes selects for rapidly proliferating population of cells and is not necessarily representative of the situation found in vivo where the majority of cells are non-proliferative [10,11]. In contrast, primary cultures of tenocytes contain all of the cells originally present in the tendon, both differentiated and undifferentiated, and would therefore seem likely to be a more realistic model of tendon metabolism. We have therefore investigated the effects of NSAIDs on both primary tenocytes and secondary and tertiary subcultures of the cells.

Methods
Isolation and culture of tendon derived cells: Tendon derived cells (TDC) were obtained from the tail tendons of 200 g male Wistar rats. Rat-tail tendons were chosen because they can be obtained in sufficient quantities to allow the extensive use of primary cells. Although they are not completely relevant to human pathologies they show similar age-related changes in their biomechanical properties to other tendons and in this laboratory rat tail TDC behave similarly to cells derived from other tendons; human and rat. The rats were maintained according to UK home office regulations and killed by a schedule 1 method. The tendons were dissected free from the tails and the TDC freed from the tendons by digesting for 18 h at 37°C in 1 mg/ml crude collagenase in culture medium.
After digestion the cells were washed, resuspended and viable cells determined.
The cells were then used immediately in primary highdensity cultures or plated out for secondary cultures.
Primary or secondary cells were plated out in 24 well plates at a density of 10,000 cells per well in DMEM containing 10% FCS, penicillin/streptomycin and glutamine. The cells were treated with indomethacin (0.1 nm -100 uM) for 6 days. The cultures were then stopped and cell number determined by either Alamar blue assay, methylene blue assay or by direct counting using a Guava PCS.

Alamar blue assay
At the end of the culture period 50 μL of Alamar blue was added to the cultures, which were then incubated at 37°C for further four hours. Cell number was then determined by analysing the supernatants spectrophotometrically at 570 and 600 nm.

Methylene blue assay
The cells were fixed with cold ethanol and then washed with borate buffer (pH 8.8, 20 mM). Cells were then stained with methylene blue (1 mg/ml in borate buffer) for 30 minutes after which they were washed three times with borate buffer. The dye was then eluted with 1% HCl in ethanol and cell number determined by measuring the absorbance at 650 nm.

Guava PCS
The cells were diluted 1 in 10 in Guava Viacount reagent (containing 7-amino-actinomycin D) and cell number and viability determined using a guava personal cytometry system according to the manufacturer's instructions.
Data handling and statistical analyses: Data are presented as group mean ± standard deviation. At least 3 replicates of each experiment were performed, and the results presented in the figures are representative of these. For each variable, effects across treatment groups were compared with one-way analysis of variance (ANOVA). If the overall difference was significant, multiple comparisons were performed between groups using Tukey's test. Differences are considered significant at a probability <0.05 on a twotailed test.

Results
Initial experiments studying the effects of indomethacin on tendon derived cell proliferation were carried out using the commonly used Alamar blue assay. However, although on visual examination an effect was evident, no effect was seen after analysis of the cell supernatants We therefore adopted the methylene blue assay to determine tendon-derived cell numbers. Although this assay has the disadvantage of detecting both live and dead cells it is thoroughly reliable and accurate. Using this assay, it was found that treating primary cells with indomethacin lead to a dose related inhibition of cell proliferation. However, rather unexpectedly the relationship was negative with the greatest inhibition being seen at 10 nM and essentially no effect at 100 μM ( Figure 3). In secondary subcultures of tendon-derived cells, the cells became relatively refractive to treatment with indomethacin showing This graph shows the relationship between increasing Indomethacin concentration and cell number of primary ten-don derived cells as measured by Alamar Blue Figure 1 This graph shows the relationship between increasing Indomethacin concentration and cell number of primary tendon derived cells as measured by Alamar Blue. no significant effect at any concentration ( Figure 4) and then by the third subculture a biphasic stimulation of proliferation were seen with a maximum at 1-10 μM (data not shown).
Because of the somewhat unexpected inhibition of proliferation with low concentrations of Indomethacin, this work was repeated using direct counts of cell number and viability (as measured by 7-amino-actinomycin D uptake). Although the concentration relationship was not as linear as with the methylene blue assay, this too showed a significant decrease in cell number at lower concentrations of indomethacin whereas treatment with high concentrations had essentially no effect ( Figure 5). Also the results with secondary sub culture of tenocytes were similar to methylene blue with no effect of indomethacin on proliferation. We did not repeat this with tertiary subculture of tenocytes.

Figure 2 This graph shows the relationship between increasing Indomethacin concentration and cell number of secondary tendon derived cells as measured by Alamar Blue.
This graph shows the relationship between increasing Indomethacin concentration and cell number of primary ten-don derived cells as measured by Methylene Blue Figure 3 This graph shows the relationship between increasing Indomethacin concentration and cell number of primary tendon derived cells as measured by Methylene Blue.

Figure 4 This graph shows the relationship between increasing Indomethacin concentration and cell number of Secondary tendon derived cells as measured by Methylene Blue.
This graph shows the relationship between increasing Indomethacin concentration and cell number of primary ten-don derived cells as measured by direct cell counting meth-ods Figure 5 This graph shows the relationship between increasing Indomethacin concentration and cell number of primary tendon derived cells as measured by direct cell counting methods.
The lack of response when measuring the tenocytes cell number using Alamar Blue was somewhat unexpected as this is a well established method of determining cell number and is used with a wide variety of cell types. An experiment relating cell number and absorption revealed this to be a cell dependent problem as Methylene blue staining gave rise to a strong cell-dependent increase in staining whereas the response using Alamar blue was essentially flat with very little response ( Figure 6). In comparison, mesenchymal stem cells produce very similar curves regardless of whether they are stained with Methylene blue or Alamar blue (data not shown). To try and determine the cause of this we stained the cells with 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (commonly known as MTT) which is metabolized from yellow MTT to an insoluble purple formazan by a similar mechanism to Alamar blue. It was found that whereas Methylene blue stained all of the cells evenly throughout the cultures, it was evident that there were 2 populations of cells in the tenocyte cultures, one staining strongly with MTT and the other barely staining at all (Figure 7).

Discussion
Tendon injuries produce considerable morbidity, and the disability that they cause may last for several months despite what is considered appropriate management [12]. The basic cell biology of tendons is still not fully understood, and the management of tendon injury poses a considerable challenge for clinicians [12]. Even though histological studies have shown absence of acute inflammatory cells in chronic tendonitis, NSAIDs are used com-monly for managing tendon injuries [13][14][15][16]. Clinical studies indicate that they decrease the pain, tenderness and stiffness associated with acute soft tissue injury 3 (4). The effect of NSAIDs on tenocytes however, remains debatable. Studies with NSAIDs have shown both beneficial [7,17] and harmful effects on tenocytes [6,8]. As the tenocyte is the major cell type in tendons and is responsible for the production and maintenance of extra cellular matrix, we investigated the effect of Indomethacin, commonly used NSAIDs for tendon injuries on tenocyte proliferation. Of particular interest to our study was the effect of primary and sub culture of tenocytes and different cell counting methods for tenocytes.
Tenocyte culture provides a uniform, controlled environment in which to study the in vitro effects of Indomethacin, with limitation of other uncontrolled variables and avoidance of the confounding influences that are present in animal models [18]. The results of our in vitro experiments showed that Indomethacin had a dose related effect on tenocyte proliferation. Indomethacin reduced the number of human tenocyte cells at a dose between 0.1 nM to 100 nM. However at higher doses it did not inhibit tenocyte proliferation. This is in contrast to some studies showing inhibition of tenocytes with therapeutic doses of Indomethacin and hence producing a negative effect on healing tendons [6,8]. Other studies have shown beneficial effect on tenocyte healing by increasing the tensile strength to failure [5,7,17]. This is due to increased maturation of collagen fibrils during healing. However it is still debatable whether this beneficial effect is due to proliferation of tenocytes or increased production of collagen and maturation. Our study indicating that Indomethacin in therapeutic doses did not inhibit tenocyte proliferation has clinical relevance. This would be beneficial in tendon healing and repair as tenocyte is the major cell type in tendons and is responsible for the production and maintenance of extracellular matrix.
The negative dose response effect of Indomethacin on tendon derived cell proliferation remains a conundrum and we have no explanation to this. It should however be noted that serum levels of Indomethacin generally reach a peak of 1-10 μg/ml after administration and due to the poor blood supply in tendons, it is likely that tenocytes are exposed to concentrations even lower than this. These concentrations are well within the range at which the above effects were seen in vitro but did not go any way towards explaining the lack of effect at higher concentrations.
Dissimilar culture conditions as in vivo or in vitro, differing species and even changes in cell culture conditions of the same species have shown different tenocyte behaviour [5][6][7][17][18][19][20]. It can thus be stated that interaction between Comparison of absorbances of Alamar blue and methylene blue obtained after culture of rat tail tendon derived cells at varying concentrations for 2 and 7 days Figure 6 Comparison of absorbances of Alamar blue and methylene blue obtained after culture of rat tail tendon derived cells at varying concentrations for 2 and 7 days. Data points are mean of 3 wells. Results were significant between cell numbers seeded for both dyes at both time points using ANOVA.
tenocytes and NSAIDS is influenced by different factors, leading to opposing results. It has been previously argued that sub culture of mesenchymal cells do not reciprocate in vivo results [11]. However this effect has not been studied or documented before, with tenocytes. We considered this as an important area of investigation and looked at role of culture conditions on effect of Indomethacin on tenocytes. We noted that sub culture of cells did not show similar results as primary tenocytes. The dose dependent effect of Indomethacin was only seen on primary tenocytes and sub culture of cells did not show this effect. Previous studies have shown Indomethacin to cause both inhibition and proliferation of tenocytes in in vitro studies [6,10,14,17]. However these studies do not clearly indicate if only primary or subcultures of cells were used. Also it has not been mentioned previously if such effect was seen. We postulate that this effect seen in our study may be due to the selection of a highly proliferative population of tenocytes that is present in the primary digest only at low levels which subsequently overgrow the slower growing cells in later passages. We consider this an important finding, as studies, which have used sub culture of cells, need to be reviewed with some caution.
Various cell-counting methods are used to measure tenocyte proliferation. Initial experiments studying the effects of Indomethacin on tendon derived cell proliferation were carried out using the commonly used Alamar blue assay. However, although on visual examination an effect was evident, no effect was seen after analysis of the cell supernatants. This conundrum was subsequently explained by the finding that tendons appear to contain 2 subpopulations of cells; one subpopulation with apparently normal metabolic activity and a second subpopulation of cells with low levels of mitochondrial enzymes and subsequently a low oxidative metabolism. As the Alamar blue assay is dependent on mitochondrial enzyme activity for the reduction of the dye resazurin to resorufin, in cells without these enzymes, no effect was seen. This was confirmed by the use of MTT which is metabolized by the same enzymes to produce a purple product and it was clearly demonstrated that 2 subpopulations of cells exist; one staining intensely indicating a high metabolic activity and one stained relatively mildly indicating a low metabolic activity. This is entirely consistent current knowledge regarding the metabolic activity of tenocytes and tenoblasts. Tenoblasts are known to contain relatively high numbers of mitochondria and tenocytes very few suggesting comparatively high and low oxidative metabolism respectively [21]. This would in turn that the response seen in primary cells is due to the presence of mature tenocytes in the cultures which would have been lost on subsequent passage. As tenocytes are thought to be non-proliferative it is obviously unlikely that this is due to an inhibition of proliferation. One possibility is a toxic effect on specific to tenocytes but not tenoblasts however, we saw no evidence of excessive cell death in these cultures. Another possibility is that the tenocytes play some Figure 7 Staining of tendon derived cells with A, methylene blue or B, MTT. Monolayer cultures of rat tail tendon cells were grown to confluence, then stained with these substances as described in materials and methods. Following staining cultures were photographed. Staining with methylene blue produces even staining whereas staining with MTT produces patchy results with some cells staining strongly and others barely staining at all.

A.
B.
form of regulatory role controlling the proliferation of the proliferative tenoblasts as is thought to be the case for osteocytes and osteoblasts [22]. This must however remain speculative until further evidence is available.
One consequence of these data is that investigations using Alamar blue or MTT to assess tenocytes numbers should be interpreted with some caution. We therefore adopted the methylene blue assay to determine tendon-derived cell numbers. Although this assay has the disadvantage of detecting both live and dead cells it is thoroughly reliable and accurate. Using this assay, it was found that treating primary cells with indomethacin led to a dose related inhibition of cell proliferation. In secondary subcultures of tendon-derived cells, the cells became relatively refractive to treatment with indomethacin showing no significant effect at any concentration ( Figure 4). Because of the somewhat unexpected inhibition of proliferation with low concentrations of Indomethacin, this work was repeated using direct counts of cell number and viability (as measured by 7-amino-actinomycin D uptake). Although the concentration relationship was not as good as with the methylene blue assay, this too showed a significant decrease in cell number at lower concentrations of indomethacin whereas treatment with high concentrations had essentially no effect ( Figure 5).
The cause of this rather unexpected dose/effect relationship is at present unclear. Indomethacin is thought to act principally by the modulation of arachidonic acid metabolism via the cyclooxygenase and lipoxygenase pathways thus blocking the production of prostaglandins and HETEs/leukotrienes respectively. The relationship is however not simple as indomethacin has been shown to stimulate [23] and inhibit [24] the production of lipoxygenase metabolites. This differential effect is likely to be both tissue and concentration dependent and is further complicated by the possibility that blocking one pathway is likely to shunt metabolites down the other. No drugs are 100% specific for any one particular mechanism of action and indomethacin is no exception giving rise to a number of effects unrelated to cyclooxygenase [25]. Lastly, endocannabinoids are now known to have a number of peripheral effects on connective tissue [26] and preliminary investigations in this laboratory indicate that this is also the case in tendons and ligaments. In relation to this, indomethacin has been shown to inhibit the enzyme fatty acid amide hydrolase (FAAH), which metabolizes endocannabinoids [27]; inhibition of FAAH would therefore result in the build of levels of endocannabinoids. The situation is therefore highly complex and is unlikely to be unraveled until further insights into the relative roles of prostaglandins, leukotrienes and endocannabinoids in tendons are obtained.
It should be stressed that in this study we have only investigated the effects of indomethacin on cell proliferation and therefore no firm conclusions can be made regarding the effects of indomethacin on other metabolic processes.
There have been few studies on tendon cells monitoring both proliferation and collagen accumulation, however, we have found that collagen accumulation in tendon cells normally parallels cell number and that specific levels of collagen synthesis remain largely unchanged [28][29][30][31]. This would suggest that indomethacin would probably produce similar effects on matrix protein synthesis to those described above, although this obviously requires confirmation.

Conclusion
In conclusion, these data show that primary tenocytes respond to Indomethacin differently compared to secondary and tertiary subcultures. This may be due to the selection of a highly proliferative population of tenocytes that is present in the primary digest only at low levels which subsequently overgrows the slower growing cells in later passages. This would suggest that where possible in vitro investigations into tenocyte metabolism should preferentially be performed using primary cells and that results obtained using subcultures should be viewed with some caution.
Furthermore we have shown that because of their lower metabolic rate tendon derived cells cannot sufficiently metabolise Resazurin, the dye used in the Alamar blue assay. Because of this we would suggest that this essay is not appropriate studying proliferation in tendon derived cells and that an alternative, such as the methylene blue assay, should be used.


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Computer Science
Biology
Medicine

text:
Ensembl’s 10th year

Ensembl (http://www.ensembl.org) integrates genomic information for a comprehensive set of chordate genomes with a particular focus on resources for human, mouse, rat, zebrafish and other high-value sequenced genomes. We provide complete gene annotations for all supported species in addition to specific resources that target genome variation, function and evolution. Ensembl data is accessible in a variety of formats including via our genome browser, API and BioMart. This year marks the tenth anniversary of Ensembl and in that time the project has grown with advances in genome technology. As of release 56 (September 2009), Ensembl supports 51 species including marmoset, pig, zebra finch, lizard, gorilla and wallaby, which were added in the past year. Major additions and improvements to Ensembl since our previous report include the incorporation of the human GRCh37 assembly, enhanced visualisation and data-mining options for the Ensembl regulatory features and continued development of our software infrastructure.

INTRODUCTION
On 27 January 2000, the Ensembl project announced the completion of 'Ensembl Milestone 1', the first complete release of the project's data and web interface. The release included gene predictions and repeat annotations across all human DNA sequence available at that time (both finished and draft) and provided supporting evidence for the gene predictions including protein homology matches. All data was accessible though the Ensembl web site and via FTP download. In the email that announced Milestone 1, we noted that work on Ensembl was still in progress and promised significant improvements in the coming months.
Ten years later, Ensembl provides a much larger and more complete genomic information resource in support of dozens of genomes including gene sets, multi-species alignments, annotations of orthologous and paralogous genes, and extensive variation and regulatory information. All data is available through a variety of visual and programmatic interfaces including the Ensembl Genome Browser, the Perl API and BioMart. We also provide a complete copy of all data and code to be used freely by the community.
Ensembl works closely with a number of other fundamental bioinformatics projects that provide resources to the wider research community to ensure data consistency and increase resource interconnectedness. Some of these projects include the Genome Browser at UCSC (1), the databases and resources of the NCBI (2), the Rat Genome Database (RGD) (3) and VEGA (4).
In this article, we provide a general overview of the data available within Ensembl and highlight some of the features and developments that have been introduced since our last report (5). Ensembl is comprehensively updated approximately five times each year and details of the new and updated data in each release are always provided on the Ensembl news pages linked from http://www.ensembl.org. Additionally, we provide more immediate information on the Ensembl blog at http://ensembl.blogspot.com/ as well as through the low volume Ensembl announce mailing list, which is open to all. To subscribe to the list, send an email to majordomo@ebi.ac.uk with the text 'subscribe ensemblannounce' as the message body.

RESULTS
Over the past year, we introduced seven new species including the anole lizard (Anolis carolinensis), the first reptile in Ensembl. Other species included the two-toed sloth (Choloepus hoffmanni), white-tufted-ear marmoset (Callithrix jacchus), the pig (Sus scrofa), the Tammar wallaby (Macropus eugenii), the zebra finch (Taeniopygia guttata) and the Western lowland gorilla (Gorilla gorilla). Of these, the anole lizard, zebra finch, marmoset and pig were high coverage genome assemblies based on 4-6 times coverage from Sanger-style sequencing reads and gorilla was the first example of an assembly that combined traditional Sanger-style sequencing at low coverage with high-throughput short-read sequencing at high coverage. Ensembl now fully supports a total of 24 high-coverage chordate genomes and 23 low-coverage chordate genomes. The lamprey (Petromyzon marinus), another high-coverage chordate genome, is currently provided with preliminary support only. An additional three non-chordate species (Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster) are included to facilitate comparative analysis.

Gene annotation
In addition to the newly supported species listed above, for each of which we released a comprehensive gene set, we released new gene sets for a number of other species. In general new gene sets are released in conjunction with each new genome assembly. Our largest single effort over the past year has been in support of the GRCh37 human assembly, which was released by the Genome Reference Consortium in early 2009 (http://www.genomereference .org). This new build includes a long list of genomic regions that have been assessed for accuracy and updated where necessary. To support projects such as ENCODE and the 1000 Genomes, we will continue to provide complete resources for the NCBI36 human assembly in the form of an enhanced Ensembl archive site including BLAT/BLAST sequence search and other features not present in standard archive sites. The site, http://ncbi36.ensembl.org, will remain active until at least Summer 2010 when, depending on usage, we intend to provide support for the NCBI36 assembly only in the form of a typical Ensembl archive site.
With the new GRCh37 assembly, a larger fraction of Ensembl genes correspond to RefSeq (6) and UniProt (7) entries suggesting continuing convergence of all of these resources [ Figure 1 and compare with Figure 1 in Birney et al. (8)]. The improved convergence level is the result of at least three components: First, the genome assembly has improved. Second, the Ensembl gene build strategy has improved including the development of a combined Ensembl/Havana merged gene set (5), which increased the number of protein-coding transcripts. Third, the other resources (i.e. RefSeq and UniProtKB) have themselves independently improved their quality and internal consistency.
Ensembl, in partnership with NCBI, UCSC and the Havana project, continues to play an active role in the CCDS consortium (9). As of Ensembl release 56 (September 2009), 19 851 Ensembl translations match human CCDS consensus coding region structures exactly, and 17 679 Ensembl translations match mouse CCDS structures exactly. In last year's report, we described in detail the creation of the extensively supported human and mouse genes sets through the merging of the Ensembl and Havana gene sets. These efforts continue in the context of the GENCODE project and have culminated in the Ensembl release 56 geneset becoming the GENCODE geneset (release 3c).
Beyond human and mouse, we released a new gene set in support of the Zv8 assembly of the zebrafish genome, which incorporates many of the new methods applied in the human and mouse builds. For the rat genome, we released a completely updated gene set using the previous assembly to incorporate the significant additional supporting information that had become available since our previous gene set was created. A number of other species, including horse and cow, received relatively minor updates. Ensembl also incorporated the data formerly held in the Alternative Splicing and Transcript Diversity (ASTD) database as part of the planned decommissioning of this database and consolidation of genomic annotation data (10).

Functional genomics and regulatory information
We have continued development of the Ensembl Regulatory Build that has been briefly described in our previous reports (5,11). Over the past year we released two updates to the set of human Ensembl regulatory features and continued our focus on CD4+T-cells by incorporating additional histone modification data from Wang et al. (12). We also released the first version of the mouse regulatory build focused on embryonic stem (ES) cells based in part on data from Mikkelson et al. (13). Additionally, Ensembl regulatory features and their supporting data such as sites of DNase I hypersensitivity and selected histone modifications are now available via BioMart to facilitate efficient data mining of the regulatory features.
Finally, in Ensembl version 56 (September 2009), we launched a dedicated visualisation of the regulatory features in the form of a Regulation Tab at the top of the page (Figure 2). The view currently provides information about the supporting features that are used to automatically assign a preliminary regulatory function to genome regions. Our regulatory feature view will be an important area of focus over the next 12 months as we incorporate data being produced by the ENCODE project.

Ensembl software and code base
As mentioned above, the Ensembl code base is being reused within the Ensembl Genomes project, which seeks to extend the Ensembl infrastructure across the taxonomic space. A number of updates to the core Ensembl infrastructure were necessary to support specific needs of Ensembl Genomes that had not been previously required by Ensembl. For example, the Ensembl core databases now support multiple species within a single core database as well as provide preliminary support for alternative transcription initiation. Support for operons is planned for the near future. The Registry component of the Ensembl API, which allows users to automatically configure database connections and other behaviours of the API, was redesigned to support connections to multiple database servers in different physical locations.

Improved data mining and analysis resources
Ensembl calculates and provides a number of key results that are useful for data integration and analyses. One of the most widely used and important examples is the identification of external references (x-refs), which was completely re-factored this year. Ensembl's x-refs associate external database identifiers to Ensembl gene and transcript IDs and serve to enable data connections between Ensembl and biological databases such as UniProt, EMBL and RefSeq. Several x-ref assignment methods are used as described here. Direct x-refs are those where a straightforward mapping between the Ensembl ID and the external ID already exists, such as when the assignment is done by the external resource. Primary x-refs are assigned by sequence matching using Exonerate (14) between the Ensembl DNA or peptide sequences and those in the external resources. Dependent x-refs are inferred from primary x-refs where the source database references other identifiers. Finally, a class of defined priority x-refs allow for prioritisation of sources that may provide several references for the same external identifier.
We have redesigned the Ensembl Ontology database and API to make access to ontology data more consistent and straightforward. For example, Gene Ontology (GO) terms (15) and their relationships to each other are now stored in a more generic and hierarchical manner; this allows more flexible querying and the ability to perform transitive closures on GO terms which was not possible before. GO slims (http://www.geneontology.org/GO .slims.shtml) are now also supported.
The Ensembl BioMart provides access to most of our data resources in a way that facilitates the creation of complex database queries in a relatively simple manner (16). In addition to their availability through the Ensembl web site, the Ensembl BioMart is also available from the main BioMart Portal (17).

Variation
Ensembl's variation data resources continue to be dominated by data imported from dbSNP (2). Over the past year we integrated data from the 1000 Genomes Project that was incorporated into dbSNP 130 and created initial SNP sets for orangutan and zebra finch in conjunction with the release of the gene sets for these species. In the variation web display, all SNPs are now provided with phylogenetic context if they map to a region included in one of Ensembl's multiple alignments. The phylogenetic context includes ancestral sequence reconstructions from Ortheus (18), allowing users to look at the ancestral alleles in an evolutionary context.
By integrating data from the NHGRI curated catalogue of SNP-trait associations (19) in addition to data provided by the European Genome-phenome Archive (EGA), we have assigned annotations to over 1100 SNPs found to be associated with nearly 200 phenotypes and provided links to the published evidence. These annotations can be found on the corresponding variation page and are available through the general Ensembl search interface by searching with the phenotype name.

Web
In our last report we extensively described the fourth major design of the Ensembl web site, which was formally launched as a part of Ensembl Release 51 (November 2008) (5). With nearly 12 months of experience of the new site, we have made a number of comparably minor changes aimed at continual performance increases and reimplementation of some displays not included in the initial release of the new web code. Performance improvements included the implementation of nginx and memcached to improve server responsiveness. Visualisations reintroduced over the course of the year included multi-species comparison and alignment views that incorporate extensive contextual annotations such as genes, repeats and other features from each of the aligned species.
We have also completed major changes to the Ensembl drawing code, which allows tracks to be configured via entries in the relevant database instead of in separate static files. Finally, we have improved the ability for users to find the specific tracks that they want to display by incorporating a search box into the AJAX control panel that provides centralised page configuration.
In parallel with the new web design, we have implemented a more comprehensive monitoring of Ensembl's performance at numerous locations around the world. We have deployed a fully functioning Ensembl mirror site to a physical location in California. This site is available at http://uswest.ensembl.org and provides an up-to-date mirror site fully monitored and maintained by Ensembl. Our tests show that users in North America and the Pacific Rim will experience faster response times from the US mirror compared to our main site in the United Kingdom and we will automatically offer users from these areas the ability to use our US mirror site as their default Ensembl. Other public Ensembl mirror sites are maintained by the user community with support from the project. Those users who take advantage of our user accounts to share settings and save sessions across multiple computers may find that the main site continues to provide faster performance due to the necessity of maintaining a single database with settings and sessions.
To address the continual growth of the size of biological databases, we have begun testing full Ensembl installations in commercial cloud computing environments. Ensembl is also currently provided as one of the free Public Data Sets on Amazon Web Services that can be integrated into any cloud based application on AWS.

Comparative genomics resources
As the number of species increases within Ensembl, our comparative genomics resources become more valuable as information sources for highly-used genomes such as human, mouse and rat. They also serve as a way to connect all aspects of the project.
One of the biggest challenges this year has been the update of the pairwise and multiple alignments to support the release of the GRCh37 human assembly. We also updated the comprehensive 31-way multi-species alignment (MSA) to include all of the low coverage mammalian genome sequences and now provide BED files for human and mouse constrained elements as determined by alignments of placental mammals. The recently published Enredo-Pecan-Ortheus (EPO) pipeline is at the heart of Ensembl's MSA computations (18,20,21).
Ensembl GeneTrees are the result of a comprehensive analysis to predict phylogeny in vertebrates and have recently been described in detail (22). The latest improvements include the use of the meta-aligner M-Coffee (23) and incorporation of information about exon boundaries into the alignments. We now restrict our calculation of pairwise dN/dS values such that they are only calculated for high-coverage species pairs, as we found the results to be more accurate.
The current GreeTree pipeline is more robust to large gene clusters, which must be built into separate trees for computational reasons. We now annotate genes in separate trees that come from the same large cluster as distant within-species paralogues. We also annotate gene-split events (which may be real or artefactually due to an assembly problem) by analysing the protein multiple alignments: when two proteins of the same species do not overlap in the alignment, we label them the result of a gene-split event.
Visually, we added clade-specific colours to the GeneTree view to help with the interpretation of the trees. It is also possible to hide or collapse genes from pre-defined clades or from the low-coverage genomes.

Outreach and user support
Ensembl has an extensive commitment to user support, outreach and training. Provided courses include browser focused workshops introducing Ensembl to users who have never visited the site before; in depth meetings attended by developers who are building bioinformatics applications based on the Ensembl code base; and courses for clinical users interested in leveraging the Ensembl resources to help understand connections between genotype and phenotype. We also participate in regular training courses such as EBI Road Shows and Wellcome Trust Open Door Workshops that incorporate information from many of the resources developed and hosted on the Wellcome Trust Genome Campus. We aim to provide on-site training for as many of our users across the world as possible and have recently conducted trained events in Europe, North and South America, Asia, Africa and the Middle East.
We invite users interested in scheduling training to contact the Ensembl helpdesk at helpdesk@ensembl.org. For those users unable to attend a workshop in person, we are developing an extensive video library of tutorials. Our current selection is available though the Ensembl YouTube channel at http://www.youtube.com/user/ EnsemblHelpdesk.

Future directions
In last year's report, we described some of the ways that we are adapting Ensembl to the data generated by the current generation of high-throughput sequencing machines (5). We continued this theme in this report with the annotation of the first genome assembly created from combined traditional long read and next-generation short read technologies. Next year we expect to release gene sets on genome assemblies created entirely with next generation sequencing data. For a number of species, we also plan to create gene sets that incorporate short read transcriptomic data, which have shown considerable potential to increase the accuracy of our gene annotations in initial experiments using RNA-seq data from a number of zebrafish tissues.
A significant focus in the next year will be the display and annotation of variation data. Through our participation in the Locus Reference Genomic (LRG) consortium (http://www.lrg-sequence.org), we plan to incorporate summary data from Locus Specific Databases (LSDBs) at the level recommended by the community (24,25). We are also developing and testing new variation displays as part of the 1000 Genomes Project, which runs a browser based on the Ensembl code at http://browser.1000genomes.org. researchers, organisations and large-scale projects that have provided data to Ensembl prior to publication under the understandings of the Fort Lauderdale meeting discussing Community Resource Projects and the Toronto meeting on prepublication data sharing.

FUNDING
The Ensembl project receives primary funding from the Wellcome Trust. Additional funding is provided by the European Union, BBSRC, NHGRI, NIH-NIAID and EMBL. Funding for open access charge: Wellcome Trust.