p300 and CREB binding protein can both activate and repress transcription. Here, we locate the CRD1 transcriptional repression domain between residues 1017 and 1029 of p300. This region contains two copies of the sequence psiKxE that are modified by the ubiquitin-like protein SUMO-1. Mutations that reduce SUMO modification increase p300-mediated transcriptional activity and expression of a SUMO-specific protease or catalytically inactive Ubc9 relieved repression, demonstrating that p300 repression was mediated by SUMO conjugation. SUMO-modified CRD1 domain bound HDAC6 in vitro, and p300 repression was relieved by histone deacetylase inhibition and siRNA-mediated ablation of HDAC6 expression. These results reveal a mechanism controlling p300 function and suggest that SUMO-dependent repression is mediated by recruitment of HDAC6.

The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21 WAF/CIP1 . Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.

Molybdenum-dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high-affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N-terminal His,-tagged derivative and characterised both with and without the Nterminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57-kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its a-helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 pM. DNA mobility-shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate K,, of 30 nM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum-dependent transcriptional repressor, DNase 1 footprinting experiments showed that the ModE-molybdenum complex binds to a single 31-bp region around the transcription start of the modABCD promoter. This region contains a 6-base palindromic sequence CGTTAT-N,,-ATAACG.

The expression of the moa locus, which encodes enzymes required for molybdopterin biosynthesis, is enhanced under anaerobiosis but repressed when the bacterium is able to synthesize active molybdenum cofactor. In addition, moa expression exhibits a strong requirement for molybdate. The molybdate enhancement of moa transcription is fully dependent upon the molybdate-binding protein, ModE, which also mediates molybdate repression of the mod operon encoding the high-affinity molybdate uptake system. Due to the repression of moa in molybdenum cofactor-sufficient strains, the positive molybdate regulation of moa is revealed only in strains unable to make the active cofactor. Transcription of moa is controlled at two sigma-70-type promoters immediately upstream of the moaA gene. Deletion mutations covering the region upstream of moaA have allowed each of the promoters to be studied in isolation. The distal promoter is the site of the anaerobic enhancement which is Fnr-dependent. The molybdate induction of moa is exerted at the proximal promoter. Molybdate-ModE binds adjacent to the −35 region of this promoter, acting as a direct positive regulator of moa. The molybdenum cofactor repression also appears to act at the proximal transcriptional start site, but the mechanism remains to be established. Tungstate in the growth medium affects moaexpression in two ways. Firstly, it can act as a functional molybdate analogue for the ModE-mediated regulation. Secondly, tungstate brings about the loss of the molybdenum cofactor repression ofmoa. It is proposed that the tungsten derivative of the molybdenum cofactor, which is known to be formed under such conditions, is ineffective in bringing about repression of moa. The complex control of moa is discussed in relation to the synthesis of molybdoenzymes in the bacterium.

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SNIP1 is a 396-amino acid nuclear protein shown to be an inhibitor of the TGF-␤ signal transduction pathway and to be important in suppressing transcriptional activation dependent on the co-activators CBP and p300. In this report we show that SNIP1 potently inhibits the activity of NF-B, which binds the C/H1 domain of CBP/ p300, but does not interfere with the activity of transcription factors such as p53, which bind to other domains of p300, or factors such as VP16, which are independent of these co-activators. Inhibition of NF-B activity is a function of the N-terminal domain of SNIP1 and involves competition of SNIP1 and the NF-B subunit, RelA/p65, for binding to p300, similar to the mechanism of inhibition of Smad signaling by SNIP1. Immunohistochemical staining shows that expression of SNIP1 is strictly regulated in development and that it colocalizes, in certain tissues, with nuclear staining for RelA/p65 and for p300, suggesting that they may regulate NF-B activity in vivo in a spatially and temporally controlled manner. These data led us to suggest that SNIP1 may be an inhibitor of multiple transcriptional pathways that require the C/H1 domain of CBP/p300.

The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1.75 and 2.1 Å, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N-and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation.

ingly important. The methods and pursuit of algae-based fuels also have applications for bioactive lipids and highvalue, lipid-based chemical precursors ( 4,5 ), including polyunsaturated fatty acids (PUFA), carotenes, antioxidants, vitamins, and pigments, which have applications in the pharmaceutical, nutraceutical, and food industries. In all such applications, the methods of analysis, access to desired composition, total yield, and effi ciency with which lipids can be converted to usable products will be the ultimate determinants of industrial success.Previous analysis of algae lipid composition using GC/MS and MALDI-TOF mass spectrometry (MS) has shown a broad variety of lipid classes: glycerolipids, such as monogalactosyl-diacylglycerol (MGDG) and digalactosyl-diacylglycerol (DGDG); phospholipids, such as phosphatidylcholine (PC); and neutral lipids (triacylglycerols) ( 6-9 ). Many reports have focused on the analysis of polar lipids, but TAG analysis and composition have become increasingly important for biofuel applications. TAG analysis of mixed samples remains a challenge because of its lower ionization efficiency relative to polar lipids (such as phospholipids) ( 10 ). Analysis of neutral lipids in microalgae often relies on lipophilic fl uorescence and colorimetric assays ( 11, 12 ); however, these assays do not provide compositional TAG characterization. Compositional analysis of microalgal TAG primarily involves the initial separation of extracts using either liquid chromatography or thin-layer chromatography followed by MS analysis or derivatization of extracts, such as transesterifi cation, followed by analysis of fatty acid methyl esters (FAME) by GC/MS ( 13-15 ). The direct analysis of algal triacylglycerol using pyrolysis GC/MS has been reported as a promising alternative, but the purchase of specialty equipment and use of higher temperature ranges may limit the broad utility of this method ( 16,17 ).Herein, we describe the compositional analysis of intact triacylglycerol of oleaginous marine algae using MALDI-TOF MS. MALDI-TOF provides a direct method Abstract We present a method for the determination of triacylglycerol (TAG) profi les of oleaginous saltwater microalgae relevant for the production of biofuels, bioactive lipids, and high-value lipid-based chemical precursors. We describe a technique to remove chlorophyll using quick, simple solid phase extraction (SPE) and directly compare the intact TAG composition of four microalgae species ( Phaeodactylum tricornutum , Nannochloropsis salina , Nannochloropsis oculata , and Microalgae have been identifi ed as an attractive source for the production of biofuels in addition to bioactive lipids and high-value, lipid-based chemical precursors based on their ability to produce 20% or more of their mass as oil. Due to the conversion of triacylglycerols (TAG) to fatty acid alkyl esters for use as biodiesel ( 1, 2 ), current research has focused on TAG production and analysis to make biofuels from microalgae a more economically feasible option ( 3 )....