The human tumour antigen PRAME (preferentially expressed antigen of melanoma) is frequently overexpressed in tumours. High PRAME levels correlate with poor clinical outcome of several cancers, but the mechanisms by which PRAME could be involved in tumourigenesis remain largely elusive. We applied protein-complex purification strategies and identified PRAME as a substrate recognition subunit of a Cullin2-based E3 ubiquitin ligase. PRAME can be recruited to DNA in vitro, and genome-wide chromatin immunoprecipitation experiments revealed that PRAME is specifically enriched at transcriptionally active promoters that are also bound by NFY and at enhancers. Our results are consistent with a role for the PRAME ubiquitin ligase complex in NFY-mediated transcriptional regulation.

Background: ATF6␣ recruits Mediator to activate endoplasmic reticulum stress response genes. Results: Mediator subunit MED25 contains an ATF6␣ binding site. Conclusion:The ATF6␣-MED25 interaction contributes to Mediator recruitment to genes. Significance: Learning how DNA binding transcription activators recruit coactivators to genes is important for understanding gene regulatory mechanisms.

Background: Transcription factor ATF6␣ is a master regulator of genes induced by endoplasmic reticulum stress. Results: ATF6␣ can recruit Mediator and histone acetyltransferase complexes to promoter DNA via interactions with overlapping sites in the activation domain of ATF6␣. Conclusion: ATF6␣ sequences essential for gene activation recruit Mediator and histone acetyltransferases. Significance: Learning how coregulators communicate with DNA binding transcription factors is important for understanding gene regulation.

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.2 is a unique extrachromosomal DNA network, found in the single mitochondrion of trypanosomatids. It consists, in the species Crithidia fasciculata, of ϳ5,000 duplex DNA minicircles of 2.5 kbp and ϳ50 maxicircles of 37 kbp that are interlocked topologically to form a DNA network. Two short sequences, the dodecameric universal minicircle sequence (UMS), GGGGTTGGTGTA, and the hexameric sequence ACGCCC, which were located at the minicircle's replication origin and implicated with its replication initiation, were conserved in all trypanosomatid species studied (recently reviewed in Refs.

Akinetes are spore-like non-motile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Here, we demonstrate variations in cellular ultrastructure during akinete formation concomitant with accumulation of cyanophycin; a copolymer of aspartate and arginine that forms storage granules. Cyanophycin accumulation is initiated in vegetative cells few days post-exposure to akinete inducing conditions. This early accumulated cyanophycin pool in vegetative cells disappears as a nearby cell differentiates to an akinete and stores large pool of cyanophycin. During the akinete maturation, the cyanophycin pool is further increased and comprise up to 2% of the akinete volume. The cellular pattern of photosynthetic activity during akinete formation was studied by a nano-metric scale secondary ion mass spectrometry (NanoSIMS) analysis in 13C-enriched cultures. Quantitative estimation of carbon assimilation in vegetative cells and akinetes (filament-attached and -free) indicates that vegetative cells maintain their basal activity while differentiating akinetes gradually reduce their activity. Mature-free akinetes practically lost their photosynthetic activity although small fraction of free akinetes were still photosynthetically active. Additional 13C pulse-chase experiments indicated rapid carbon turnover during akinete formation and de novo synthesis of cyanophycin in vegetative cells 4 days post-induction of akinete differentiation.