Crystal optimization and preliminary diffraction data analysis of the SCAN domain of Zfp206.

Zfp206 (also named Zscan10) is a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells. Zfp206 is a member of the SCAN-domain family of C(2)H(2) zinc-finger transcription factors. The SCAN domain is a highly conserved motif of 84 residues which mediates the self-association of and heterodimerization between SCAN-domain family transcription factors. The SCAN domain may therefore be the key to the selective oligomerization of and may combinatorially enhance the regulatory versatility of C(2)H(2) zinc fingers. This paper describes crystallization attempts with the SCAN domain of Zfp206 (Zfp206SCAN) and optimization strategies to obtain diffraction-quality crystals. The best diffracting crystal was grown in a solution consisting of 0.3 M ammonium sulfate, 0.1 M Tris-HCl pH 8.6, 25% PEG 3350, 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) using the hanging-drop vapour-diffusion technique. Optimized crystals diffracted to 1.85 Å resolution and belonged to space group I422, with unit-cell parameters a = 67.57, c = 87.54 Å. A Matthews analysis indicated the presence of one Zfp206SCAN molecule per asymmetric unit.

Non-Coding RNAs in Neural Networks, REST-Assured.

In the nervous system, several key steps in cellular complexity and development are regulated by non-coding RNAs (ncRNAs) and the repressor element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF). REST recruits gene regulatory complexes to regulatory sequences, among them the repressor element-1/neuron-restrictive silencer element, and mediates developmental stage-specific gene expression or repression, chromatin (re-)organization or silencing for protein-coding genes as well as for several ncRNAs like microRNAs, short interfering RNAs or long ncRNAs. NcRNAs are far from being just transcriptional noise and are involved in chromatin accessibility, transcription and post-transcriptional processing, trafficking, or RNA editing. REST and its cofactor CoREST are both highly regulated through various ncRNAs. The importance of the correct regulation within the ncRNA network, the ncRNAome, is demonstrated when it comes to a deregulation of REST and/or ncRNAs associated with molecular pathophysiology underlying diverse disorders including neurodegenerative diseases or brain tumors.

The transcription factor Zfp281 controls embryonic stem cell pluripotency by direct activation and repression of target genes.

Oct4, Sox2, and Nanog are key components of a core transcriptional regulatory network that controls the ability of embryonic stem cells to differentiate into all cell types. Here we show that Zfp281, a zinc finger transcription factor, is a key component of the network and that it is required to maintain pluripotency. Zfp281 was shown to directly activate Nanog expression by binding to a site in the promoter in very close proximity to the Oct4 and Sox2 binding sites. We present data showing that Zfp281 physically interacts with Oct4, Sox2, and Nanog. Chromatin immunoprecipitation experiments identified 2,417 genes that are direct targets for regulation by Zfp281, including several transcription factors that are known regulators of pluripotency, such as Oct4, Sox2, and Nanog. Gene expression microarray analysis indicated that some Zfp281 target genes were activated, whereas others were repressed, upon knockdown of Zfp281. The identification of both activation and repression domains within Zfp281 suggests that this transcription factor plays bifunctional roles in regulating gene expression within the network. Disclosure of potential conflicts of interest is found at the end of this article.

Zfp206 is a transcription factor that controls pluripotency of embryonic stem cells.

Zfp206 (ZNF206 in human) encodes a zinc finger- and SCAN domain-containing protein that is highly expressed in pluripotent ESC. Upon differentiation of human and mouse ESC, Zfp206 expression is quickly repressed. Zfp206 was found to be expressed throughout embryogenesis but absent in adult tissues except testis. We have identified a role for Zfp206 in controlling ESC differentiation. ESC engineered to overexpress Zfp206 were found to be resistant to differentiation induced by retinoic acid. In addition, ESC with knocked-down expression of Zfp206 were more sensitive to differentiation by retinoic acid treatment. We found that Zfp206 was able to enhance expression from its own promoter and also activate transcription of the Oct4 and Nanog promoters. Our results show that Zfp206 is an embryonic transcription factor that plays a role in regulating pluripotency of embryonic stem cells.

Crystal structure of THEP1 from the hyperthermophile Aquifex aeolicus: a variation of the RecA fold.

BACKGROUND: aaTHEP1, the gene product of aq_1292 from Aquifex aeolicus, shows sequence homology to proteins from most thermophiles, hyperthermophiles, and higher organisms such as man, mouse, and fly. In contrast, there are almost no homologous proteins in mesophilic unicellular microorganisms. aaTHEP1 is a thermophilic enzyme exhibiting both ATPase and GTPase activity in vitro. Although annotated as a nucleotide kinase, such an activity could not be confirmed for aaTHEP1 experimentally and the in vivo function of aaTHEP1 is still unknown. RESULTS: Here we report the crystal structure of selenomethionine substituted nucleotide-free aaTHEP1 at 1.4 A resolution using a multiple anomalous dispersion phasing protocol. The protein is composed of a single domain that belongs to the family of 3-layer (alpha/beta/alpha)-structures consisting of nine central strands flanked by six helices. The closest structural homologue as determined by DALI is the RecA family. In contrast to the latter proteins, aaTHEP1 possesses an extension of the beta-sheet consisting of four additional beta-strands. CONCLUSION: We conclude that the structure of aaTHEP1 represents a variation of the RecA fold. Although the catalytic function of aaTHEP1 remains unclear, structural details indicate that it does not belong to the group of GTPases, kinases or adenosyltransferases. A mainly positive electrostatic surface indicates that aaTHEP1 might be a DNA/RNA modifying enzyme. The resolved structure of aaTHEP1 can serve as paradigm for the complete THEP1 family.

Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase.

BACKGROUND: To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1). RESULTS: aaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80 degrees C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + Pi with kcat = 5 x 10(-3) s(-1) and Km = 5.5 x 10(-6) M. In addition, the enzyme exhibits GTPase activity (kcat = 9 x 10(-3) s(-1) and Km= 45 x 10(-6) M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally. CONCLUSION: Since aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases.

The telomerase-associated protein p43 is involved in anchoring telomerase in the nucleus.

Telomere replication of eukaryotic chromosomes is achieved by a specialized enzyme, the telomerase. Although the biochemistry of end-replication is well understood, little is known about the organization of the end-replication machinery, its regulation throughout the cell cycle or the biological function of the telomerase-associated proteins. Here we investigate the function of the telomerase-associated protein p43 within the macronucleus of the ciliated protozoa Euplotes. It has been shown that p43 binds in vitro to the RNA subunit of telomerase and shares homology with the La autoantigen family. It therefore has been suggested that it is involved in the assembly and/or nuclear retention of telomerase. We show that the p43-telomerase complex is bound to a subnuclear structure in vivo and is resistant to electroelution. Upon inhibition of p43 or telomerase expression by RNAi, which in this study was used for the first time in spirotrichs, this complex is no longer retained in the nucleus. Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.