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BBA - Gene Regulatory Mechanisms (v.1789, #3)

Editorial Board (pp. i).

Specific variants of general transcription factors regulate germ cell development in diverse organisms by Richard N. Freiman ⁎ (pp. 161-166).

Through the reductive divisions of meiosis, sexually reproducing organisms have gained the ability to produce specialized haploid cells called germ cells that fuse to establish the diploid genome of the resulting progeny. The totipotent nature of these germ cells is highlighted by their ability to provide a single fertilized egg cell with all the genetic information necessary to develop the complete repertoire of cell types of the future organism. Thus, the production of these germ cells must be tightly regulated to ensure the continued success of the germ line in future generations. One surprising germ cell development mechanism utilizes variation of the global transcriptional machinery, such as TFIID and TFIIA. Like histone variation, general transcription factor variation serves to produce gonadal-restricted or -enriched expression of selective transcriptional regulatory factors required for establishing and/or maintaining the germ line of diverse organisms. This strategy is observed among invertebrates and vertebrates, and perhaps plants, suggesting that a common theme in germ cell evolution is the diversification of selective promoter initiation factors to regulate critical gonadal-specific programs of gene expression required for sexual reproduction. This review discusses the identification and characterization of a subset of these specialized general transcription factors in diverse organisms that share a common goal of germ line regulation through transcriptional control at its most fundamental level.

Keywords: Transcription; TFIID; TFIIA; Oogenesis; Spermatogenesis; Meiosis

Sites of 18S rRNA contacting mRNA 3′ and 5′ of the P site codon in human ribosome: A cross-linking study with mRNAs carrying 4-thiouridines at specific positions by Konstantin Bulygin; Soria Baouz-Drahy; Dmitri Graifer; Alain Favre; Galina Karpova ⁎ (pp. 167-174).

Long synthetic mRNAs were used to study the positioning of the E site codon, the 2nd and 3rd nucleotides of the A site bound codon and a nucleotide 3′ of this codon with respect to the 18S rRNA in the human 80S ribosome. The mRNAs contained a GAC triplet coding for Asp and a single 4-thiouridine residue (s⁴U) upstream or downstream of the GAC codon. In the presence of tRNA^Asp, the GAC codon of the mRNAs was targeted to the ribosomal P site thus placing s⁴U in one of the following positions −3, −2, −1, +5, +6 or +7 with respect to the first nucleotide of the P site bound codon. It was found that mRNAs that bore s⁴U in positions +5 to +7 cross-linked to the 18S rRNA nucleotides C1696, C1698 and 1820–1825, the distribution of cross-links among these targets depending on the position of s⁴U. Cross-links of mRNAs containing s⁴U in positions −3 to −1 were found in the region 1699–1704 of the 18S rRNA. In the absence of tRNA, all mRNAs cross-linked only to C1696 and C1698. Absence of the cross-linked nucleotides C1696 and C1698 in the case of mRNAs containing s⁴U in positions −3 to −1 confirmed that tRNA^Asp actually phased the mRNA on the ribosome.

Keywords: 80S ribosome; mRNA binding site; 4-thiouridine photocross-linking; 18S rRNA

Transcriptional control of occludin expression in vascular endothelia: Regulation by Sp3 and YY1 by Hadassah Sade; Karen Holloway; Ignacio A. Romero; David Male ⁎ (pp. 175-184).

Endothelium differentiates in response to tissue-specific signals; brain endothelium expresses tight junctions and transporters which are absent from other endothelia. The promoter of the tight junction protein occludin exhibited strong activity in a brain endothelial cell line, hCMEC/D3 but was inactive in lung endothelial cells. Expression of occludin in brain endothelium corresponded with binding of Sp3 to a minimal promoter segment close to the transcription-start site. However, in lung endothelium Sp-transcription factors did not bind to this site although they are present in the cell nucleus. In contrast, repression of occludin in lung endothelium was associated with the binding of YY1 to a remote site in the promoter region, which was functionally inactive in brain endothelium. The work identified a group of transcription factors including Sp3 and YY1, which differentially interact with the occludin promoter to induce expression of occludin in brain endothelium and repression in other endothelia. The mechanism controlling occludin expression is similar to that which controls tissue-specific expression of the transferrin receptor in brain endothelium, leading to a scheme for endothelial differentiation, in which activation or repression of tissue-specific proteins is maintained by a set of transcription factors which include Sp3 and YY1.

Keywords: Abbreviations; BMEC; brain microvascular endothelial cells; ChIP; Chromatin immunoprecipitation; EMSA; Electrophoretic mobility shift assay; hCMEC/D3; human cerebral microvascular endothelial cell line — D3; LMVEC; Lung microvascular endothelial cells; TFR; transferrin receptorEndothelium; Occludin; Tight junction; Differentiation; YY1; Sp3

T box transcription antitermination riboswitch: Influence of nucleotide sequence and orientation on tRNA binding by the antiterminator element by Hamid Fauzi; Akwasi Agyeman; Jennifer V. Hines ⁎ (pp. 185-191).

Many bacteria utilize riboswitch transcription regulation to monitor and appropriately respond to cellular levels of important metabolites or effector molecules. The T box transcription antitermination riboswitch responds to cognate uncharged tRNA by specifically stabilizing an antiterminator element in the 5′-untranslated mRNA leader region and precluding formation of a thermodynamically more stable terminator element. Stabilization occurs when the tRNA acceptor end base pairs with the first four nucleotides in the seven nucleotide bulge of the highly conserved antiterminator element. The significance of the conservation of the antiterminator bulge nucleotides that do not base pair with the tRNA is unknown, but they are required for optimal function. In vitro selection was used to determine if the isolated antiterminator bulge context alone dictates the mode in which the tRNA acceptor end binds the bulge nucleotides. No sequence conservation beyond complementarity was observed and the location was not constrained to the first four bases of the bulge. The results indicate that formation of a structure that recognizes the tRNA acceptor end in isolation is not the determinant driving force for the high phylogenetic sequence conservation observed within the antiterminator bulge. Additional factors or T box leader features more likely influenced the phylogenetic sequence conservation.

Keywords: T box; Transcription antitermination; In vitro selection; Binding; Riboswitch; RNA

Characterization of functional heterodimer partners in brain for a bHLH-PAS factor NXF by Norihisa Ooe; Koichi Saito; Hideo Kaneko (pp. 192-197).

NXF, a brain-specific bHLH-PAS transcription factor, can regulate the transcription of target genes forming heterodimer complexes, along with several other bHLH-PAS family members (Arnt1, Arnt2, BMAL1) in vitro. To characterize its dimerization partner protein(s) in vivo, we performed a co-immunoprecipitation analysis of whole brain extracts using anti-NXF IgG. In the protein fraction co-precipitating with the NXF protein, in addition to the major precipitate of Arnt2 protein, a faint protein band of Arnt1 protein was consistently observed. The following in vitro co-precipitation analysis with recombinant proteins and yeast-two-hybrid analysis confirmed the specific physical associations. Reporter gene analyses further revealed comparable levels of transcriptional activity with Arnt1:NXF and Arnt2:NXF combinations. mRNA expression for Arnt1 was found in several NXF-containing regions in brain, even an example with no Arnt2 expression. The data thus suggest that Arnt1, as well as Arnt2, could have the separate significance for NXF signaling, with Arnt1:NXF heterodimer complexes in vivo.

Keywords: NXF; bHLH-PAS; Arnt; Arnt1; Transcription factor; Heterodimer

Differential CpG island methylation of murine adenine nucleotide translocase genes by Jeffrey V. Brower; Chae Ho Lim; Chul Han; Katherine E. Hankowski; Takashi Hamazaki; Naohiro Terada ⁎ (pp. 198-203).

Adenine nucleotide translocase (Ant) mediates the exchange of ADP and ATP across the inner mitochondrial membrane in eukaryotes. Mice possess three distinct but highly homologous Ant isoforms, encoded by independent genes, whose transcription depends upon tissue type . Ant1 is expressed selectively in heart and skeletal muscles, Ant2 is ubiquitously expressed in most tissues but lower in skeletal muscle and testis, while Ant4 is exclusively expressed in the testis. Of interest, each of these Ant genes contains CpG islands in their proximal promoter regions. We investigated the methylation status of the three Ant genes in various tissues with active and inactive transcription. In contrast to the Ant4 gene in which CpG island methylation is essential for gene repression, the CpG islands of Ant1 and Ant2 are hypomethylated regardless of the gene expression status throughout the tissues of male mice. Despite the tissue specific expression profile of Ant1, CpG methylation is unlikely involved in the regulation of the gene. Consistent with these findings, addition of a CpG-demethylating agent, 5-aza-2′-deoxycitidine, to fibroblasts increased the expression of Ant4 but not Ant1 or Ant2 genes. This study provides insight regarding the differential regulation of Ant isoforms in mammals, whereby both the Ant1 and Ant2 genes are capable of expression, but the Ant4 gene is completely repressed throughout somatic tissues. To the best of our knowledge, this is a first example to clearly demonstrate a differential usage of CpG island methylation within a family of genes.

Keywords: Adenine nucleotide translocase; ATP; Gene expression; Transcription; CpG island; DNA methylation

Sp1 and Sp3 regulate transcription of the cyclin-dependent kinase 5 regulatory subunit 2 (p39) promoter in neuronal cells by Alvaro Valin; Julie D. Cook; Sarah Ross; Christi L. Saklad; Grace Gill (pp. 204-211).

Cyclin-dependent kinase 5 (cdk5) activity is critical for development and function of the nervous system. Cdk5 activity is dependent on association with the regulators p35 and p39 whose expression is highly regulated in the developing nervous system. We have identified a small 200 bp fragment of the p39 promoter that is sufficient for cell type-specific expression in neuronal cells. Mutational analysis revealed that a cluster of predicted binding sites for Sp1, AP-1/CREB/ATF and E box-binding transcription factors is essential for full activity of the p39 promoter. Electrophoretic mobility shift assays revealed that Sp1 and Sp3 bound to sequences required for p39 promoter function and chromatin immunoprecipitation assays confirmed binding of these proteins to the endogenous p39 promoter. Furthermore, depletion of either Sp1 or Sp3 by siRNA reduced expression from the p39 promoter. Our data suggest that the ubiquitously expressed transcription factors Sp1 and Sp3 regulate transcription of the cdk5 regulator p39 in neuronal cells, possibly in cooperation with tissue-specific transcription factors.

Keywords: p39; cdk5; GC box; E box; Transcription factor; Promoter; Sp1; Sp3; Neuron

Dynamics of backtracking long pauses of RNA polymerase by Ping Xie (pp. 212-219).

The dynamics of backtracking long pauses by RNA polymerase is quantitatively studied based on our proposed model. Analytical formulas are obtained for the dependence of mean backtracking time and mean lifetime of backtracking long pauses on the binding affinity V₀ of RNAP to the DNA and RNA lattices, an important parameter for the transcription elongation complex. By comparison of the theoretically with experimentally obtained mean backtracking times, the value of binding affinity V₀ is predicted. Using the predicted V₀, the effects of external load on the mean backtracking time, on the lifetime of the backtracking long pauses and on the exit from the backtracking long pauses are studied. The results are in agreement with the available experimental data and, moreover, some predicted results are presented. In addition, using this V₀ we study the processivity of RNA polymerase under no and sideways forces.

Keywords: Transcription elongation; Sequence-dependent; Molecular motor

Phosphorylated H3S10 occurs in distinct regions of the nucleolus in differentiated leaf cells by Gila Granot; Noga Sikron-Persi; Yan Li; Gideon Grafi ⁎ (pp. 220-224).

Serine 10 phosphorylation of histone H3 (H3S10ph) has long been considered a mitotic marker, which is often associated with chromosome condensation both in plants and animals. Yet, in animal cells, H3S10ph was found associated with transcriptional activation of genes. Here we extend this view to plant cells showing that H3S10ph not only occurs in dividing cells during mitosis, but also in differentiated mesophyll cells. In these cells H3S10ph displayed a peculiar localization within the nucleolus where it was restricted to specific domains reminiscent of fibrillar centers. Chromatin immunoprecipitation analysis showed that H3S10ph is associated with ribosomal DNAs. Thus, in plants H3S10ph appears to be associated with two structurally differing nuclear sites engaged in gene silencing (mitotic centromeres) and in gene transcription (nucleolus).

Keywords: Histone modification; H3S10 phosphorylation; Nucleolus; Chromatin; Ribosomal RNA; Nicotiana tabacum

Involvement of Oct-1 in the regulation of CDKN1A in response to clinically relevant doses of ionizing radiation by Mitsuru Nenoi; Kazuhiro Daino; Tetsuo Nakajima; Bing Wang; Keiko Taki; Ayana Kakimoto (pp. 225-231).

CDKN1A is a cyclin-dependent kinase inhibitor that plays a critical role in cell cycle checkpoint regulation. It is transcriptionally induced by TP53 (p53) following exposure to ionizing radiation (IR). Induction of CDKN1A after irradiation is closely related to IR-sensitivity of tumor cells, but the underlying mechanisms remain obscure because conventional reporter gene systems respond poorly to IR unless hyperlethal doses are used. Here, we performed a promoter analysis of the CDKN1A gene following irradiation with clinically relevant doses of IR using the adeno-associated virus-mediated reporter system which we have recently shown to be highly responsive to IR. We demonstrate that there are regulatory elements at −1.1 kb, −1.4 kb, and −1.8 kb, and deletion of these elements attenuate induction of the CDKN1A gene promoter in response to 0.2–2.0 Gy of IR. EMSA and ChIP assays showed that Oct-1 binds constitutively to the elements at −1.1 kb and −1.8 kb. Functional involvement of Oct-1 was confirmed by RNA interference targeting the Oct-1 gene, which suppressed both the basal and IR-inducible components of the CDKN1A expression. Thus, our results reveal that Oct-1 is crucial to the TP53-mediated regulation of the CDKN1A gene promoter following exposure to clinically relevant doses of IR.

Keywords: CDKN1A/p21/WAF1/CIP1; Ionizing radiation; Oct-1; TP53; Adeno-associated virus vector

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors by Yi Cai; Allen Laughon (pp. 232-245).

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor β ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. Here we show that this repression domain functions through interaction with two corepressors, dCtBP and dSin3A, and that either interaction is sufficient for repression. We also report that Schnurri contains additional repression domains that function through interaction with dCtBP, Groucho, dSin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype we provide evidence that these diverse repression domains are both cooperative and partially redundant. In addition we find that Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor.

Keywords: Schnurri; Corepressor; CtBP; Sin3A; Groucho; SMRTER

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BBA - Gene Regulatory Mechanisms (v.1789, #3)