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BBA - Gene Regulatory Mechanisms (v.1779, #5)

Editorial Board (pp. ii).

Transcriptional regulation of human small nuclear RNA genes by Gauri W. Jawdekar; R. William Henry (pp. 295-305).

The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated.

Keywords: Human snRNA gene; Transcription; Retinoblastoma protein; p53 tumor suppressor; RNA polymerase II; RNA polymerase III

The expression patterns of genes involved in the RNAi pathways are tissue-dependent and differ in the germ and somatic cells of mouse testis by Emilio González-González; Pedro P. López-Casas; Jesús del Mazo (pp. 306-311).

Different RNA interference (RNAi) components participate in post-transcriptional regulation via RNA silencing. The expression pattern of the genes Drosha and Dicer and the members of the Argonaute family Ago1, Ago2, Ago3 and Ago4, all elements participating in the RNAi pathways, were investigated in mouse somatic tissues and testis using quantitative RT-PCR. Expression patterns of different testis cells and those emerging during testis development were also investigated. The differential patterns of expression seen suggest potential pleiotropic roles for certain components of the RNAi machinery. Both spermatocytes and spermatids showed a defined gene expression pattern. The strong expression of Ago4 in germ cells suggests that this protein plays a key role in germ-cell differentiation in the seminiferous epithelium.

Keywords: RNAi; Drosha; Dicer; Argonaute; Spermatogenesis; Germ cell; Sertoli cell

Activation properties of GAGA transcription factor by Alejandro Vaquero; Marta Blanch; Espinas M. Lluïsa Espinás; Bernues Jordi Bernués (pp. 312-317).

GAGA is a Drosophila transcription factor that has been involved in many nuclear activities. We present evidence that GAGA factor enhances transcription by stabilizing pre-initiation complex (PIC) and promoting reinitiation. Formation of PIC prior to GAGA addition prevents activation suggesting that GAGA is required early in the formation of activated complexes. GAGA stimulation of transcription can be attributed in part to a stabilization of PIC. All these properties depend on the GAGA C-terminal glutamine-rich domain and, in addition to other roles and together with previous data, support a role of GAGA as a transcription factor.

Keywords: GAGA factor; Mechanism of activation; TATA-box mutant; Drosophila; Glutamine-rich domain

Transcriptional regulation of the 5′-flanking region of the human transcription factor Sp3 gene by NF-1, c-Myb, B-Myb, AP-1 and E2F by Alicia Tapias; Carlos J. Ciudad; Noe Véronique Noé (pp. 318-329).

We analyzed in detail the proximal promoter of transcription factor Sp3, which expands 281 bp from the translational start. This sequence contains putative binding sites for Sp1, NF-Y, NF-1, Myb, AP-1 and E2F transcription factors. In this work, we further explored the role of these boxes on the regulation of the Sp3 gene. Gel-shift and competition assays showed specific binding of NF-1, Myb, AP-1 and E2F. Furthermore, chromatin immunoprecipitation assays demonstrated that Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb, c-Jun and E2F1 actually occupied the Sp3 promoter in HeLa cells. Transient transfections and luciferase assays revealed activation of the Sp3 proximal promoter upon overexpression of NF-1, c-Myb, B-Myb, c-Jun and c-Fos, and repression after overexpression of E2F/DP1. Point mutation of the binding sites for NF1, Myb, AP1 and E2F and cell incubation with specific siRNAs further confirmed the role of these transcription factors in the regulation of the Sp3 promoter. The regulation of the endogenous Sp3 gene was also observed at the mRNA level when the studied transcription factors were overexpressed or knocked down by siRNA incubation. These results help to explain the complex regulation of the Sp3 gene, which depends, at least in part, on the relative amount of Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb, AP-1, and E2F proteins in the cell.

Keywords: Sp3 promoter; Sp1; Myb; AP-1; NF-1; NF-Y; E2F

A microarray approach for comparative expression profiling of the discrete maturation zones of mouse growth plate cartilage by Daniele Belluoccio; Bianca C. Bernardo; Lynn Rowley; John F. Bateman (pp. 330-340).

In vertebrates, longitudinal bone growth is the consequence of a complex series of events that take place in a specialized structure, the growth plate cartilage. Within the growth plate chondrocytes undergo a sequential maturation program from resting cells to proliferative, pre-hypertrophic, and ultimately hypertrophic end-stage chondrocytes. This process of chondrocyte maturation is under the control of the temporally and spatially regulated expression of a myriad of signaling molecules, transmembrane receptors, transcription factors, and structural extracellular matrix (ECM) proteins. One approach to the comprehensive definition of the key components of such complex interrelated pathways is the use of microarray expression profiling to catalogue transcriptome changes during chondrocyte maturation in the individual developmental zones of the mouse growth plate cartilage. However, this has not been achieved because of the difficulty in obtaining sufficient quantities of the individual growth plate cartilage zones to all microarray analysis. In this study we describe the development of microdissection methods for the isolation of tissue from the proliferative, pre-hypertrophic, and proliferative zone from one single mouse femur, RNA extraction and linear amplification of the RNA to allow interrogation of NIA 15k microarrays to generate comparative expression profiles. Verification of a subset of differentially expressed genes by RT-PCR and by in situ hybridization confirmed the reliability of this approach.

Keywords: Microarray; Microdissection; Growth plate cartilage; Endochondral bone; Gene expression

RNA interference-mediated inhibition of hepatocyte nuclear factor 1α identifies target genes by Joanne R. Evans; David L. Kelly; Kirsten J. Morris; Elsa M. Arvide; Ann Harris (pp. 341-346).

Hepatocyte nuclear factor 1α (HNF1α) is a homeodomain transcription factor that is central to co-ordinated differentiation of a number of cell lineages, including hepatocytes in the liver and islet cells in the pancreas. HNF1α interacts directly with other transcription factors and co-factors and is involved in chromatin modification to alter gene expression. To further investigate the pivotal role of HNF1α in transcriptional control pathways we utilized RNA interference. An siRNA oligonucleotide specific for HNF1α reduced HNF1α protein levels by up to 70% in transient transfections of Caco2 cells. The same sequence incorporated into an shRNAi reduced protein levels by up to 90% in stable transfections. Microarray analysis of RNA from cell lines with stable RNAi-mediated down-regulation of HNF1α, identified genes known to be regulated by this transcription factor and also novel genes.

Keywords: Hepatocyte nuclear factor α; RNA interference; Transcriptional network

Genomic structure, alternative splicing and expression of TG-interacting factor, in human myeloid leukemia blasts and cell lines by Rizwan Hamid; Johnequia Patterson; Stephen J. Brandt (pp. 347-355).

TG-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer, including leukemias. In this study, we provide the first detailed description of the TGIF locus characterizing 12 TGIF splice isoforms. These isoforms have similar open reading frames but different 5′ untranslated regions. TGIF expression data are presented from multiple tissues, cell lines and primary leukemia cells. Isoform-specific real-time PCR analysis showed that even though these isoforms were broadly expressed all except isoform 4, had very low level of expression. In fact, isoform 4 was the predominant TGIF isoform expressed in all tissues analyzed. Since TGIF, levels have recently implicated to play a role in acute myelogenous leukemia we proceeded to characterize the minimal promoter region of isoform 4 as a first step in understanding mechanisms of TGIF expression. As expected for homeobox genes, the minimal promoter region for isoform 4 has multiple Sp1 binding sites and a CpG island raising the possibility that the low TGIF expression seen in some AML patients and leukemia cell lines may be secondary to methylation. Further characterization of expression from this promoter using 5-Aza-2′-deoxycytidine treatment and transient expression assays showed that decreased TGIF expression is likely secondary to active repression and not because of promoter methylation. A detailed characterization of this complex locus is important as it may help to clarify the functions of this gene in brain development and leukemia biology.

Keywords: TGIF; Leukemia; AML; Splice isoforms; Expression; Acute myelogenous leukemia

Regulation of the human mitotic centromere-associated kinesin (MCAK) promoter by the transcription factors Sp1 and E2F1 by Do Youn Jun; Hae-Sun Park; Ji-Young Lee; Young Ho Kim (pp. 356-361).

To understand transcriptional regulation of the human mitotic centromere-associated kinesin (MCAK) promoter, the 1,151-bp promoter region of the human MCAK gene in Jurkat T cells was cloned by polymerase chain reaction (PCR). Although a bioinformatic analysis of the promoter sequence predicted several putative transcription factor binding sites for E2F, Sp1, c-Myb, p53, p300, NF-1, AML-1a, Ap-1, E-box factor, and C/EBPα/β with no consensus TATA-box motif, deletion constructs of the promoter region revealed that the core positive promoter activity resided at −266/−66, containing three GC-motifs for binding Sp1. Site-directed disruption and chromatin immunoprecipitation analysis indicated that Sp1-binding to the GC-motifs was crucial for promoter activation, but the E2F1-binding to the E2F-motif (−57/−50) was crucial for promoter repression. Cotransfection of the luciferase reporter with either Sp1- or E2F1-expression plasmid further verified the role of Sp1 as a transcriptional activator and E2F1 as a transcriptional repressor in the human MCAK promoter.

Keywords: Human; MCAK; promoter; Transcriptional regulation; Activator Sp1; Repressor E2F1; HEK-293 cells

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BBA - Gene Regulatory Mechanisms (v.1779, #5)