Nuclear Respiratory Factor 1 Plays an Essential Role in Transcriptional Initiation from the Hepatitis B Virus X Gene Promoter

The X gene of hepatitis B virus (HBV) is one of the major factors in HBV-induced hepatocarcinogenesis and is essential for the establishment of productive HBV replication in vivo. Recent studies have shown that the X gene product targets mitochondria and induces calcium flux, thereby activating Ca(+)-dependent signal transduction pathways. However, regulatory mechanisms of X gene expression have remained unclear. Previous studies had localized a minimal promoter activity to a 21-bp GC-rich sequence located 130 bp upstream of the X protein coding region and showed that there was a cellular protein bound to this DNA. Interestingly, the 21-bp sequence identified as an X gene minimal promoter does not contain any previously identified core promoter elements, such as a TATA box. To better understand the mechanisms of transcriptional initiation of the X gene, we set out to biochemically purify the binding protein(s) for the 21-bp DNA. We report here the identification of the X gene minimal promoter-binding activity as nuclear respiratory factor 1 (NRF1), a previously known transcription factor that activates the majority of nucleus-encoded mitochondrial genes and various housekeeping genes. Primer extension analyses of the X mRNAs show that mutations at the binding site specifically inactivate transcription from this promoter and that a dominant-negative NRF1 mutant and short interfering RNAs inhibit transcription from this promoter. Therefore, NRF1 specifically binds the 21-bp minimal promoter and positively contributes to transcription of the X gene. Simultaneous activation of the X gene and mitochondrial genes by NRF1 may allow the X protein to target mitochondria most efficiently

Gene Regulatory Networks Controlling Hematopoietic Progenitor Niche Cell Production and Differentiation in the <em>Drosophila</em> Lymph Gland

<div><p>Hematopoiesis occurs in two phases in <em>Drosophila</em>, with the first completed during embryogenesis and the second accomplished during larval development. The lymph gland serves as the venue for the final hematopoietic program, with this larval tissue well-studied as to its cellular organization and genetic regulation. While the medullary zone contains stem-like hematopoietic progenitors, the posterior signaling center (PSC) functions as a niche microenvironment essential for controlling the decision between progenitor maintenance versus cellular differentiation. In this report, we utilize a PSC-specific GAL4 driver and UAS-gene RNAi strains, to selectively knockdown individual gene functions in PSC cells. We assessed the effect of abrogating the function of 820 genes as to their requirement for niche cell production and differentiation. 100 genes were shown to be essential for normal niche development, with various loci placed into sub-groups based on the functions of their encoded protein products and known genetic interactions. For members of three of these groups, we characterized loss- and gain-of-function phenotypes. Gene function knockdown of members of the BAP chromatin-remodeling complex resulted in niche cells that do not express the <em>hedgehog</em> (<em>hh</em>) gene and fail to differentiate filopodia believed important for Hh signaling from the niche to progenitors. Abrogating gene function of various members of the insulin-like growth factor and TOR signaling pathways resulted in anomalous PSC cell production, leading to a defective niche organization. Further analysis of the <em>Pten</em>, <em>TSC1</em>, and <em>TSC2</em> tumor suppressor genes demonstrated their loss-of-function condition resulted in severely altered blood cell homeostasis, including the abundant production of lamellocytes, specialized hemocytes involved in innate immune responses. Together, this cell-specific RNAi knockdown survey and mutant phenotype analyses identified multiple genes and their regulatory networks required for the normal organization and function of the hematopoietic progenitor niche within the lymph gland.</p> </div

Alteration of insulin-like growth factor signaling pathway gene functions.

<p>Expression of the PSC cell-specific markers <i>hhF4f-GFP</i> and Antp was assessed in lymph glands of the following loss- or gain-of-function genotypes: (A) wild-type, (B) <i>col>dAkt1</i> RNAi, (C) <i>col>dAkt1</i> cDNA, (D) <i>col>PDK1</i> RNAi, (E) <i>col>InR^DN</i> cDNA, (F) <i>col>InR</i> cDNA, (G) <i>col>Pi3K92E</i> RNAi, (H) <i>col>Pi3K92E^DN</i> cDNA, and (I) <i>col>Pi3K92E^CA</i> cDNA. Arrows in panels C, F, and I point out expanded populations of PSC niche cells, with an abnormal niche organization caused by PSC cell-specific expression of the <i>InR</i> cDNA. Arrowheads in panels B, D, E, G, and H point out reduced populations of PSC niche cells. Scale bar indicates 20 µm.</p

Nontransmissible Virus-Like Particle Formation by F-Deficient Sendai Virus Is Temperature Sensitive and Reduced by Mutations in M and HN Proteins

The formation of nontransmissible virus-like particles (NTVLP) by cells infected with F-deficient Sendai virus (SeV/ΔF) was found to be temperature sensitive. Analysis by hemagglutination assays and Western blotting demonstrated that the formation of NTVLP at 38°C was about 1/100 of that at 32°C, whereas this temperature-sensitive difference was only moderate in the case of F-possessing wild-type SeV. In order to reduce the NTVLP formation with the aim of improving SeV for use as a vector for gene therapy, amino acid substitutions found in temperature-sensitive mutant SeVs were introduced into the M (G69E, T116A, and A183S) and HN (A262T, G264R, and K461G) proteins of SeV/ΔF to generate SeV/M(ts)HN(ts)ΔF. The use of these mutations allows vector production at low temperature (32°C) and therapeutic use at body temperature (37°C) with diminished NTVLP formation. As expected, the formation of NTVLP by SeV/M(ts)HN(ts)ΔF at 37°C was decreased to about 1/10 of that by SeV/ΔF, whereas the suppression of NTVLP formation did not cause either enhanced cytotoxicity or reduced gene expression of the vector. The vectors showed differences with respect to the subcellular distribution of M protein in the infected cells. Clear and accumulated immunocytochemical signals of M protein on the cell surface were not observed in cells infected by SeV/ΔF at an incompatible temperature, 38°C, or in those infected by SeV/M(ts)HN(ts)ΔF at 37 or 38°C. The absence of F protein in SeV/ΔF and the additional mutations in M and HN in SeV/M(ts)HN(ts)ΔF probably weaken the ability to transport M protein to the plasma membrane, leading to the diminished formation of NTVLP

Quantification of hemolymph lamellocyte numbers.

<p>Quantification of hemolymph lamellocyte numbers.</p

Alteration of TOR signaling pathway gene functions.

<p>Expression of the PSC cell-specific markers <i>hhF4f-GFP</i> and Antp was assessed in lymph glands of the following loss-of-function genotypes: (A) <i>col>Tor</i> RNAi, (B) <i>col>raptor</i> RNAi, (C) <i>col>rictor</i> RNAi, (D) <i>col>TSC1</i> RNAi, (G) <i>col>TSC2</i> RNAi, (J) <i>S6K^L-1/S6K^L-1</i> (Antp only), and (K) <i>Thor⁰⁶²⁷⁰/Thor⁰⁶²⁷⁰</i>. Arrows in panels G and K point out expanded populations and abnormal organization of PSC niche cells. Arrowheads in panels A, B, C, and J point out severely reduced populations of PSC niche cells. (E) Expanded expression of the <i>hhF4f-GFP</i> transgene in <i>TSC1^f01910/TSC1^f01910</i> lymph glands. (F) Increase in plasmatocyte number (detected by P1 antibody) and supernumerary lamellocyte production (detected by <i>MSNF9mCherry</i> activity) in <i>TSC1^f01910/TSC1^f01910</i> lymph glands. (H) Expanded expression of the <i>hhF4f-GFP</i> transgene in <i>TSC2¹⁰⁹/TSC2¹⁰⁹</i> lymph glands. (I) Increase in plasmatocyte number and supernumerary lamellocyte production in <i>TSC2¹⁰⁹/TSC2¹⁰⁹</i> lymph glands. Scale bar indicates 20 µm.</p

Lymph gland domains, cell markers, and strategy for a PSC-specific gene function knockdown analysis.

<p>(A) Dissected dorsal vessel and associated lymph glands assayed for DAPI (DNA), Antp protein (PSC cells), and <i>eater-GFP</i> transgene activity (plasmatocytes). Abbreviations: CZ, cortical zone; PSC, posterior signaling center. (B) Enlargement of a primary lymph gland lobe stained for Su(H) protein expressed in hematopoietic progenitors of the medullary zone (MZ) and crystal cells of the cortical zone (CZ). Also highlighted are <i>hhF4f-GFP</i>-positive niche cells of the PSC. (C) Focus on a lymph gland PSC niche assayed for nuclear Antp protein and membrane-associated GFP due to expression of the <i>col>UAS-gapGFP</i> combination. The arrow points out a filopodia extending from a niche cell. (D) Strategy for the PSC-specific gene function knockdown analysis undertaken in this study.</p

Summary of findings of a PSC-specific gene function knockdown analysis of 820 <i>Drosophila</i> loci.

<p>Center: <i>hhF4f-GFP</i> transgene activity (hh in green circle) was the primary marker assessed in the RNAi-based gene function knockdown analysis, while expression of Antp protein (Antp in yellow circle) and filopodia formation based on <i>col>UAS-gapGFP</i> expression were monitored as secondary markers. Periphery: Grouping of genes (based on their encoded protein products), that when functionally altered by RNAi expression in PSC cells, resulted in abnormalities in <i>hh-GFP</i>, Antp, and/or <i>col>UAS-gapGFP</i> expression. Negative (blue circle) and positive (red circle) regulators are indicated. A negative regulator is defined as a gene whose loss-of-function condition leads to increased numbers of <i>hhF4f-GFP</i>-positive cells and/or enhanced transgene expression, while a positive regulator is defined as a gene whose loss-of-function condition leads to decreased numbers of <i>hhF4f-GFP</i>-positive cells and/or decreased transgene expression. Those loss-of-function conditions that resulted in lamellocyte induction (#) or absence of filopodia (*) are also indicated.</p

Alteration of <i>Pten</i> and <i>dFOXO</i> gene functions.

<p>Expression of the PSC cell-specific markers <i>hhF4f-GFP</i> and Antp was assessed in lymph glands of the following loss- or gain-of-function genotypes: (A) wild-type, (B) <i>Pten¹⁰⁰/Pten¹¹⁷</i>, (C) <i>P85col>Pten</i> cDNA, (G) <i>dFOXO²¹/dFOXO²⁵</i>, (H) <i>col>dFOXO</i> cDNA, and (I) <i>Pten¹¹⁷/+;dFOXO²¹/+</i>. Arrows in panels B, G, and I point out expanded populations and abnormal organization of niche cells. Arrowheads in panels C and H point out severely reduced populations of PSC niche cells. (D) Wild-type lymph gland assayed for DAPI (DNA), P1 antigen (plasmatocytes), and <i>dome-lacZ</i> expression (hematopoietic progenitors). (E) <i>Pten¹⁰⁰/Pten¹¹⁷</i> lymph gland assayed for DAPI, P1 antigen, and <i>dome-lacZ</i> expression. The arrow points out the substantial increase in plasmatocyte number. (F) <i>Pten¹⁰⁰/Pten¹¹⁷</i> lymph gland assayed for <i>MSNF9mCherry</i> transgene activity (lamellocytes). The arrow points out the de novo production of lamellocytes. Scale bar indicates 20 µm.</p