This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Gene expression in Plasmodium parasites undergoes significant changes in each developmental stage, but the transcription factors (TFs) regulating these changes have not been identified. We report here a Plasmodium TF (AP2-O) that activates gene expression in ookinetes, the mosquito-invasive form, and has a DNA-binding domain structurally related to that of a plant TF, Apetala2 (AP2). AP2-O mRNA is pre-synthesized by intraerythrocytic female gametocytes and translated later during ookinete development in the mosquito. The Plasmodium TF activates a set of genes, including all genes reported to be required for midgut invasion, by binding to specific six-base sequences on the proximal promoter. These results indicate that AP2 family TFs have important roles in stage-specific gene regulation in Plasmodium parasites.

SummaryThe malarial sporozoite is the stage that infects the liver, and genes expressed in this stage are potential targets for vaccine development. Here, we demonstrate that specific gene expression in this stage is regulated by an AP2-related transcription factor, designated AP2-Sp (APETALA2 in sporozoites), that is expressed from the late oocyst to the salivary gland sporozoite. Disruption of the AP2-Sp gene did not affect parasite replication in the erythrocyte but resulted in loss of sporozoite formation. The electrophoretic mobility-shift assay showed that the DNAbinding domain of AP2-Sp recognizes specific eightbase sequences, beginning with TGCATG, which are present in the proximal promoter region of all known sporozoite-specific genes. Promoter assays demonstrated that these sequences act as cis-acting elements and are critical for the expression of sporozoite-specific genes with different expression profiles. In transgenic parasites that express endogenous AP2-O (APETALA2 in ookinetes), but whose AP2 domain had been swapped with that of AP2-Sp, several target genes of AP2-Sp were induced in the ookinete stage. These results indicate that AP2-Sp is a major transcription factor that regulates gene expression in the sporozoite stage.

Stage-specific transcription is a fundamental biological process in the life cycle of the Plasmodium parasite. Proteins containing the AP2 DNA-binding domain are responsible for stage-specific transcriptional regulation and belong to the only known family of transcription factors in Plasmodium parasites. Comprehensive identification of their target genes will advance our understanding of the molecular basis of stage-specific transcriptional regulation and stage-specific parasite development. AP2-O is an AP2 family transcription factor that is expressed in the mosquito midgut-invading stage, called the ookinete, and is essential for normal morphogenesis of this stage. In this study, we identified the genome-wide target genes of AP2-O by chromatin immunoprecipitation-sequencing and elucidate how this AP2 family transcription factor contributes to the formation of this motile stage. The analysis revealed that AP2-O binds specifically to the upstream genomic regions of more than 500 genes, suggesting that approximately 10% of the parasite genome is directly regulated by AP2-O. These genes are involved in distinct biological processes such as morphogenesis, locomotion, midgut penetration, protection against mosquito immunity and preparation for subsequent oocyst development. This direct and global regulation by AP2-O provides a model for gene regulation in Plasmodium parasites and may explain how these parasites manage to control their complex life cycle using a small number of sequence-specific AP2 transcription factors.

Gametocytes are nonreplicative sexual forms that mediate malaria transmission to a mosquito vector. They are generated from asexual blood-stage parasites that proliferate in the circulation. However, little is known about how this transition is genetically regulated. Here, we report that an Apetala2 (AP2) family transcription factor, AP2-G2, regulates this transition as a transcriptional repressor. Disruption of AP2-G2 in the rodent malaria parasite Plasmodium berghei did not prevent commitment to the sexual stage but did halt development before the appearance of sex-specific morphologies. ChIP-seq analysis revealed that AP2-G2 targeted ∼1,500 genes and recognized a five-base motif in their promoters. Most of these target genes are required for asexual proliferation of the parasites in the blood, suggesting that AP2-G2 blocks the program that precedes asexual replication to promote conversion to the sexual stage. Microarray analysis showed that the identified targets constituted ∼70% of the up-regulated genes in AP2-G2-depleted parasites, suggesting that AP2-G2 actually functions as a repressor in gametocytes. A promoter assay using a centromere plasmid demonstrated that the binding motif functions as a cis-acting negative regulatory element. These results suggest that global transcriptional repression, which occurs during the initial phase of gametocytogenesis, is an essential step in Plasmodium sexual development. malaria | sexual development | transcription factor | gametocytogenesis

SummaryThe liver stage is the first stage of the malaria parasite that replicates in the vertebrate host. However, little is known about the interplay between the parasite liver stage and its host cell, the hepatocyte. In this study, we identified an exported protein that has a critical role in parasite development in host hepatocytes. Expressed sequence tag analysis of Plasmodium berghei liverstage parasites indicated that transcripts encoding a protein with an N-terminal signal peptide, designated liver-specific protein 2 (LISP2), are highly expressed in this stage. Expression of LISP2 was first observed 24 h after infection and rapidly increased during the liver-stage schizogony. Immunofluorescent staining with anti-LSP2 antibodies showed that LISP2 was carried to the parasitophorous vacuole and subsequently transported to the cytoplasm and nucleus of host hepatocytes. Gene targeting experiments demonstrated that majority of the LISP2-mutant liver-stage parasites arrested their development during formation of merozoites. These results indicate that exported LISP2 is involved in parasite-host interactions required for the development of liver-stage parasites inside hepatocytes. This study demonstrated that midto-late liver-stage malarial parasites have a system for exporting proteins to the host cell as intraerythrocytic stages do and presumably to use the proteins to modify the host cell and improve the environment.

Liver-stage malaria parasites are a promising target for drugs and vaccines against malaria infection. However, little is currently known about gene regulation in this stage. In this study, we used the rodent malaria parasite Plasmodium berghei and showed that an AP2-family transcription factor, designated AP2-L, plays a critical role in the liver-stage development of the parasite. AP2-L-depleted parasites proliferated normally in blood and in mosquitoes. However, the ability of these parasites to infect the liver was approximately 10,000 times lower than that of wild-type parasites. In vitro assays showed that the sporozoites of these parasites invaded hepatocytes normally but that their development stopped in the middle of the liver schizont stage. Expression profiling using transgenic P. berghei showed that fluorescent protein-tagged AP2-L increased rapidly during the liver schizont stage but suddenly disappeared with the formation of the mature liver schizont. DNA microarray analysis showed that the expression of several genes, including those of parasitophorous vacuole membrane proteins, was significantly decreased in the early liver stage of AP2-L-depleted parasites. Investigation of the targets of this transcription factor should greatly promote the exploration of liver-stage antigens and the elucidation of the mechanisms of hepatocyte infection by malaria parasites.

Abstract. It has been postulated that protein filtered through glomeruli activates tubular epithelial cells, which secrete vasoactive and inflammatory substances including chemokines, leading to tubulointerstitial renal injury. The present study was designed to investigate the role of monocyte chemoattractant protein-1 (MCP-1) in this process and to evaluate the effectiveness of a kidney-targeted gene transfer technique using hydrodynamic pressure. Naked plasmid encoding 7ND (an MCP-1 antagonist) or a control plasmid was introduced into the left kidney of rats. Three days after gene transfer (day 0), intraperitoneal administration of bovine serum albumin (10 mg/g body wt per day) was started and continued for 14 or 21 d. RT-PCR showed that 7ND mRNA was expressed only in the gene-transfected kidney. Immunostaining showed that 7ND protein was localized in the interstitial cells. Macrophage infiltration was significantly reduced in the left kidney of rats treated with 7ND on days 14 and 21. In the right kidney, such effects were not observed. 7ND also attenuated tubular damage and decreased the number of apoptotic cells. Computer-assisted analysis revealed that the areas positively stained for ␣-smooth muscle actin (␣SMA), fibronectin-EDA, type I collagen, and collagen fibrils were significantly reduced in the 7ND-treated kidney on day 21. Furthermore, 7ND gene therapy significantly reduced MCP-1 and TGF-␤1 mRNA expression. These results demonstrate that MCP-1 plays an important role in the development of tubulointerstitial inflammation, tubular damage, and fibrosis induced by proteinuria. The fact that 7ND gene therapy had little effect on the contralateral kidney indicates that 7ND acted locally. This strategy may have a potential usefulness as a gene therapy against tubulointerstitial renal injury.Besides being a hallmark of glomerular disease, proteinuria has been shown to be an independent factor that induces and maintains renal damage. Cellular infiltration and fibrosis in the interstitium are common characteristics for virtually all progressive renal diseases with proteinuria. It has been postulated that protein filtered through glomeruli activates tubular epithelial cells, induces cellular infiltration into the interstitium, and subsequently causes interstitial fibrosis (1). Secretion of vasoactive mediators (e.g., endothelins), and chemokines, (e.g., monocyte chemoattractant protein-1 [MCP-1] and osteopontin) are induced in the tubules by protein-overload proteinuria (2,3). These substances are considered to promote inflammation and fibrosis of the interstitium, resulting in renal scarring (4,5). From among these mediators, we focused on MCP-1, a C-C chemokine with potent monocyte chemotactic and activating properties. It has been shown that MCP-1 is upregulated in various experimental and human renal diseases (6,7). MCP-1 expression is enhanced in tubular cells by bovine serum albumin (BSA) via a NF-B-dependent pathway (8). MCP-1 can also induce fibrosis through recruitment and activation of macrophages t...