Early Gene Cluster Research Articles

ZIC2 Is Essential for Maintenance of Latency and Is a Target of an Immediate Early Protein during Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivation.

ABSTRACTBivalent histone modifications are defined as repressive and activating epigenetic marks that simultaneously decorate the same genomic region. The H3K27me3 mark silences gene expression, while the H3K4me3 mark prevents the region from becoming permanently silenced and prepares the domain for activation when needed. Specific regions of Kaposi's sarcoma-associated herpesvirus (KSHV) latent episomes are poised to be activated by the KSHV replication and transcription activator (K-Rta). How KSHV episomes are prepared such that they maintain latent infection and switch to lytic replication by K-Rta remains unclear. K-Rta transactivation activity requires a protein degradation function; thus, we hypothesized that identification of cellular substrates of K-Rta may provide insight into the maintenance of KSHV latent infection and the switch to lytic replication. Here we show that a zinc finger protein, ZIC2, a key regulator for central nervous system development, is a substrate of K-Rta and is responsible for maintaining latency. K-Rta directly interacted with ZIC2 and functioned as an E3 ligase to ubiquitinate ZIC2. ZIC2 localized at immediate early and early gene cluster regions of the KSHV genome and contributed to tethering of polycomb repressive complex 2 through physical interaction, thus maintaining H3K27me3 marks at the K-Rta promoter. Accordingly, depletion of ZIC2 shifted the balance of bivalent histone modifications toward more active forms and induced KSHV reactivation in naturally infected cells. We suggest that ZIC2 turnover by K-Rta is a strategy employed by KSHV to favor the transition from latency to lytic replication.IMPORTANCE Posttranslational histone modifications regulate the accessibility of transcriptional factors to DNA; thus, they have profound effects on gene expression (e.g., viral reactivation). KSHV episomes are known to possess bivalent chromatin domains. How such KSHV chromatin domains are maintained to be reactivatable by K-Rta remains unclear. We found that ZIC2, a transcriptional factor essential for stem cell pluripotency, plays a role in maintaining KSHV latent infection in naturally infected cells. We found that ZIC2 degradation by K-Rta shifts bivalent histone marks to a more active configuration, leading to KSHV reactivation. ZIC2 interacts with and maintains polycomb repressor complex 2 at the K-Rta promoter. Our findings uncover (i) a mechanism utilized by KSHV to maintain latent infection, (ii) a latency-lytic cycle switch operated by K-Rta, and (iii) a molecular mechanism of ZIC2-mediated local histone modification.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

Characterization of an unusual bipolar helicase encoded by bacteriophage T5

Bacteriophage T5 has a 120 kb double-stranded linear DNA genome encoding most of the genes required for its own replication. This lytic bacteriophage has a burst size of ∼500 new phage particles per infected cell, demonstrating that it is able to turn each infected bacterium into a highly efficient DNA manufacturing machine. To begin to understand DNA replication in this prodigious bacteriophage, we have characterized a putative helicase encoded by gene D2. We show that bacteriophage T5 D2 protein is the first viral helicase to be described with bipolar DNA unwinding activities that require the same core catalytic residues for unwinding in either direction. However, unwinding of partially single- and double-stranded DNA test substrates in the 3′–5′ direction is more robust and can be distinguished from the 5′–3′ activity by a number of features including helicase complex stability, salt sensitivity and the length of single-stranded DNA overhang required for initiation of helicase action. The presence of D2 in an early gene cluster, the identification of a putative helix-turn-helix DNA-binding motif outside the helicase core and homology with known eukaryotic and prokaryotic replication initiators suggest an involvement for this unusual helicase in DNA replication initiation.

A module of negative feedback regulators defines growth factor signaling

Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

Genes and Regulatory Sites of the “Host-Takeover Module” in the Terminal Redundancy ofBacillus subtilisBacteriophage SPO1

Early in infection ofBacillus subtilisby bacteriophage SPO1, the synthesis of most host-specific macromolecules is replaced by the corresponding phage-specific biosyntheses. It is believed that this subversion of the host biosynthetic machinery is accomplished primarily by a cluster of early genes in the SPO1 terminal redundancy. Here we analyze the nucleotide sequence of this 11.5-kb “host-takover module,” which appears to be designed for particularly efficient expression. Promoters, ribosome-binding sites, and codon usage statistics all show characteristics known to be associated with efficient function inB. subtilis.The promoters and ribosome-binding sites have additional conserved features which are not characteristic of their host counterparts and which may be important for competition with host genes for the cellular biosynthetic machinery. The module includes 24 genes, tightly packed into 12 operons driven by the previously identified early promoters PE1 to PE12. The genes are smaller than average, with half of them having fewer than 100 codons. Most of their inferred products show little similarity to known proteins, although zinc finger, trans-membrane, and RNA polymerase-binding domains were identified. Transcription–termination and RNase III cleavage sites were found at appropriate locations.

Transcription map of the early region of the Streptomyces bacteriophage φC31

Streptomyces coelicolor A3(2), lysogenised by the temperature-sensitive c ts1 mutant of φC31, can be synchronously induced into the lytic cycle by heat treatment. A transcription map of 10 kb of the φC3l early gene cluster was deduced using low-resolution S1 nuclease mapping of RNA prepared 10 min after induction. At least nine early transcripts, early (e)RNAs 1-9, were localised reading exclusively rightwards with respect to the standard physical map of φC31. The mRNAs were extensively overlapping, frequently initiating at the same place but terminating at different sites, and vice versa. Gene expression during the lytic cycle was tightly regulated; no transcription was observed before induction. Transcription was maximal at 10 min post-induction, and at 20 min, eRNAs 5 and 6 persisted whilst eRNAs 7–9 were severely reduced or absent. The pattern of transcription of the early region is consistent with the simultaneous activation of a large number of promoters and differential termination efficiency.

Clusters of genes for the biosynthesis of antibiotics: regulatory genes and overproduction of pharmaceuticals.

In the last decade numerous genes involved in the biosynthesis of antibiotics, pigments, herbicides and other secondary metabolites have been cloned. The genes involved in the biosynthesis of penicillin, cephalosporin and cephamycins are organized in clusters as occurs also with the biosynthetic genes of other antibiotics and secondary metabolites (see review by Martín and Liras [65]). We have cloned genes involved in the biosynthesis of beta-lactam antibiotics from five different beta-lactam producing organisms both eucaryotic (Penicillium chrysogenum, Cephalosporium acremonium (syn. Acremonium chrysogenum) Aspergillus nidulans) and procaryotic (Nocardia lactamdurans, Streptomyces clavuligerus). In P. chrysogenum and A. nidulans the organization of the pcbAB, pcbC and penDE genes for ACV synthetase, IPN synthase and IPN acyltransferase showed a similar arrangement. In A. chrysogenum two different clusters of genes have been cloned. The cluster of early genes encodes ACV synthetase and IPN synthase, whereas the cluster of late genes encodes deacetoxycephalosporin C synthetase/hydroxylase and deacetylcephalosporin C acetyltransferase. In N. lactamdurans and S. clavuligerus a cluster of early cephamycin genes has been fully characterized. It includes the lat (for lysine-6-aminotransferase), pcbAB (for ACV synthase) and pcbC (for IPN synthase) genes. Pathway-specific regulatory genes which act in a positive (or negative) form are associated with clusters of genes involved in antibiotic biosynthesis. In addition, widely acting positive regulatory elements exert a pleiotropic control on secondary metabolism and differentiation of antibiotic producing microorganisms. The application of recombinant DNA techniques will contribute significantly to the improvement of fermentation organisms.

Organization of RNA transcripts from a vaccinia virus early gene cluster.

The detailed organization of the RNAs transcribed from an early gene cluster encoded by vaccinia virus has been determined from the information derived from several complementary techniques. These include hybrid selection coupled with cell-free translation to locate DNA sequences complementary to mRNAs encoding specific polypeptides; RNA filter hybridization to size and locate on the DNA mature RNAs as well as higher-molecular-weight RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; S1 nuclease mapping to precisely locate the 5' and 3' ends of the RNAs; and fractionation of hybrid-selected mRNAs in an agarose gel containing methyl mercury hydroxide followed by the cell-free translation of these mRNAs to definitively ascertain the size of the mRNA encoding each polypeptide. The early gene cluster is located between 21 and 26 kilobases from the left end of the vaccinia virus genome and is encoded by a 5.0-kilobase EcoRI fragment which spans the HindIII-N, -M, and -K fragments. Transcribed towards the left terminus are four mature mRNAs, 1,450, 950, 780, and 400 nucleotides in size, encoding polypeptides of 55, 30, 20, and 10 kilodaltons, respectively. These mRNAs are colinear with the DNA template and are closely spaced such that the 5' terminus of one mRNA is within 50 base pairs of the 3' terminus of the adjacent RNA. In addition to the mature size mRNAs, there are higher-molecular-weight RNAs, 5,000, 3,300, 2,350, 2,300, 1,800, 1,700, and 1,350 nucleotides in size. The 5' and 3' termini of the high-molecular-weight RNAs are coterminal with the 5' and 3' termini of the mature size mRNA. The implications of this arrangement and the biogenesis of these early mRNAs are discussed.

Transcriptional and translational analysis of a strongly expressed early region of the vaccinia virus genome.

A transcriptional and translational map was obtained for a 7-kilobase pair EcoRI fragment of vaccinia virus DNA containing 4% of the viral genome. This particular region contains a cluster of early genes which are transcribed in viral cores in vitro and in infected cells before uncoating of the viral DNA. Expression of this region was characterized by using vaccinia virus DNA sequences cloned in phage and plasmid vectors. Polypeptides encoded on the 7-kilobase pair fragment were identified by cell-free translation of viral mRNA selected by hybridization to immobilized DNA fragments. Early RNA programmed the synthesis of six proteins having apparent molecular weights of 63,000, 38,000, 37,000, 25,000, 15,000, and 13,000. The same result was obtained with RNA synthesized in vitro. A new species of 40 kilodaltons was synthesized in response to late RNA. Of the six "early" polypeptides, only the 13-kilodalton component was synthesized from late mRNA. RNA derived from the 7-kilobase pair region was analyzed by a variety of methods including hybridization blot, in vitro recapping, and S1 nuclease techniques. A surprisingly complex pattern of early transcription was found, indicating the existence of families of overlapping RNA species which share common 5'-proximal sequences. In addition, larger RNAs were identified spanning two or more adjacent genes. These RNAs appear to possess a common 5' terminus with transcripts derived from the first gene and are coterminal at the 3' end with RNAs from the downstream gene. Late RNA encoding the 40-kilodalton protein was shown to be heterogeneous in size. A single 5' terminus but no unique 3' terminus was identified for this class of transcripts. RNA species synthesized by cores in vitro were of similar size to authentic transcripts isolated from infected cells at early times.

Sequence homology in DNA molecules of temperate phages phi81, phi80 and lambda.

Homology maps between bacteriophages phi81, phi80 and lambda were constructed on the basis of electron microscopy observation of DNA heteroduplexes. In phi81/phi80 heteroduplex, the left half and the right terminal region of 13% the total molecular length were highly homologous, while the remaining region covering the early gene cluster was entirely nonhomologous. In phi81/lambda heteroduplex, high-degree homologies were detected at the left 14% terminal region covering the head gene cluster, the central 3.8% region covering the att-int-xis region and the 1.3% Q homology region. Low-degree homologies of shorter length were scattered at the tail gene cluster, b2 region, cIII region, PQ region and SR region. Comparing our results with the homology maps of other lambdoid phages reported by Simon et al. (1971) and Fiandt et al. (1971), a phylogenic relation of phi81 to other lambdoid phages and the role of recombination in the course of divergence of lambdoid phages are discussed.

Congruent transcriptional controls and heterology of base sequences in coliphages λ and ∅80

The reciprocal DNA-DNA and mRNA-DNA homologies were determined for two lambdoid phages, λ and ∅80, and for the hybrid λ-∅80 phages. There are no base sequence homologies between λ and ∅80 on the right arms and about a 20% homology on the left arms of the genomes, as determined by a technique based on buoyant density measurements on heteroduplex DNA molecules. The reciprocal mRNA-DNA homologies are observed only for the late mRNA's synthesized on the r strand of the left arm and amount to 5–11%. Even with such a low degree of base sequence homology, the timing, control, orientation, and regions of transcription are practically identical for both phages, when one allows for the lower rate of ∅80 development. In ∅80, as in λ (see Szybalski, 1969), transcription is restricted to the inner right quarter of strand l and to the c 80 I-proximal segment of the outer right quarter of the r strand (Fig. 1), during the early period of phage development. At later times, the terminal portion of the right arm and the whole left arm are transcribed from the r strand at a progressively increasing rate, whereas the transcription rate for the l strand does not change greatly. One may conclude that the observed congruency of transcriptional patterns need not be correlated with the gross base-sequence homologies. The contributions of the ∅80 and λ genomes in the hybrid phages were mapped (Fig. 1). The λi 80 hybrid appears to contain the left arm and the central b2 region of λ and the right arm of ∅80. The ∅80 i λ hybrid is composed mainly of ∅80 DNA, with only the central part of the right arm, containing the cluster of early genes, belonging to the λ genome.