Poly-glycine-alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3.

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine-arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine-alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Target capture during Mos1 transposition.

DNA transposition contributes to genomic plasticity. Target capture is a key step in the transposition process, because it contributes to the selection of new insertion sites. Nothing or little is known about how eukaryotic mariner DNA transposons trigger this step. In the case of Mos1, biochemistry and crystallography have deciphered several inverted terminal repeat-transposase complexes that are intermediates during transposition. However, the target capture complex is still unknown. Here, we show that the preintegration complex (i.e., the excised transposon) is the only complex able to capture a target DNA. Mos1 transposase does not support target commitment, which has been proposed to explain Mos1 random genomic integrations within host genomes. We demonstrate that the TA dinucleotide used as the target is crucial both to target recognition and in the chemistry of the strand transfer reaction. Bent DNA molecules are better targets for the capture when the target DNA is nicked two nucleotides apart from the TA. They improve strand transfer when the target DNA contains a mismatch near the TA dinucleotide.

Polymorphic core promoter GA-repeats alter gene expression of the early embryonic developmental genes.

Protein complexes that bind to 'GAGA' DNA elements are necessary to replace nucleosomes to create a local chromatin environment that facilitates a variety of site-specific regulatory responses. Three to four elements are required for the disruption of a preassembled nucleosome. We have previously identified human protein-coding gene core promoters that are composed of exceptionally long GA-repeats. The functional implication of those GA-repeats is beginning to emerge in the core promoter of the human SOX5 gene, which is involved in multiple developmental processes. In the current study, we analyze the functional implication of GA-repeats in the core promoter of two additional genes, MECOM and GABRA3, whose expression is largely limited to embryogenesis. We report a significant difference in gene expression as a result of different alleles across those core promoters in the HEK-293 cell line. Across-species homology check for the GABRA3 GA-repeats revealed that those repeats are evolutionary conserved in mouse and primates (p<1 × 10(-8)). The MECOM core promoter GA-repeats are also conserved in numerous species, of which human has the longest repeat and complexity. We propose a novel role for GA-repeat core promoters to regulate gene expression in the genes involved in development and evolution.

Implication of CA repeated tracts on post-transcriptional regulation in Trypanosoma cruzi.

In Trypanosoma cruzi gene expression regulation mainly relays on post-transcriptional events. Nevertheless, little is known about the signals which control mRNA abundance and functionality. We have previously found that CA repeated tracts (polyCA) are abundant in the vicinity of open reading frames and constitute specific targets for single stranded binding proteins from T. cruzi epimastigote. Given the reported examples of the involvement of polyCA motifs in gene expression regulation, we decided to further study their role in T. cruzi. Using an in silico genome-wide analysis, we identify the genes that contain polyCA within their predicted UTRs. We found that about 10% of T. cruzi genes carry polyCA therein. Strikingly, they are frequently concurrent with GT repeated tracts (polyGT), favoring the formation of a secondary structure exhibiting the complementary polydinucleotides in a double stranded helix. This feature is found in the species-specific family of genes coding for mucine associated proteins (MASPs) and other genes. For those polyCA-containing UTRs that lack polyGT, the polyCA is mainly predicted to adopt a single stranded structure. We further analyzed the functional role of such element using a reporter approach in T. cruzi. We found out that the insertion of polyCA at the 3' UTR of a reporter gene in the pTEX vector modulates its expression along the parasite's life cycle. While no significant change of the mRNA steady state of the reporter gene could be detected at the trypomastigote stage, significant increase in the epimastigote and reduction in the amastigote stage were observed. Altogether, these results suggest the involvement of polyCA as a signal in gene expression regulation in T. cruzi.

A Type III restriction-modification system in Mycoplasma mycoides subsp. capri.

The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction-modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.MmyCI) was shown to recognize the sequence 5'-TGAG-3' and methylate the adenine. The coding region of the methyltransferase gene contains 12 consecutive AG dinucleotide repeats that result in a translational termination at a TAA codon immediately beyond the repeat region. This strain does not have MmyCI activity. A clone was found with 10 AG repeats such that the gene is in frame, and this strain has MmyCI activity, suggesting that the expression of the MmyCI methyltransferase may be phase variable.

Di-nucleotide repeat polymorphisms of the insulin-like growth factor-1 gene and their association with IGF-1, insulin-like growth factor-binding protein-1 and birth size in a Sri-Lankan cohort.

BACKGROUND: Insulin-like growth factor (IGF)-I is implicated in fetal growth. Ethnic variations of IGF-1 have also been suggested. Di-nucleotide repeat polymorphisms of the IGF-1 gene or their association with IGF-1 levels and birth size have not been studied in Sri Lankans. OBJECTIVES: To describe IGF-1 di-nucleotide repeat polymorphisms and their association with IGF-1 and IGF-binding protein (IGFBP)-1 levels and birth size in a cohort of Sri Lankans. METHODS: A cross-sectional study on 200 mother-newborn pairs was carried out. Maternal and cord blood levels of IGF-1 and IGFBP-1 were measured by enzyme-linked immunosorbent assay. Three di-nucleotide repeat polymorphisms of the IGF-1 gene [cytosine-adenine (CA) repeats in the promoter and 3' regions and intron 2 cytosine-thymine (CT) repeat] were studied using PCR amplification and fragment analysis. RESULTS: Cord blood IGFBP-1 levels correlated negatively with birth weight (p < 0.01) and crown-heel length (p < 0.05). Wild-type alleles of the CA repeat polymorphisms differed from those reported in other populations. Newborn and maternal intron 2 CT repeat polymorphism showed a significant effect on birth weight (p < 0.01 and p < 0.001, respectively), crown-heel length (p < 0.01) and head circumference (p < 0.01 and p < 0.05, respectively). Promoter region CA repeat polymorphism and intron 2 CT repeat polymorphism in the newborns were significantly (p < 0.05) associated with cord blood IGF-1 levels. Almost all these effects were limited to primiparous pregnancies. CONCLUSIONS: In Sri Lankans intron 2 CT repeat polymorphism of the IGF-1 gene appears to be a significant contributor to IGF-1 levels and birth size in primiparous pregnancies.

IGF(CA)19 and IGFBP-3-202A/C gene polymorphism in patients with acromegaly.

OBJECTIVE: We aimed to investigate IGF-1 and IGFBP-3 gene polymorphisms in patients with acromegaly. DESIGN: We included 34 patients with acromegaly and 37 healthy subjects to study. At baseline examinations, antropometric measurements were done. Genomic DNA from the patients and controls were prepared. Serum, glucose, insulin, total cholesterol, triglyceride, HDL cholesterol, LDL cholesterol, growth hormone (GH), Insulin-like growth factor I (IGF-I) and IGFBP-3 levels of subjects were analyzed. RESULTS: The frequency of genotype IGF-1(CA)19 and IGFBP3-202 A/C gene was significantly different between control and patients. In acromegalic patients, a significant difference in the serum IGF-1 levels and LDL cholesterol levels among the three IGF(CA)19 genotype. LDL levels were positively correlated with IGF-1. Subjects having >194 bp genotype had higher IGF-1 and LDL cholesterol levels. We observed that the patients with 194 bp genotype have more invasive and bigger tumors and they require adjunctive therapies. Clinical characteristics among the three IGFBP3-202 A/C genotype, AA, AC and CC, did not display any significant difference. CONCLUSIONS: In our study, 194 bp allele (20 CA repeats) of the IGF-I promoter have higher circulating IGF-I levels than others. We have found that the patients with 194 bp genotype are the resistant patients with active disease and they required high dose medication. We think this study may help to define the patients, who are resistant to drug therapy, and possible cardiovascular disease.

Increasing the physical markers of wheat chromosomes using SSRs as FISH probes.

In plants the marker sequences used to identify chromosomes are mainly repetitive DNA probes. Simple sequence repeats (SSRs) are major components of many plant genomes and could be good markers for chromosome identification. In a previous work, we reported the physical distribution of 4 oligonucleotides, (AG)12, (CAT)5, (AAC)5, and (AAG)5, on Triticum aestivum L. chromosomes. The distinctive distribution pattern found suggested that SSR in situ hybridization is useful as a diagnostic tool in wheat cytogenetics. To check whether that finding is generally applicable, we analyzed the chromosomal distribution of the rest of the 14 possible classes of di- and tri-nucleotide repeats by FISH. A detailed knowledge of the sequence content of hexaploid wheat chromatin was acquired based on the hybridization signals, which also provide a rich set of chromosome markers for chromosome identification. Except for (AT)10 and (GC)10, for which the chromosomal distribution could not be accurately determined, and (AC)8 and (GCC)5, which were found dispersed throughout the chromosomes, the remaining repeats were observed as clusters on specific chromosome sites. (AGG)5, (CAC)5, (ACG)5, (AAT)5, and (CAG)5 exhibited a preferential distribution in the pericentromeric regions of the B genome chromosomes. The richest patterns of intercalary signals on several A and B genome chromosomes were produced by (ACT)5. A karyotype based on the SSR probes providing the best FISH patterns was constructed for T. aestivum 'Chinese Spring'.

An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein.

Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif.

Subtelomeric factors antagonize telomere anchoring and Tel1-independent telomere length regulation.

Yeast telomeres are anchored at the nuclear envelope (NE) through redundant pathways that require the telomere-binding factors yKu and Sir4. Significant variation is observed in the efficiency with which different telomeres are anchored, however, suggesting that other forces influence this interaction. Here, we show that subtelomeric elements and the insulator factors that bind them antagonize the association of telomeres with the NE. This is detectable when the redundancy in anchoring pathways is compromised. Remarkably, these same conditions lead to a reduction in steady-state telomere length in the absence of the ATM-kinase homologue Tel1. Both the delocalization of telomeres and reduction in telomere length can be induced by targeting of Tbf1 or Reb1, or the viral transactivator VP16, to a site 23 kb away from the TG repeat. This correlation suggests that telomere anchoring and a Tel1-independent pathway of telomere length regulation are linked, lending a functional significance to the association of yeast telomeres with the NE.

Estrogen receptor-alpha thymidine and adenine repeat polymorphism and endothelial fibrinolytic regulation in postmenopausal women.

OBJECTIVE: We hypothesized that the capacity of the endothelium to release tissue-type plasminogen activator is blunted in postmenopausal women with long (TA)(n) repeat alleles (> or = 18 repeats). STUDY DESIGN: Forty-two healthy postmenopausal women were studied: 10 women with short allele genotypes (both alleles, <18 repeats; age, 59 +/- 2 years), 8 women with long allele genotypes (both alleles, > or = 18 repeats; age, 59 +/- 3 years), and 24 women with mixed allele genotypes (1 short and 1 long allele; age, 56 +/- 1 years). Net endothelial tissue-type plasminogen activator release was determined in response to intra-arterial bradykinin and sodium nitroprusside. RESULTS: Tissue-type plasminogen activator release in response to bradykinin was highest in homozygotes for the short allele. The total amount of tissue-type plasminogen activator antigen that was released was significantly higher (>55%) in the short (452 +/- 68 ng/100 mL tissue) compared with the mixed (248 +/- 27 ng/100 mL tissue) and long allele (290 +/- 53 ng/100 mL tissue) groups. CONCLUSION: Our results demonstrate that the long (TA)n dinucleotide repeat allele is associated with blunted endothelial tissue-type plasminogen activator release in healthy postmenopausal women.

eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides.

beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.

A nonsense mutation (E72X) in growth hormone releasing hormone receptor (GHRHR) gene is the major cause of familial isolated growth hormone deficiency in Western region of India: founder effect suggested by analysis of dinucleotide repeat polymorphism close to GHRHR gene.

An identical nonsense mutation (E72X) in growth hormone releasing hormone receptor (GHRHR) gene was identified in 17 patients with isolated GH deficiency belonging to one Muslim and four Hindu families residing in the Western part of India. Analysis of two dinucleotide repeat polymorphism, one at 6 kb downstream and the other at 13 kb downstream of GHRHR gene, revealed that all the patients shared the same homozygotic alleles at both loci. These results strongly indicate that the nonsense mutation occurred in a single ancestor and was subsequently transmitted to the descendants. This GHRHR mutation may be an important cause of familial IGHD in Western India and Sindh area of Pakistan as previous studies have also reported the same mutation.

Decreased ethanol preference and wheel running in Nurr1-deficient mice.

Nurr1 (Nr4a2) is a transcription factor expressed in dopamine cells from early development and throughout life. Null mutants for Nurr1 lack the ventral midbrain dopamine neurons and die soon after birth. Animals with a heterozygous deletion are viable and display no apparent abnormality. We have investigated the impact of heterozygous deletion of Nurr1 on ethanol consumption in adult mice as a model for drug-induced reward and on wheel running as a model for natural reward. Interestingly, Nurr1 heterozygous mice never developed high ethanol consumption nor did they develop as much running behaviour as did the wild-type animals. Thus, Nurr1 appears to have a key role for the reinforcing properties of ethanol and running that underlies the development of excessive reward-seeking behaviours characteristic for addiction. Quantitative trait loci mapping using C57Bl/6 and DBA/2 mice describe a locus for ethanol preference on chromosome 2, wherein Nurr1 is located. We found two dinucleotide repeats in the Nurr1 promoter that were longer in mice with low preference for ethanol (DBA/2 and 129/Sv) than in mice with high preference for ethanol (C57Bl/6J and C57Bl/6NIH). These sequential data are compatible with Nurr1 as a candidate gene responsible for the quantitative trait loci for ethanol preference on mouse chromosome 2. Together, our data thus imply involvement of Nurr1 in the transition to a state of high ethanol consumption as well as in the development of a high amount of wheel running in mice.

Identification and characterization of an angiogenin-binding DNA sequence that stimulates luciferase reporter gene expression.

Angiogenin undergoes nuclear translocation in endothelial and smooth muscle cells where it accumulates in the nucleolus and binds to DNA. Nuclear translocation of angiogenin is necessary for its biological activity and is mediated by an endocytotic pathway that is independent of the microtubule system and lysosomal processing. Because the nucleolus is a subnuclear organelle containing clusters of transcriptionally active ribosomal RNA genes, we studied the binding of angiogenin to the intergenic spacer of the ribosomal RNA gene where many of the transcription regulatory elements are located. Here we report that angiogenin binds to CT repeats that are abundant in the nontranscribed region of the ribosomal RNA gene. An angiogenin-binding DNA sequence (CTCTCTCTCTCTCTCTCCCTC) has been identified and designated angiogenin-binding element (ABE). ABE binds angiogenin specifically and exhibits angiogenin-dependent promoter activity in a luciferase reporter system. CT repeats, or inverted GA box, which are abundantly distributed in the eukaryotic genome and are often located in the 5'-flanking region, have been implicated in regulating gene expression. We have previously shown that angiogenin stimulates rRNA synthesis. The present results suggest that the nuclear function of angiogenin may not only be related to rRNA production but also play a role in regulating expression of genes containing CT repeats.

Heme oxygenase-1 gene promoter polymorphism is associated with coronary artery disease in Japanese patients with coronary risk factors.

OBJECTIVE: Heme oxygenase (HO) is important in the defense against oxidative stress and as a factor in an antiatherogenic mechanism. Compared with long (GT)(n) repeats, short (GT)(n) repeats in the human HO-1 gene promoter were shown to have higher transcriptional activity in response to oxidative stress. There is a strong link between oxidative stress and the pathogenesis of coronary artery disease (CAD). METHODS AND RESULTS: We screened the allelic frequencies of (GT)(n) repeats in the HO-1 gene promoter in 577 patients who underwent coronary angiography. Because the distribution of numbers of (GT)(n) repeats was bimodal, we divided the alleles into 2 subclasses: class S included shorter (<27) repeats, and class L included longer (> or =27) repeats. Multivariate logistic regression models including standard coronary risk factors revealed that the genotypes were significantly related to CAD status in hypercholesterolemic, diabetic patients or in smokers. In this study, the patients with shorter GT repeats were less likely to have CAD. CONCLUSIONS: Length polymorphism in the HO-1 gene promoter is related to CAD susceptibility in Japanese people who also have coronary risk factors such as hypercholesterolemia, diabetes, and smoking. HO-1 may play an antiatherogenic role in Japanese patients with these coronary risk factors.

Association of estrogen receptor-alpha gene polymorphisms with coronary artery disease in patients with familial hypercholesterolemia.

To investigate the association of estrogen receptor (ER)-alpha gene polymorphisms with coronary artery disease (CAD), we studied 197 men and 98 postmenopausal women with heterozygous familial hypercholesterolemia. We examined the known polymorphisms, including PvuII, XbaI, TA repeat, and CA repeat, and identified 6 novel polymorphisms in the ER-alpha gene. The distributions of -1989T/G (a novel polymorphism in promoter B) and XbaI in intron 1 were associated with CAD in postmenopausal women and in men, with a higher frequency of the G/G genotype (P=0.03) or X1/X1 genotype (P=0.02) in the CAD group. The frequency of alleles of TA repeats >17 was found to be significantly higher in postmenopausal women with CAD than in those without CAD (P=0.04), but not in men. Logistic regression analysis with all coronary risk factors as covariates showed that the G/G genotype was a higher risk for CAD (odds ratio 4.5, 95% CI 1.0 to 19.5;P=0.04) but that X1/X1 was not. We conclude that -1989T/G or its linked polymorphisms in the ER-alpha gene may confer risk for CAD and that the G/G genotype may be an independent predictor for CAD in patients with familial hypercholesterolemia.

Physical and functional characterization of the human LGI1 gene and its possible role in glioma development.

The human gene termed LGI1 (leucine-rich gene - glioma inactivated) has been isolated recently, and is supposed to be an additional candidate tumor suppressor gene involved in the formation and progression of glioblastoma multiforme [Chernova et al. (1998) Oncogene 17:2873-2881]. To test this hypothesis and to complete the characterization of the gene, we performed various detailed studies on the genomic structure, the mRNA expression level, the integrity of the cDNA, and retroviral gene transfer into LGI1-deficient cell lines. Two single nucleotide polymorphisms in the promotor region and a highly polymorphic intragenic microsatellite repeat between exon 4 and 5 were found. Phylogenetic sequence analysis techniques were applied, which showed functional relationships between LGI1 and TRK and SLIT protein families that are known to be involved in development and maintenance of the nervous system. Fluorescence in situ hybridization (FISH) analysis showed LGI1 to be present on 10q24 in each of 11 glioma-derived cell lines evaluated. Sequence analysis of the LGI1 transcript did not detect any mutation. Relative amounts of LGI1 mRNA copy numbers as measured by the real-time fluorescence detection LightCycler technology differed more than three orders of magnitude and were significantly reduced in 10 of 11 cell lines. Retroviral gene transfer into LGI1-deficient glioma-derived cell lines could not substantiate any difference to control infected cultures regarding growth rate, S phase transition, and maintenance of marker gene expression. The strong homology to proteins involved in development, differentiation, or maintenance of the nervous system provides evidence for a function of the LGI1 protein in neural tissue. The observation that translocation or deletion of the LGI1 locus or mutation of the coding sequence of the LGI1 mRNA is not a frequent event in malignant glioma cell lines suggests that epigenetic factors lead to substantial differences in the amount of LGI1 mRNA expression. In addition, that the effect is lacking after retroviral gene transfer in cell culture suggests that binding of some kind of a ligand is essential for its biological activity.

Expression of TNF-alpha by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by NF-kappa B and activating transcription factor 2/Jun and translational regulation through the AU-rich region of the 3' untranslated region.

Here we have investigated the regulation of TNF-alpha expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-alpha mRNA present in resting macrophages, no TNF-alpha protein is detectable. HSV-2 infection marginally increases the level of TNF-alpha mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-gamma-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-alpha promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-alpha protein, indicating that the infection lifts a block on translation of TNF-alpha mRNA. EMSA analysis showed that specific binding to the kappaB#3 site of the murine TNF-alpha promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-alpha expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-alpha promoter. Inhibitors against either NF-kappaB activation or the activating transcription factor 2 kinase p38 abrogated TNF-alpha expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-alpha, the AU-rich sequence in the 3' untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-alpha production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.