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What is a regulable promoter? And how does one regulate it?

What is a regulable promoter? And how does one regulate it?



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I'm reading a patent where they (in S. cerevisae YNP5 strain):

downregulate the ERG9 gene by replacing the native ERG9 promoter with the regulable MET3 promoter

What's a regulable promoter and how does one regulate it? Must one add something to the broth to down-regulate the expression?

I don't see anything special in the culture medium.


Regulatable promoter means that the expression of the gene downstream of this promoter can be either induced or repressed. Met3 is an example of such regulatable promoters and it is regulated by the addition of methionine to the media. In yeast, Met3 gene encodes for ATP sulphurylase, an enzyme in the methionine biosynthetic pathway. Upon the addition of methionine, the gene driven by Met3 promoter is repressed, as its expression level is reversely correlated with the concentration of methionine added to the media (Figure from this paper):


What is a regulable promoter? And how does one regulate it? - Biology

In bacteria and archaea, structural proteins with related functions—such as the genes that encode the enzymes that catalyze the many steps in a single biochemical pathway—are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter. This forms a polycistronic transcript (Figure 1). The promoter then has simultaneous control over the regulation of the transcription of these structural genes because they will either all be needed at the same time, or none will be needed.

Figure 1. In prokaryotes, structural genes of related function are often organized together on the genome and transcribed together under the control of a single promoter. The operon’s regulatory region includes both the promoter and the operator. If a repressor binds to the operator, then the structural genes will not be transcribed. Alternatively, activators may bind to the regulatory region, enhancing transcription.

French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the lac operon of E. coli. They found that in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. For this work, they won the Nobel Prize in Physiology or Medicine in 1965.

Although eukaryotic genes are not organized into operons, prokaryotic operons are excellent models for learning about gene regulation generally. There are some gene clusters in eukaryotes that function similar to operons. Many of the principles can be applied to eukaryotic systems and contribute to our understanding of changes in gene expression in eukaryotes that can result pathological changes such as cancer.

Each operon includes DNA sequences that influence its own transcription these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.

In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers.


13.1: Regulation of the GAL1 promoter

  • Contributed by Clare M. O&rsquoConnor
  • Associate Professor Emeritus (Biology) at Boston College

In yeast, glycolysis plays a major role in energy production, and glucose is far and away its preferred carbon source. Genes involved in the metabolism of other carbon sources are usually repressed when glucose is available. When glucose is not available, however, yeast activate genes that metabolize other available energy sources, such as galactose. Galactose increases the transcription of several genes for enzymes that transport galactose into cells and ultimately convert it into glucose-6-phosphate (G6P), an intermediate in glycolysis. The first gene in the pathway induced by galactose, GAL1, encodes galactokinase, which phosphorylates galactose to galactose-1-phosphate. (Check out the GAL1 pathways link in SGD.) The GAL1 promoter has been incorporated upstream of the ORF site in both the pBG1805 and pYES2.1 plasmids and therefore controls transcription of plasmid-encoded MET/Met and lacZ genes in transformed cells.

The figure on the opposite page provides a simple overview of gene expression from theGAL1 promoter in the presence of glucose, raffinose and galactose. The promoter contains both negative and positive regulatory sites encoded within its DNA sequence. In the presence of glucose, repressor proteins bind to the negative regulatory sites and repress transcription. The Gal4p transcriptional activator binds to positive regulatory sites. Gal4p is a transcription factor that binds to DNA as a dimer. (The figure at the beginning of this chapter shows the crystal structure of the DNA binding and dimerization domains of Gal4p complexed with DNA.) In the presence of glucose, Gal4p is inactive, because it is bound to the repressor protein, Gal80p.

Glucose repression can be relieved by growing cells in a poor carbon source, such as raffinose. Raffinose is a trisaccharide composed of galactose, fructose and glucose. Raffinose is not able to induce high levels of GAL1 expression, which requires galactose. In the presence of galactose, expression of the GAL1 gene increases

1000-fold above the level observed in the presence of glucose. This stimulation is primarily due to the activity of Gal4p, which is no longer bound to the inhibitory Gal80p protein. Gal4p acts as a master regulator of galactose metabolism. In addition to activating GAL1 transcription, Gal4p also binds to the promoters of the GAL7 and GAL10 genes, which are situated adjacent to the GAL1 gene on yeast chromosome 2. Like GAL1, the GAL7 and GAL10 genes encode proteins involved in galactose metabolism.

Regulation of the GAL1promoter. In the presence of glucose, transcription is repressed because repressor proteins bind to regulatory sites
in the DNA and to the Gal4p transcriptional activator. Glucose repressionis relieved in the presence of raffinose, but Gal4p remains inactive.
Gal4p activates transcription in the presence of galactose due to the removal of the Gal80p protein.


Expression using the T7 RNA polymerase/promoter system

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (p(T7)). In the Basic Protocol, two plasmids are maintained within the same E. coli cell. One (the expression vector) contains p(T7) upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat-inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of p(T7). If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.


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What is Promoter

Promoter is one of the main regulatory element of the gene which initiates the transcription. It is located near the gene, upstream to the codon sequence. The size of the promoter can be 100-1000 bp. The specific DNA sequences called response elements provide initial binding sites for both RNA polymerase and transcription factors which recruit RNA polymerase. RNA polymerase is the enzyme responsible for the transcription, polymerizing complementary RNA nucleotides to synthesize a mRNA molecule.

Figure 2: Promoter

Bacterial RNA polymerase associated with sigma factor can bind to the promoter. Sigma factor is a bacterial transcription initiation factor. In eukaryotes, around 7 different basal transcription factors have to be bound to the promoter to recruit RNA polymerase.


Relationship between promoter sequence and its strength in gene expression

Promoter strength, or activity, is important in genetic engineering and synthetic biology. A constitutive promoter with a certain strength for one given RNA can often be reused for other RNAs. Therefore, the strength of one promoter is mainly determined by its nucleotide sequence. One of the main difficulties in genetic engineering and synthetic biology is how to control the expression of a certain protein at a given level. One usually used way to achieve this goal is to choose one promoter with a suitable strength which can be employed to regulate the rate of transcription, which then leads to the required level of protein expression. For this purpose, so far, many promoter libraries have been established experimentally. However, theoretical methods to predict the strength of one promoter from its nucleotide sequence are desirable. Such methods are not only valuable in the design of promoter with specified strength, but also meaningful to understand the mechanism of promoter in gene transcription. In this study, through various tests, a theoretical model is presented to describe the relationship between promoter strength and nucleotide sequence. Our analysis shows that promoter strength is greatly influenced by nucleotide groups with three adjacent nucleotides in their sequences. Meanwhile, nucleotides in different regions of promoter sequence have different effects on promoter strength. Based on experimental data for E. coli promoters, our calculations indicate that nucleotides in the −10 region, the −35 region, and the discriminator region of a promoter sequence are more important for determining promoter strength than those in the spacing region. With model parameter values obtained by fitting to experimental data, four promoter libraries are theoretically built for the corresponding experimental environments under which data for promoter strength in gene expression has been measured previously.

Graphical abstract

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Results

Conditional depletion of Lhx8 by Gdf9Cre causes massive primordial follicle activation

We previously reported that global knockout of Lhx8 causes infertility and loss of oocytes by postnatal day 7 (PD7) [14]. In the global knockout of Lhx8, primordial-like follicles form (oocytes less than 20 μm in diameter and surrounded by flat granulosa cells), but oocytes do not grow. Since Lhx8 is expressed in both embryonic and postnatal female germ cells, it is possible that global knockout of Lhx8 disrupts early embryonic pathways that lead to postnatal oocyte depletion. We therefore investigated the postnatal functions of Lhx8 by generating a conditional knockout mouse, using a floxed Lhx8 allele (Lhx8 flx/flx ) [15] and a Gdf9Cre transgenic mouse [16]. The Gdf9Cre transgene will inactivate Lhx8 specifically in primordial oocytes. Gdf9Cre is highly efficient in oocytes and, when present in either Lhx8 flx/flx or Lhx8 flx/- animals, displayed the same ovarian phenotype. We used Lhx8 flx/flx Gdf9Cre to study the effects of Lhx8 conditional deficiency in primordial follicles on ovarian development.

At PD7 and PD14, LHX8 protein was depleted in Lhx8 flx/flx Gdf9Cre ovaries and massive oocyte activation occurred in the primordial follicles, as manifested by oocytes reaching a diameter greater than 20 μm without significant transformation of the surrounding flat granulosa cells (Fig. 1a–f). At PD7, Lhx8 flx/flx Gdf9Cre mice had 398 ± 53 activated primordial follicles per ovary compared to 16 ± 2 per ovary in Lhx8 flx/flx controls. The number of primordial follicles was 1917 ± 23 per ovary in Lhx8 flx/flx controls and was significantly reduced to 1043 ± 119 per ovary in Lhx8 flx/flx Gdf9Cre mice (Fig. 1c). We detected a decline in primary follicles in Lhx8 flx/flx Gdf9Cre mice, implying a block in the transition from activated primordial follicles to primary follicles. There were a negligible number of advanced follicle types (oocytes surrounded by multiple layers of granulosa cells) in PD7 Lhx8 flx/flx Gdf9Cre ovaries, compared to the Lhx8 flx/flx controls.

Postnatal inactivation of Lhx8 causes premature activation of primordial follicles and ovarian failure. a and b Anti-LHX8 antibodies were used to detect oocytes in paraformaldehyde-fixed and hematoxylin-counterstained ovaries taken at postnatal day 7 (PD7). Ovaries were derived from control (Lhx8 flx/flx , A) and Lhx8 conditional knockout (Lhx8 flx/flx Gdf9Cre, B) mice. Arrowheads in the inset of panel A indicate primordial follicles that stain with anti-LHX8 antibodies (brown signal). Arrowheads in the inset in panel B show activated primordial follicles (oocytes larger than 20 μm without cuboidal granulosa cells). d, e, g and h Periodic acid–Schiff (PAS) staining of PD14 (D and E) and PD21 (G and H) ovaries derived from control (D and G) and Lhx8 conditional knockout (E and H) mice. Arrowheads in the inset of panels D and E indicate primordial activated primordial follicles. c, f and i Quantification of ovarian follicle types in Lhx8 flx/flx and Lhx8 flx/flx Gdf9Cre mice. Five pairs of ovaries from PD7 (C), PD14 (F), and PD21 (I) were embedded in paraffin and serially sectioned at 5 μm thickness, and the follicles were counted. Anti-NOBOX antibodies stain oocyte nuclei throughout folliculogenesis and were used to identify oocytes in our counts. Every fifth section was counted. We scored primordial follicles (PF), activated primordial follicles (Act. PF, oocyte diameter greater than 20 μm without cuboidal granulosa cells), primary follicles (PrF) and secondary/antral follicles (SF/AF). *P < 0.05, **P < 0.01. Scale bars: 100 μm (A, B, D, E, G and H) 20 μm (inset in A, B, D and E)

At PD14, Lhx8 flx/flx Gdf9Cre ovaries contained 847 ± 82 activated primordial follicles per ovary, compared to 15 ± 7 per ovary in Lhx8 flx/flx controls. The number of primordial follicles significantly diminished from 1753 ± 204 in Lhx8 flx/flx controls to 353 ± 58 in Lhx8 flx/flx Gdf9Cre ovaries (Fig. 1f). By PD21, the total number of follicles from primordial to secondary was greatly diminished in the Lhx8 flx/flx Gdf9Cre ovary (Fig. 1g–i). By PD35, there were barely any oocytes and follicles detected in the Lhx8 flx/flx Gdf9Cre ovary (see Additional file 1: Figure S1C) and Lhx8 flx/flx Gdf9Cre females were sterile (see Additional file 2: Figure S2A). Our studies show that postnatal inactivation of Lhx8 within oocytes of primordial follicles leads to massive oocyte activation, decoupling of oocyte activation from somatic cell transformation, oocyte death, and infertility.

LHX8 interacts with PI3K-AKT pathway

We examined RNA expression of genes that encode important members of the PI3K-AKT-mTOR pathways in PD7 Lhx8 flx/flx Gdf9Cre oocytes. Real-time polymerase chain reaction (PCR) did not detect significant changes in the expression of Pten, Akt, Foxo3, Pdk1, Mtor, Rps6, Deptor, Rictor, or Tsc1/2 (some of these are shown in Fig. 2). These results indicate that LHX8 does not directly affect transcription of a subset of genes known to encode proteins in the PI3K-AKT-mTOR pathways. Then we assessed whether the PI3K-AKT pathway was activated at the protein level in Lhx8 flx/flx Gdf9Cre oocytes. AKT is a serine/threonine-specific protein kinase that plays a key role in apoptosis and PFA. Phosphorylated AKT activates multiple downstream pathways, including FOXO3 phosphorylation, resulting in oocyte activation [8]. Western blot analyses on oocytes from PD7 ovaries showed that phosphorylation of AKT at two sites, S473 and T308, was higher in Lhx8 flx/flx Gdf9Cre oocytes than in controls (Fig. 2h). But interestingly, only p-AKT (T308) was detected in the activated primordial follicles (see Additional file 3: Figure S3).

AKT is activated in Lhx8 flx/flx Gdf9Cre oocytes. ag Oocytes were isolated from PD7 control (Lhx8 flx/flx , Ctrl) and Lhx8 flx/flx Gdf9Cre (G9cKO) mouse ovaries and RNA was extracted for cDNA conversion and real-time quantitative polymerase chain reaction (RT-qPCR). Data were normalized to Gapdh expression and are given as the mean relative quantity (compared with control), with error bars representing the standard error of the mean. Student’s t-test was used to calculate P values. The only significant difference was noted in the expression of Lhx8, as expected. ** P < 0.01. h Oocytes were isolated from PD7 control and Lhx8 flx/flx Gdf9Cre ovaries, protein was extracted, and a Western blot test was performed on three independent samples, using antibodies against AKT and its two phosphorylated forms (S473 and T308). Histone H3 immunoreactivity was used as a control

Previous studies have shown FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation via the PI3K-AKT-mTOR pathways to be associated with PFA [5, 6, 8]. We examined FOXO3 nucleocytoplasmic translocation and rpS6 phosphorylation in PD7 Pten and Lhx8 conditional knockouts (Fig. 3 and Additional file 4: Figure S4). FOXO3 nucleocytoplasmic translocation was prominent in oocytes larger than 30 μm but did not show obvious translocation among oocytes between 20 and 30 μm in Lhx8 flx/flx Gdf9Cre mice (Fig. 3d–f, f', p). Oocytes of primordial follicles in Pten and Lhx8 conditional single knockouts as well as corresponding controls did not show FOXO3 nucleocytoplasmic translocation. However, FOXO3 nucleocytoplasmic translocation was significantly induced in the primordial (<20 μm) oocytes (Fig. 3j–l, l', p) of the PD7 double Lhx8/Pten conditional knockouts (Lhx8 flx/flx Pten flx/flx Gdf9Cre). These data indicate a synergistic action of the LHX8 and PTEN proteins on FOXO3 nucleocytoplasmic translocation.

Lhx8 and Pten conditional knockout effects on FOXO3 localization. ac In control mice (Lhx8 flx/flx ), FOXO3 is expressed in the nucleus and cytoplasm of primordial oocytes (PF, arrows in c'). df In Lhx8 flx/flx Gdf9Cre mice, the extensive nucleocytoplasmic translocation is not observed in activated primordial follicles between 20 and 30 μm (aPF, arrow in f') but is noted in activated primordial follicles larger than 30 μm (aPF, arrow in F'). di A similar expression pattern of FOXO3 localization exists in Pten conditional knockout (Pten flx/flx Gdf9Cre) mice. The arrows in i' represent primordial follicles (PF) with both nuclear and cytoplasm expression of FOXO3 and cytoplasm expression of FOXO3 in activated primordial follicles (aPF) below 30 μm. jl However, in mice that are conditionally deficient in both Lhx8 and Pten (Lhx8 flx/flx Pten flx/flx Gdf9Cre), FOXO3 nucleocytoplasmic translocation is present in primordial, activated, and primary oocytes. The negative control is immunofluorescence in the presence of secondary antibodies and is shown in mo. The boxed areas in C, F, I, L, and O are shown magnified in C', F', I', L', and O'. p Graphic representation of FOXO3 distribution (cytoplasm only or nucleus and cytoplasm). Oocytes were grouped by size (diameter) as less than 20 μm, between 20 and 30 μm, and greater than 30 μm. Only oocytes with clear DAPI nuclear staining were counted. Scale bars: 50 μm (A–C, D–F, G–I, J–L, and M–O) 20 μm (C', F', I', L', and O')

We also examined the effects of double Pten and Lhx8 deficiency on rpS6. mTORC1 promotes protein translation and cell growth, in part, through activation of S6K1 (by phosphorylation of its threonine 389) and through phosphorylation and inactivation of eIF4E-binding proteins. S6K1 is responsible for phosphorylation and activation of rpS6, which leads to enhanced protein translation and ribosome biogenesis. In control, Lhx8 flx/flx Gdf9Cre, and Pten flx/flx Gdf9Cre ovaries, rpS6 was only significantly activated in growing follicles at PD7 (see Additional file 4: Figure S4). However, the PD7 Lhx8 flx/flx Pten flx/flx Gdf9Cre ovaries showed significant activation of rpS6 in primordial (<20 μm) oocytes (see Additional file 4: Figure S4D, D'). These results further indicate that the Lhx8 and Pten pathways synergistically interact to accelerate two events associated with PFA—FOXO3 nucleocytoplasmic translocation and phosphorylation of rpS6.

Lin28a RNA and protein expression are upregulated in Lhx8flx/flxGdf9Cre oocytes

LHX8 is a transcription factor, and we expect that its major effect will be at the RNA level. We therefore analyzed the transcriptome of Lhx8 flx/flx Gdf9Cre ovaries via high-throughput RNA sequencing (RNA-seq). We sequenced 180 million tags in each sample and compared the relative abundance of RNA tags encoded by genes in the PI3K-AKT-mTOR pathways derived from PD7 Lhx8 flx/flx Gdf9Cre and control ovaries. No significant differences in RNA expression of PI3K-AKT-mTOR pathway genes (Pik3r1, Pik3ca, Kit, Kitl, Gsk3b, Cdnd1, Wee1, Cdkn1a/b, Rheb, Mlst8, Mapkap1, Prr5, and Eif4ebp1) were evident in the RNA-seq experiment (see Additional file 5: Table S1).

In addition to analyzing the expression of known PI3K-AKT-mTOR genes, we studied global differences in the transcriptome of Lhx8 flx/flx Gdf9Cre and control ovaries. We detected a sixfold increase in Lin28a RNA transcripts in Lhx8 flx/flx Gdf9Cre compared to control ovaries (Fig. 4b and Additional file 5: Table S1). Immunofluorescence and Western blot analysis with anti-LIN28A antibodies showed that LIN28A protein was expressed at a significantly higher level in Lhx8 flx/flx Gdf9Cre oocytes compared to the controls (Fig. 4a, c).

Lhx8 suppresses Lin28a expression. a Immunofluorescence with anti-LIN28A antibodies shows that LIN28A is preferentially expressed in oocytes within the ovary. LIN28A abundance is higher in Lhx8 conditionally deficient oocytes (Lhx8 flx/flx Gdf9Cre) compared to controls (Lhx8 flx/flx ). b and c Lin28a transcripts and protein are significantly more highly expressed in Lhx8 flx/flx Gdf9Cre oocytes (G9cKO) unlike controls (Ctrl). af Chromatin immunoprecipitation (ChIP) assays with anti-LHX8 affinity purified antibodies on oocytes. d A putative LHX8 DNA binding site, TGATTG [22], which perfectly fits the LHX8 binding consensus sequence, was identified at position −536 to −531 relative to the Lin28a transcription initiation site. e Anti-LHX8 antibodies precipitate genomic DNA containing the TGATTG binding sequence from the Lin28a promoter region as shown by ChIP-quantitative PCR (qPCR). Immunoglobulin G (IgG) antibodies served as control. The percentage input method is used to analyze the qPCR data. “Input” is the PCR product from chromatin pellets before immunoprecipitation. A triplicate average Ct normalized to an adjusted input was used for the calculation of percentage input. Two sets of primers (F1 and R1) and (F2 and R2) were used to perform ChIP-qPCR. f PCR amplification of the oocyte input DNA, as well as DNA precipitated by normal guinea pig IgG and anti-LHX8 antibodies by the F1/R1 and F2/R2 primer sets. Scale bars: 50 μm (A)

LIN28A is an RNA-binding protein that blocks biogenesis of let-7 microRNAs and a known regulator of mammalian body size and metabolism, including onset of menarche [17, 18]. Lin28a is preferentially expressed in oocytes and embryo stem cells [19, 20]. Moreover, the PI3K-AKT-mTOR pathways can be activated by LIN28A [21]. LIN28A, therefore, may play a role in oocyte activation and growth.

LHX8 can directly bind to the LHX8 DNA binding motif in the Lin28a promoter

We tested whether LHX8 can bind to the Lin28a promoter. LHX8 is an oocyte-specific LIM homeodomain transcriptional regulator that is predicted to bind DNA. A previous study showed that the LHX8 homeodomain preferentially binds a TGATTG DNA motif [22]. We identified a single TGATTG DNA motif −531 to −536 bp upstream of the putative transcriptional initiation site in the Lin28a gene (Fig. 4d). The Lin28a TGATTG motif is conserved in other mammals, including humans. We performed a chromatin immunoprecipitation (ChIP) experiment on wild-type oocytes, using our highly specific and affinity purified anti-LHX8 antibodies [13]. Anti-LHX8 antibodies preferentially immunoprecipitated the Lin28a promoter DNA fragment containing the TGATTG motif (Fig. 4e, f). These data further suggest that LHX8 represses Lin28a expression by directly binding to the Lin28a promoter.

Lin28a deficiency partially rescues the Lhx8 flx/flx Gdf9Cre phenotype

Our data indicates that LHX8 suppresses Lin28a expression. Since LIN28A is a growth-promoting factor [17, 23] preferentially expressed in oocytes, we hypothesized that Lin28a deficiency will rescue Lhx8 flx/flx Gdf9Cre-induced PFA. We bred Lin28a and Lhx8 floxed mice with Gdf9Cre to generate Lhx8/Lin28a double conditional knockouts (Lhx8 flx/flx Lin28a flx/flx Gdf9Cre).

We performed ovarian morphometric analyses to determine the effects of the double knockout on PFA. No morphometric difference was observed between Lin28a flx/flx Gdf9Cre and control females at PD7 (Fig. 5a, c, e). As expected, we observed significantly fewer activated primordial follicles in Lhx8 flx/flx Lin28a flx/flx Gdf9Cre compared to Lhx8 flx/flx Gdf9Cre ovaries, but they were not completely normal compared with the control (Fig. 5a, b, d, f).

Lin28a deficiency rescues Lhx8 flx/flx Gdf9Cre-induced PFA. ad Representative histology of control (Lhx8 flx/flx ), Lhx8 conditional knockout (Lhx8 flx/flx Gdf9Cre), Lin28a conditional knockout (Lin28 flx/flx Gdf9Cre), and double Lhx8/Lin28a conditional knockout (Lhx8 flx/flx Lin28 flx/flx Gdf9Cre) ovaries. Double Lhx8/Lin28a conditional knockouts show a diminished number of activated primordial follicles. Anti-Nobox antibodies were used to label oocyte nuclei (dark brown). PF: primordial follicle aPF: activated primordial follicle. e The Lin28 flx/flx Gdf9Cre (Lin28G9cKO) mice have a similar phenotype as control (Ctrl) mice and the primordial follicle count is expressed as the percentage of total follicles. f Quantitation of activated primordial follicles in control, Lhx8 conditional, and double Lhx8/Lin28a conditional knockouts. The percentage of primordial follicles (PF) and activated primordial follicles (aPF) is shown. Three pairs of ovaries from Lhx8 conditional knockout and double Lhx8/Lin28a conditional knockouts mice were serially sectioned, and every fifth section was counted. g AKT phosphorylation is diminished in double Lhx8/Lin28a conditional knockouts. We quantitated by fluorescence p-AKT (T308) expression in the primordial and activated primordial follicles of the Lhx8 flx/flx Gdf9Cre (G9cKO) and Lhx8 flx/flx Lin28a flx/flx Gdf9Cre (G9dKO) ovaries at PD7. There was a significant decrease in AKT phosphorylation in Lhx8 flx/flx Lin28a flx/flx Gdf9Cre primordial and activated primordial follicles compared to Lhx8 flx/flx Gdf9Cre. Student’s t-test was used to calculate P values. *P < 0.05, **P < 0.01. Scale bars: 50 μm (A–D)

The partial rescue of Lhx8 flx/flx Gdf9Cre-induced PFA by Lin28a deficiency argues that Lin28a is a regulator of oocyte growth. The diminished number of activated primordial follicles in Lhx8 flx/flx Lin28a flx/flx Gdf9Cre ovaries suggests that AKT pathway activation is also diminished. We assayed the p-AKT (T308) signal in primordial and activated primordial follicles of Lhx8 flx/flx Lin28a flx/flx Gdf9Cre and Lhx8 flx/flx Gdf9Cre ovaries (Fig. 5g) and found its expression was significantly reduced in the Lhx8 flx/flx Lin28a flx/flx Gdf9Cre ovary compared to the ovaries of Lhx8 flx/flx Gdf9Cre mice.

Lhx8 regulates the primary to secondary follicle transition

Previous studies have shown that PTEN-regulated pathways are important in primordial oocyte activation, but not in primary oocytes [24]. We studied the role of Lhx8 in primary oocytes by generating Lhx8 flx/flx Zp3Cre mice. Zp3Cre is specifically expressed in primary oocytes, and Lhx8 flx/flx Zp3Cre ovaries continue to express LHX8 in primordial, but not primary, oocytes. Morphometric analyses revealed that the Lhx8 flx/flx Zp3Cre conditional knockout ovaries did not significantly differ from Lhx8 flx/flx (control) mice at PD0 and PD7 (Fig. 6a–f). However, at PD14, we counted 159 ± 23 primary follicles and 29 ± 7 secondary/antral follicles per ovary in the Lhx8 flx/flx Zp3Cre mice, compared to 79 ± 6 primary follicles and 108 ± 7 secondary/antral follicles per ovary in the control mice (Fig. 6g–i). The relative increase of primary follicles at PD14 and the relative decrease of multilayer follicles in the Lhx8 flx/flx Zp3Cre ovary indicated that the transition from primary follicles to secondary follicles was blocked, which was consistent with the observation in Lhx8 flx/flx Gdf9Cre mice (Fig. 1f). At PD21 and PD30, we observed that many primary follicles were devoid of oocytes (Fig. 6k, n). We stained for LIN28A in PD21 ovaries and found that LIN28A was strongly expressed in Lhx8 deficient oocytes of the Lhx8 flx/flx Zp3Cre ovary, but no expression was detected in the empty follicles (see Additional file 6: Figure S5). However, excluding these empty primary follicles, the number of primary follicles between control and Lhx8 flx/flx Zp3Cre ovaries was not significantly different at PD21 or PD30 (Fig. 6l, o). For secondary/antral follicles, the number sharply dropped to 17 ± 3 at PD21 and to 2 ± 1 at PD30 in Lhx8 flx/flx Zp3Cre ovaries, compared to 170 ± 2 and 104 ± 4, respectively, in control mice. These findings imply that the growing follicle pool continued to be eliminated from the Lhx8 flx/flx Zp3Cre mice. Lhx8 flx/flx Zp3Cre mice were infertile (see Additional file 2: Figure S2A) and superovulation treatment of Lhx8 flx/flx Zp3Cre mice did not produce oocytes (see Additional file 2: Figure S2B). This result was in accord with the sharp fall of secondary/antral follicles in the Lhx8 flx/flx Zp3Cre ovary at PD21. Taken together, these data show that the folliculogenesis of Lhx8 flx/flx Zp3Cre mice is blocked in the transition from the primary to secondary follicle stage and results in primary oocyte death and infertility. The relative stability of the primordial follicle pool from PD14 to PD30 suggests that the PFA into primary follicles was not affected by the diminution in the number of secondary and more advanced ovarian follicles in Lhx8 flx/flx Zp3Cre ovaries.

Lhx8 inactivation in primary follicles (Lhx8 flx/flx Zp3Cre) abolishes follicle growth. Histomorphological analysis was done on control (Lhx8 flx/flx ) and Lhx8 deficient ovaries (Lhx8 flx/flx Zp3Cre) at various stages of postnatal ovarian development ranging from newborn (PD0) to postnatal day 30 (PD30). af Periodic acid–Schiff (PAS) staining and counting of different follicle types in the newborn and PD7 ovaries showed no significant differences between control and Lhx8 deficient ovaries. go Anti-NOBOX antibodies were used to stain oocytes (brown immunoreactivity) in ovaries from PD14 (G and H), PD21 (J and K), and PD30 (M and N) mice. At PD14, the Lhx8 flx/flx Zp3Cre ovaries showed a significantly higher number of primary follicles (PrF) and significantly diminished number of secondary/preantral (SF/AF) follicles characterized by two or more layers of granulosa cells. At PD21 and PD30, the primary follicle pool did not differ significantly between Lhx8 flx/flx Zp3Cre and control ovaries however, there was a marked decrease in the number of secondary and more advanced ovarian follicles in conditional knockouts including degenerating follicles without oocytes (marked by asterisks in insets in K and N). The primordial follicle (PF) pool remained relatively stable between PD14 and PD30, with no significant difference between Lhx8 flx/flx Zp3Cre and control ovaries. **P < 0.01. Scale bars: 100 μm (A, B, D, and E) 200 μm (G, H, J, K, M, and N) 50 μm (insets of K and N)


Tetracycline-inducible Empty Backbones

Find a construct that will allow you to insert and induce your gene of interest.

ID Plasmid Description Co-expressed tTA, rtTA, or TetR On or Off PI
21916 Tet-pLKO-neo 3rd generation lentiviral plasmid for inducible expression of shRNA neomycin selection plasmid 21915 has puromycin selection TetR On Wiederschain
41393 pCW57.1 Lentiviral vector for inducible expression for Gateway cloning selection cassette in format: PGK-rtTA-2A-puro see article for tagged insert options rtTA On Root
11651 pLVUT-tTR-KRAB Lentiviral vector for inducible expression of transgene or shRNA see article for detailed information about cloning and additional plasmids tetR-KRAB On Aebischer & Trono
16542 pBI-MCS-EGFP Expression of your gene of interest (MCS with a &beta-globin poly A) & EGFP from a bidirectional tet-responsive promoter (Pbi) Pbi contains a TRE between two minimal CMV and is silent in absence of binding of tTA or rtTA None On Vogelstein
25735 pSLIK-Neo Lentiviral vector for inducible expression of a miR-shRNA selection cassette in format: rtTA3+IRES+Neo see article for additional selection options third-generation rtTA On Fraser
44012 pInducer20 Lentiviral vector for shRNA expression see article for additional tookit plasmids third-generation rtTA On Elledge
19407 pTREtight2 Promoter contains a modified TRE that is silent in absence of binding tTA or rTA has high copy E. coli origin None Either Ralser
64238 pTet-IRES-EGFP Lentiviral plasmid for inducible expression of transgene of interest and EGFP None Either Lung
11662 pPRIME-TET-GFP-FF3 Lentiviral, miRNA expression (PRIME) system for application in knockdown of gene expression at a single copy in mammalian cells Expresses firefly luciferase hairpin and GFP under pTREtight promoter None Either Elledge
35625 pAAV-Ptet-RFP-shR-rtTA AAV shRNA cloning vector for evaluation of shRNA efficacy using fluorescence use with pGFPns-reporter for cDNA target rtTA On Gu
60495 pSBtet-GP Sleeping Beauty transposon system has luciferase in cloning site see article for additional selection and FP option rtTA On Kowarz
16623 pBI-GFP Expression of your gene of interest & GFP from a bidirectional tet-responsive promoter (Pbi) Pbi contains a TRE between two minimal CMV and is silent in absence of binding of tTA or rtTA None Either Vogelstein
100521 pCW57.1-MAT2A Lentiviral Tet-Off all in one plasmid derived from pCW57.1. rtTA was replaced with tTA, and Puromycin was replaced with Blasticidin selection. Please NOTE, this vector contains an insert (MAT2A) which would need to be replaced by the gene of interest. tTA Off Sabatini
92099 AAVS1_Puro_Tet3G_3xFLAG_Twin_Strep Bidirectional promoter controls expression of gene of interest with Strep-Tag and Tet-On 3G transactivator, creating an auto-regulated Tet-On 3G System. Contains homology arms for integration into AAVS1 Genomic Safe Harbor Locus. Tet-On 3G On Doyon
58245 pGLTR-X-GFP Single vector lentiviral Gateway RNAi system for conditional cell line generation contains expression cassette for TetR-P2A-GFP see article for additional constructs TetR On Geley


Example 4

In experiments using S. aureus cells grown in culture, intracellularly expressed ssJT01ss bound to and inhibited a specific essential cellular target in a manner similar to that of an antimicrobial drug. Hence induction of expression of this peptide during an infection should have the effect of an antibiotic. An established animal infection model was used to test this concept (Onyegji, C. O. et al., Antimicrobial Agents and Chemotherapy 38:112-117, 1994).

Six groups of CD-1 female mice (5 mice per group, Charles River Laboratories, Wilmington, Ma.) weighing 20-24 grams were used in this experiment. The inoculum was prepared from CYL316tt/pC 3 883 (encoding a ssJT01ss peptide-GST fusion protein under the control of the tet operon) which was cultured at 37° C. for 17 hours in TS broth containing erythromycin and kanamycin. 1.6×10 10 cfu (colony-forming units) of S. aureus CYL316tt/pC 3 883 (OD 600 of 0.1=10 8 cfu/ml) from the overnight culture were diluted to 20 ml with 0.01 M PBS (Sigma P-0261) containing 8% hog gastric mucin (Sigma M-2378) as well as 50 μg/ml kanamycin and 10 μg/ml erythromycin. Each mouse of groups 1 through 4 was injected with 0.5 ml of the inoculum intraperitoneally (i.p.), equivalent to 4×10 8 cfu/mouse (lethal dose). Groups 5 and 6 served as vector controls. Each mouse of these two groups was injected with 4×10 8 cfu of CYL316tt/pC 3 875, which was cultured and processed the same way as CYL316tt/pC 3 883. One half hour and four hours after the inoculation, groups 1 and 5 received a saline injection i.p. at 10 ml/kg groups 2 and 6 received i.p. injections of tetracycline (Sigma T-3383) at 8 mg/kg group 3 received i.p. injections of tetracycline at 4 mg/kg group 4 received i.p. injections of ciprofloxacin (Bayer 851510, dissolved in water) at 50 mg/kg. The injection volume for all the animals was 10 ml/kg. Surviving mice were counted at 7 days post inoculation. Ciprofloxacin given at 50 mg/kg protected all infected animals from lethal infection.

The data summarized in Table 2 demonstrate that induction of intracellular expression of the ssJT01ss peptide can be achieved in an animal infection. Inhibition of S. aureus MetRS by the intracellularly expressed ssJT01ss peptide cured a lethal infection in a mouse model.

TABLE 2
Inhibition of S. aureus growth in mice by intracellular production
of S. aureus MetRS inhibitor
# of Mice# of Mice
Experimental ConditionTestedSurvival
M1 Peptide
Saline, 10 mg/kg C250
Inducer, mg/kg C255
Expression Control
Saline, 10 mg/kg C250
Inducer, mg/kg C250

The relevant teachings of all references cited herein are hereby incorporated by reference herein in their entirety.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

28 1 33 DNA Artificial Sequence Oligonucleotide 1 acgggtcgac tcatatcttt tattcaataa tcg 33 2 32 DNA Artificial Sequence Oligonucleotide 2 ccggaaagct tacttattaa ataatttata gc 32 3 34 DNA Artificial Sequence Oligonucleotide 3 taagtaagct taaggaggaa ttaatgatgt ctag 34 4 34 DNA Artificial Sequence Oligonucleotide 4 acgggtcgac ttaagaccca ctttcacatt taag 34 5 45 DNA Artificial Sequence Oligonucleotide 5 ctcggtaccg agctaaaatt cggaggcata tcaaatgagc tctgg 45 6 44 DNA Artificial Sequence Oligonucleotide 6 ggcatatcaa atgagctctg gaggtggagg catgtcccct atac 44 7 31 DNA Artificial Sequence Oligonucleotide 7 aggcctaggt taatccgatt ttggaggatg g 31 8 42 DNA Artificial Sequence Oligonucleotide 8 ctgatccgaa tacgtggcag ttgcggtggc ctatgcatag ct 42 9 42 DNA Artificial Sequence Oligonucleotide 9 atgcataggc caccgcaact gccacgtatt cggatcagag ct 42 10 48 DNA Artificial Sequence Oligonucleotide 10 tcgagttcat gaaaaactaa aaaaaatatt gacatcccta tcagtgat 48 11 45 DNA Artificial Sequence Oligonucleotide 11 agagataatt aaaataatcc ctatcagtga tagagagctt gcatg 45 12 29 DNA Artificial Sequence Oligonucleotide 12 caagctctct atcactgata gggattatt 29 13 56 DNA Artificial Sequence Oligonucleotide 13 ttaattatct ctatcactga tagggatgtc aatatttttt ttagtttttc atgaac 56 14 58 DNA Artificial Sequence Oligonucleotide 14 aataaaaaac tagtttgaca aataactcta tcaatgatag agtgtcacaa aaaggagg 58 15 56 DNA Artificial Sequence Oligonucleotide 15 gatagagtgt caacaaaaag gaggaattaa tgatgtcccc tatactaggt tattgg 56 16 36 DNA Artificial Sequence Oligonucleotide 16 ggattaaggt aaccttaatc cgattttgga ggatgg 36 17 12 PRT Artificial Sequence Synthetic Peptide 17 Asp Pro Asn Thr Trp Gln Leu Arg Trp Pro Met His 1 5 10 18 6 PRT Artificial Sequence Synthetic Peptide 18 Gly Gly Arg Gly Gly Met 1 5 19 54 DNA Artificial Sequence Oligonucleotide 19 gatcctaata catggcagtt gaggtggcct atgcatggcg gccgcggagg tatg 54 20 66 DNA Artificial Sequence Oligonucleotide 20 agctctgatc ctaatacatg gcagttgagg tggcctatgc attcttcagg cggccgcgga 60 ggtatg 66 21 21 PRT Artificial Sequence peptide 21 Ser Arg Trp Glu Lys Tyr Ile Asn Ser Phe Glu Leu Asp Ser Arg Gly 1 5 10 15 Gly Arg Gly Gly Met 20 22 63 DNA Artificial Sequence Oligonucleotide 22 tctagatggg aaaaatatat taattctttt gaattagatt ctcgaggtgg tagaggtgga 60 atg 63 23 21 PRT Artificial Sequence peptide 23 Ser Ser Gln Gly Thr Met Arg Trp Phe Asp Trp Tyr Arg Ser Arg Gly 1 5 10 15 Gly Arg Gly Gly Met 20 24 63 DNA Artificial Sequence Oligonucleotide 24 agctctcaag gtactatgag atggtttgat tggtatagat ctcgaggtgg tagaggtgga 60 atg 63 25 49 DNA Artificial Sequence Oligonucleotide 25 agcaccttgg cggccgcgga ggtgctagca aaggagaaga actcttcac 49 26 37 DNA Artificial Sequence Oligonucleotide 26 aactgaggta acctcagttg tacagttcat ccatgcc 37 27 52 DNA Artificial Sequence Oligonucleotide 27 tttaccttgg cggccgcgga ggtaaactga agaaggtaaa ctggtaatct gg 52 28 40 DNA Artificial Sequence Oligonucleotide 28 acttagggta accttaagtc tgcgcgtctt tcagggcttc 40


Identification of the Omega4514 regulatory region, a developmental promoter of Myxococcus xanthus that is transcribed in vitro by the major vegetative RNA polymerase

Omega4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and sigma(A) RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Omega4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac Omega4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Omega4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Omega4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.

Figures

Physical map of the Ω4514…

Physical map of the Ω4514 insertion region and summary of upstream segments tested…

Developmental expression of lacZ under…

Developmental expression of lacZ under the control of the Ω4514 promoter. Developmental β-galactosidase…

Localization of an mRNA 5′…

Localization of an mRNA 5′ end within the Ω4514 upstream region. Low-resolution S1…

Primer extension analysis of Ω4514…

Primer extension analysis of Ω4514 mRNA. RNA was isolated from wild-type DK1622 cells…

Developmental expression of lacZ from…

Developmental expression of lacZ from a small segment containing the Ω4514 promoter. Developmental…

In vitro transcription from the…

In vitro transcription from the Ω4514 promoter. DNA fragments containing the Ω4514 promoter…

Western blot analysis of σ…

Western blot analysis of σ A in growing and developing M. xanthus cells.…

Effect of orf1 and orf2…

Effect of orf1 and orf2 mutations on developmental lacZ expression under the control…

Level of ORF1 protein and…

Level of ORF1 protein and β-galactosidase specific activity during growth and development. (A)…

Sequences of promoters transcribed in…

Sequences of promoters transcribed in vitro by M. xanthus σ A RNAP. The…