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Team:Freiburg/Project/effector
From 2013.igem.org
Effectors
Activation
Introduction
The aim of this subproject was to engineer a new form of activation system based on a CRISPR RNA (crRNA)-guided dCas9-VP16 fusion protein which is able to activate gene expression of a reporter construct.
Therefore, we used our double mutated Cas9 (BBa_K1150000 ) impaired in its cleavage activity and fused it -to the 5’ end of the sequence coding for the transactivation domain of VP16 (BBa_K1150001 ). To ensure nuclear localization of the whole expressed construct a nuclear localization signal (NLS) was fused to the 5’ end of Cas9-VP16. For detection of protein expression the whole construct was tagged with a HA-epitope coding sequence (BBaa_K1150016) and its expression was set under control of the SV40/CMV promoter (BBa_K1150011/BBa_K747096) and BGH terminator (BBa_K1150012). Figure 1 illustrates the detailed design of the whole device.
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| Figure 1: Design of the dCas9-VP16 fusion constuct. Cas9 was fused via a 3 amino acid linker to VP16. The resulting fusion protein was flanked by NLS sequences and tagged by a HA epitope. The SV40/CMV promoter and BGH terminator were chosen to control gene expression. BBa_K1150019 is set under the control of the SV40 promotor, BBa_K1150020 is under the control of the CMV promotor. |
The Virus Protein 16 (VP16) is a transcription factor of Herpes simplex virus-1.
Through complex formation with cellular host factors VP16 can bind to a common regulatory element in the upstream promotor region of immediate-early genes [Weir, 2001]. Through the transactivating function of VP-16 the expression of these genes will be enhanced.
VP16 consists of 490 amino acids with two important functional domains: a core domain in its central region which is necessary for the indirect DNA binding and a carboxy-terminal transactivation domain (Greaves and O’Hare, 1989; Triezenberg et al., 1988) The transactivation domain of VP16 can be fused to a DNA-binding domain of another protein in order to increase expression of a desired target gene [Hirai et al., 2010].
Mechanism:
By co-transfecting our RNA plasmid (BBa_K1150034) which includes the tracrRNA and the separately integrated, desired crRNA, the Cas9 specifically binds to the targeted DNA sequence. With the help of the transactivation domain of VP16, transcription factors are recruited and the pre-initiation complex can be built. By targeting this construct upstream of a promotor regionany gene of interest can be activated.
| Figure 2: Principle of transactivation of mammalian gene expression by the fusion protein Cas9-VP19 The double mutated Cas9 (D10A; H840A) fused to the herpes simplex virus (HSV) derived VP16 activation domain can serve as a crRNA-guided DNA-binding and transactivating protein. If a PAM sequence is present at the 5’ end of the crRNA binding site almost any DNA sequence can be targeted. |
Repression
Transcriptional Repression via uniCAS-KRAB
Krüppel-associated Box (KRAB) repressor domains are highly conserved polypeptide motifs and were first functionally characterized in 1991 [1]. Their occurence in about one third of all human zinc finger transcription factors suggests key regulatory features in higher eukaryotic transcriptomics [2]. In terms of tetrapod evolution, the role of their great abundance has been extensively discussed [3]. Even though KRAB minimal domains are usually no longer than ~ 50-75 amino acids, their mechanism of function remains complex. Common biochemical models suggest a key role in epigenetic silencing, by recruiting a scaffold of diverse proteins to the zinc fingers‘ binding site - amongst others histone deacetylases and histone methyltransferases [4]. Til date, KRAB domains were attached to several DNA binding proteins such as lacR and tetR, thereby silencing gene expression downstream of designed reporter targets.
In this work, KRAB was fused to enzymatically inoperable dCas9. Thus, a transcriptional repressor with the flexibility to target almost any DNA sequence of interest was yielded. Transient SEAP expression could thus be reduced by almost 60 %. In a second attempt, CMV-driven expression of the signaling scaffold protein CNK1 was targeted over 36h - being partially knocked down to background amounts [5]. Furthermore, GFP reporter expression was shown to be drastically reduced by dCas9-KRAB in both Fluorescence Microscopy and Flow Cytometry data. Endogenous levels of VEGF-A, a key factor in hypoxic tumor angiogenesis [6], were also successfully reduced and quantified through an Enzyme-Linked Immunosorbent Assay.
References
Epigenetics
Histone modification by dCas9-G9a
Introduction
For organisms it is crucial to have a tight control over their transcriptional machinery. As every cell has basically the same genetic information, different tissues have to be formed by differentially regulating the expression of this information. One of the most prominent mechanisms that give rise to this differential expression of genes are epigenetic modifications. Epigenetics are, by definition, inheritable changes in gene expression, that cannot be traced back to changes in the nucleotide sequence [7].
There are several types of epigenetic modifications that may have severe impact on gene expression [8]. Basically there are two main types of modifications. The first type are the chemical modification of cytosine residues of nucleotides, better known as DNA methylation. At so-called CpG islands, that can be found clustered in front of promoters the nucleotides can be altered by methylation. This methylation is a hallmark of repressive transcriptional states. But not only the DNA may be altered, but also the protein-nucleotide complex, called the chromatin, that forms the highly variable system that has massive impact on the differential regulation of expression. The probably most prominent epigenetic modifications are histone modifications.
Histones are proteins that work as multi-histone complexes and form a backbone for the DNA to be wound around. The termini of these nucleosomes are protruding from the histone core complex and can be targets for a lot of different modifications, which influence the chromatin state. One very prominent modification is methylation at lysine 9 of histone 3 (H3K9me) which renders before-open chromatin inactive. Such methylations are a hallmark of gene repression. One interesting fact about histone modification is the capability to spread the activity state over the surrounding chromatin via reader proteins. So the information of e.g. "repressed state" can, once specifically introduced, be propagated over a whole locus.
This is the point where the uniCAS system becomes interesting. By introducing specific histone modifications at several loci we should be able to regulate several genes at once using a dCas9 fusion with a methyltransferase that is known to specifically perform certain histone tail methylations.
For our device we used a part of the murine EHMT2 gene, the G9a. It is described in literature that, when targeted to an open locus via zinc finger proteins, G9a is able to repress expression of this locus [9] .
We sought to test and improve this system by fusing G9a to dCas9 and assayed the function as an epigenetic repressor. As a test subject we chose the VEGF locus, as it is
- well characterized
- easy to measure by ELISA methods
- VEGF is known to be involved in tumorgenesis and therefore an interesting target for testing our system
- HEK293T cells have an open VEGF locus, so we do not have to artificially open the locus, before testing the system
Results
HEK293T cells were seeded into 24-well plates and transfected with our constructs, that were targeted to open regions [10] of the VEGF locus. By specific histone modification through G9a we should see repression of VEGF secretion into the medium. Twelve hours after transfection the medium was change and 24 hours after medium change we harvested the supernatant and performed VEGF measurments by ELISA. Additionally we co-transfected a constitutive reporter (SEAP) as an internal standard to reduce mistake of non-transfected cells and cell number. A major problem of working with endogenous loci is the background of non-transfected cells which will display VEGF secretion, even though we have strong repression in other cells.
For having a control, that our protein does not sterically block the transcription we designed a mutated version of the G9a (dG9a), that has no catalytic activity. So every detectable difference is due to the G9a targeted to this locus. [11] As we can see in the graph, we achieved a strong reduction in VEGF expression, especially when targeting the -475 locus. This is in line with literature results and plausible, when having in mind the fact, that the promoter region is targeted here and dense chromatin in promoters leads to repression.
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| Figure 3: Endogenous, stable repression by dCas9-G9a Chromatin remodeling, resulting in repression of endogenous genes, is possible by fusing the histone methyltransferase G9a to dCas9. |
Discussion
Our results obviously show a strong repression of VEGF expression, depending on the locus targeted. We can conclude from our results and literature data that we were able to specifically methylate histones and change the transcriptional state of the VEGF locus.This results in a valuable repressor tool that is able to specifically change histone methylation patterns and can change transcriptional states. This leads to many possible applications such as cancer research, fundamental epigenetical science or even tissue engineering.
References
