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The immune system is the human body's first line of defense against invading pathogens. It is what is responsible for the targeting and destruction of foreign agents and prevention of further invasion. The immune system is not a specific organ, but rather a collective of cellular responses. These cellular responses are comprised of specialized cells that are targeted and activated by a specific foreign agent, or a generalized response activated by any foreign stimulus. The former cellular immune response is generally referred to as adaptive immunity.
An important component of the adaptive immunity is the T cell. T cells are the orchestrators of the adaptive immune response. These cells recognize foreign antigen and once activated, go on to activate other cells of the immune system, including macrophages and B cells. Another component of the adaptive immune system is the professional antigen presenting cell, which brings foreign agents to the regulators of adaptive immunity. Examples of professional antigen presenting cells include dendritic cells and macrophages. These cells can detect foreign agents on its surface, internalize these agents, and display fragments of the foreign peptides on its surface for immune surveillance. The professional antigen presenting cells display these foreign peptides to T cells on molecules known as Major Histocompatibility Complex Class I and II (Ting and Trowsdale, 2002).
MHC Class II molecules are surface proteins that contain a binding pocket for foreign peptides. MHC II molecules bind to the T cell receptor and can activate it with a costimulatory molecule. They are constitutively expressed on professional antigen presenting cells, whose main job is to present antigens to T cells. All other nucleated cells can be induced to express MHC class II molecules with cytokines such as Interferon-γ. The MHC Class II molecule has been shown to be controlled by a master regulator known as Class II Transactivator (CIITA), which binds to the CIITA promoter with the help of a complex of transcription factors (Figure 1). W
Figure . MHC Class II Promoter and Transcription Factors
CIITA is the master regulator of MHC Class II transcription. It has been shown that the upregulation of MHC Class II parallels the upregulation of CIITA levels in the cell. CIITA is constitutively expressed in B cells and dendritic cells and is inducible in all nucleated cells. Currently, it is widely accepted that there are four promoters for CIITA expression. Dendritic cells use CIITA promoter I for expression, B cells use CIITA promoter III for expression and promoter IV is used for inducible expression in all other nucleated cells. Recently, it has been shown that epigenetic regulation of CIITA can prove to be useful in the regulation of surface expression of MHC class II (Harton and Ting, 2000).
Epigenetic control of a gene involves post translational modification of histones to either bind the histones tightly together, or open up the chromatin structure by decreasing the association between histones. Unpublished data in the Greer lab has shown that Histone 3 Lysine 27 trimethylation is extremely dynamic in cells when CIITA transcription is turned on and off. Upon stimulation to display Major Histocompatibility Complex II by interferon gamma, cells dramatically downregulate the trimethylation of H3K27me3. This is in coordination with an opening up of the promoter for transcription availability. Also, the opposite effect is shown in cells that do not constitutively express Major Histocompatibility Complex II and are not stimulated to produce this protein; high levels of this repressive mark are found at the CIITA promoter. This in turn would suggest that this histone repressive modification is what is in control of the expression of this protein (Wright and Ting, 2006).
The polycomb group proteins are extremely important in regulation of gene transcription. They have been shown to regulate the repression of HOX genes in insects and mammalian cells and have also been implicated in the direction of differentiation in pluripotent cells (Schuettengruber et al., 2007). It is argued that in the core complex of the Polycomb Repressive Complex 2 there is a group of 4 or 5 of these polycomb group proteins make up a complex called the Polycomb Repressive Complex 2. This is a complex that is responsible for trimethylating Histone 3 at lysine 27, which is a mechanism that allows the histones to associate with each other in a much tighter complex and leaves the chromatin, and ultimately the gene of interest, inaccessible to transcription factors. This causes a repression of that gene expression because transcription factors are unable to bind and RNA polymerase is never recruited to the gene promoter (Guenther and Young, 2010).
The most important protein in this complex is EZH2, which is the histone methyltransferase that is responsible for the catalytic activity of the Polycomb repressive complex 2. Mutations in EZH2 have shown that it is essential for normal cellular functions; when mutated in a cell, the cell generally shows many cancerous phenotypes. Although it is hard to pinpoint the exact mechanism for these cancerous properties, it is generally believed that the regulation of gene suppression through Polycomb Repressive Complex 2 and EZH2 is extremely important to most cell types (Sneeringer et al., 2010). In embryonic stem cells, which are pluripotent cells, the deregulation of Polycomb Repressive Complex 2 or EZH2 causes abnormal differentiation or an absence of differentiation altogether (Peng et al., 2009).
Although much is known about polycomb group proteins and the polycomb repressive complex 2, it is still unclear what sequences of DNA the polycomb complex is recruited to; PREs, or polycomb responsive elements are the homologous sequences on that the Polycomb Repressive Complex 2 complex binds with the help of a recruiter protein. These are studied and relatively well-known in Drosophila, but are a topic of continued debate in mammalian cells (Simon and Kingston, 2009.) There are no DNA binding motifs in any of the members of the polycomb repressive complex 2, and therefore it is thought that this complex does not directly bind to DNA. Taking this in mind, it is currently a topic of much speculation as to how this complex is recruited (Villa et al., 2007).
A possible suspect for the recruitment of the PRC2 to the CIITA pIV in mammalian cells is YY1. YY1 is a homolog of PHO, which is a protein in Drosophila known to bind to PREs in DNA and recruit the PRC2 to the promoter. It has been shown that YY1 can replace PHO in Drosophila and perform the same function (Srinivasan et al., 2005).
To begin the investigation as whether YY1 is a possible recruiter for the PRC2 in mammalian cells, a Chromatin Immunoprecipitation (ChIP) was performed to determine whether YY1 is present at the CIITA Promoter IV in HeLa cells. First, HeLa cells were plated on a 15 cm plate with a density of 2 million cells per plate. The cells were stimulated with IFN γ with the appropriate time course, and after the time course was finished, the cells were harvested and cross-linked. The first crosslinking reagent used was DSG, to crosslink protein-protein interaction. The second crosslinking reagent used was formaldehyde, to crosslink protein-DNA interaction. After this, the cells were lysed on ice in SDS buffer solution. Then, after cells were lysed, the cells were sonicated to fragment the DNA in the 750 base pair fragments. The lysate was run on an agarose gel to view sonication and if the sonication was effective, the lysates were pre-cleared with salmon sperm protein A beads to minimize the background. Once the samples were pre-cleared, 15 microliters were taken out as inputs and frozen. The remaining lysates were split into two, one sample was incubated with antibody for YY1 and the other was incubated with nonspecific rabbit IgG overnight. The next day, salmon sperm protein A beads were added to the lysates to pull out everything bound to the antibodies. The samples were then eluted, and the remaining protein was dissolved overnight. After, the DNA was isolated and purified and quantified using Real time PCR using primers and probes specific for CIITA promoter IV. Using this method, in theory, the antibodies specific for YY1 would pull out YY1 bound to DNA, and once the DNA is quantified it will be apparent how much YY1 was bound to the CIITA pIV in each instance.
Figure - YY1 ChIP with EZH2 control
In Figure 2, EZH2 is the control for the experiment because it is known that EZH2 decreases its presence at the CIITA promoter after IFN stimulation because the promoter is opened up for transcription and EZH2 is a repressive protein. Here it appears as high binding in unstimulated cells, and after 60 minutes there is a sharp drop in EZH2 binding, and at 18 hours there is a slight increase. The trend is the same in the YY1 samples. When looking at IgG however, it nullifies most of the results because there is a high background noise with IgG.
Figure - YY1 ChIP with H3K27me3 control
Figure 3 is the same experiment with different controls shown. Here, the control is the repressive histone modification Histone 3 Lysine 27 trimethylation, which is the target of EZH2 catalyzation. Here it is also expected that there be high levels of H3K27me3 in unstimulated cells, with this modification decreasing upon IFN stimulation as the CIITA promoter is opened up for transcription. This control is more in line with what was expected, although the 60 minute timepoint again does not show the decrease that was expected.
This preliminary data does not show us what was expected, but it is clear that the data is largely uninterpretable, with the background IgG controls being so high and nullifying the rest of the data. This is most likely because of a protocol error found in the dual crosslinking protocol. I believe that the cells were cross-linked too long with DSG and that they were cross-linked to each other with cell surface proteins interacting. This makes it much harder for the samples to be sonicated and separated out into what is truly binding to the DNA. What is probably happening as indicated by the high IgG controls is that there are giant complexes of cells and proteins cross-linked to each other and it is impossible to pull out individual proteins bound to the DNA. So for future experiments this protocol has been corrected and will be repeated. Once this is repeated and YY1 does actually show what we would expect, with less binding to the promoter upon stimulation with IFN, the next step would be to use siRNA to knock down YY1 and observe the effects on the cell involving CIITA transcription at the level of mRNA.