The immune system is the human organic structure ‘s first line of defence against occupying pathogens. It is what is responsible for the targeting and devastation of foreign agents and bar of farther invasion. The immune system is non a specific organ, but instead a collective of cellular responses. These cellular responses are comprised of specialised cells that are targeted and activated by a specific foreign agent, or a generalised response activated by any foreign stimulation. The former cellular immune response is by and large referred to as adaptative unsusceptibility.
An of import constituent of the adaptative unsusceptibility is the T cell. T cells are the orchestrators of the adaptative immune response. These cells recognize foreign antigen and one time activated, travel on to trip other cells of the immune system, including macrophages and B cells. Another constituent of the adaptative immune system is the professional antigen showing cell, which brings foreign agents to the regulators of adaptative unsusceptibility. Examples of professional antigen showing cells include dendritic cells and macrophages. These cells can observe foreign agents on its surface, internalise these agents, and show fragments of the foreign peptides on its surface for immune surveillance. The professional antigen showing 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 trip it with a costimulatory molecule. They are constitutively expressed on professional antigen showing cells, whose chief occupation is to show antigens to T cells. All other nucleated cells can be induced to show MHC category II molecules with cytokines such as Interferon-? . The MHC Class II molecule has been shown to be controlled by a maestro regulator known as Class II Transactivator ( CIITA ) , which binds to the CIITA booster with the aid of a composite of written text factors ( Figure 1 ) . W
Figure. MHC Class II Promoter and Transcription Factors
CIITA is the maestro regulator of MHC Class II written text. It has been shown that the upregulation of MHC Class II parallels the upregulation of CIITA degrees in the cell. CIITA is constitutively expressed in B cells and dendritic cells and is inducible in all nucleated cells. Presently, it is widely accepted that there are four boosters for CIITA look. Dendritic cells use CIITA booster I for look, B cells use CIITA booster III for look and booster IV is used for inducible look in all other nucleated cells. Recently, it has been shown that epigenetic ordinance of CIITA can turn out to be utile in the ordinance of surface look of MHC category II ( Harton and Ting, 2000 ) .
Epigenetic control of a cistron involves post translational alteration of histones to either adhere the histones tightly together, or open up the chromatin construction by diminishing the association between histones. Unpublished informations in the Greer lab has shown that Histone 3 Lysine 27 trimethylation is highly dynamic in cells when CIITA written text is turned on and off. Upon stimulation to expose Major Histocompatibility Complex II by interferon gamma, cells dramatically downregulate the trimethylation of H3K27me3. This is in coordination with an opening up of the booster for written text handiness. Besides, the opposite consequence is shown in cells that do non constitutively express Major Histocompatibility Complex II and are non stimulated to bring forth this protein ; high degrees of this inhibitory grade are found at the CIITA booster. This in bend would propose that this histone inhibitory alteration is what is in control of the look of this protein ( Wright and Ting, 2006 ) .
The polycomb group proteins are highly of import in ordinance of cistron written text. They have been shown to modulate the repression of HOX cistrons in insects and mammalian cells and have besides been implicated in the way of distinction in pluripotent cells ( Schuettengruber et al. , 2007 ) . It is argued that in the nucleus composite of the Polycomb Repressive Complex 2 there is a group of 4 or 5 of these polycomb group proteins make up a composite 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 tie in with each other in a much tighter composite and leaves the chromatin, and finally the cistron of involvement, unaccessible to written text factors. This causes a repression of that cistron look because written text factors are unable to adhere and RNA polymerase is ne’er recruited to the cistron booster ( Guenther and Young, 2010 ) .
The most of import protein in this composite is EZH2, which is the histone methyltransferase that is responsible for the catalytic activity of the Polycomb repressive composite 2. Mutants in EZH2 have shown that it is indispensable for normal cellular maps ; when mutated in a cell, the cell by and large shows many cancerous phenotypes. Although it is difficult to nail the exact mechanism for these cancerous belongingss, it is by and large believed that the ordinance of cistron suppression through Polycomb Repressive Complex 2 and EZH2 is highly of import to most cell types ( Sneeringer et al. , 2010 ) . In embryologic root cells, which are pluripotent cells, the deregulating of Polycomb Repressive Complex 2 or EZH2 causes unnatural distinction or an absence of distinction wholly ( Peng et al. , 2009 ) .
Although much is known about polycomb group proteins and the polycomb repressive composite 2, it is still ill-defined what sequences of DNA the polycomb composite is recruited to ; PREs, or polycomb antiphonal elements are the homologous sequences on that the Polycomb Repressive Complex 2 complex binds with the aid of a recruiter protein. These are studied and comparatively well-known in Drosophila, but are a subject of continued argument in mammalian cells ( Simon and Kingston, 2009. ) There are no DNA adhering motives in any of the members of the polycomb repressive complex 2, and therefore it is thought that this composite does non straight adhere to DNA. Taking this in head, it is presently a subject of much guess as to how this composite is recruited ( Villa et al. , 2007 ) .
A possible suspect for the enlisting 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 adhere to PREs in DNA and enroll the PRC2 to the booster. It has been shown that YY1 can replace PHO in Drosophila and execute the same map ( Srinivasan et al. , 2005 ) .
To get down the probe as whether YY1 is a possible recruiter for the PRC2 in mammalian cells, a Chromatin Immunoprecipitation ( ChIP ) was performed to find whether YY1 is present at the CIITA Promoter IV in HeLa cells. First, HeLa cells were plated on a 15 centimeter home base with a denseness of 2 million cells per home base. The cells were stimulated with IFN ? with the appropriate clip class, and after the clip class was finished, the cells were harvested and cross-linked. The first crosslinking reagent used was DSG, to crosslink protein-protein interaction. The 2nd 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 break up the Deoxyribonucleic acid in the 750 base brace fragments. The lysate was run on an agarose gel to see sonication and if the sonication was effectual, the lysates were pre-cleared with pink-orange sperm protein A beads to minimise the background. Once the samples were pre-cleared, 15 microliters were taken out as inputs and frozen. The staying lysates were split into two, one sample was incubated with antibody for YY1 and the other was incubated with nonspecific coney IgG overnight. The following twenty-four hours, pink-orange sperm protein A beads were added to the lysates to draw out everything edge to the antibodies. The samples were so eluted, and the staying protein was dissolved overnight. After, the Deoxyribonucleic acid was isolated and purified and quantified utilizing Real clip PCR utilizing primers and investigations specific for CIITA booster IV. Using this method, in theory, the antibodies specific for YY1 would draw out YY1 edge to DNA, and one time the Deoxyribonucleic acid is quantified it will be evident how much YY1 was bound to the CIITA pIV in each case.
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 booster after IFN stimulation because the booster is opened up for written text and EZH2 is a inhibitory protein. Here it appears every bit high binding in unstimulated cells, and after 60 proceedingss there is a crisp bead in EZH2 binding, and at 18 hours there is a little addition. The tendency is the same in the YY1 samples. When looking at IgG nevertheless, it nullifies most of the consequences 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 inhibitory histone alteration Histone 3 Lysine 27 trimethylation, which is the mark of EZH2 catalyzation. Here it is besides expected that there be high degrees of H3K27me3 in unstimulated cells, with this alteration diminishing upon IFN stimulation as the CIITA booster is opened up for written text. This control is more in line with what was expected, although the 60 minute timepoint once more does non demo the lessening that was expected.
This preliminary information does non demo us what was expected, but it is clear that the information is mostly uninterpretable, with the background IgG controls being so high and invalidating the remainder of the information. This is most likely because of a protocol mistake found in the double crosslinking protocol. I believe that the cells were cross-linked excessively 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 genuinely adhering to the Deoxyribonucleic acid. What is likely go oning as indicated by the high IgG controls is that there are elephantine composites of cells and proteins cross-linked to each other and it is impossible to draw out single 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 really demo what we would anticipate, with less adhering to the booster upon stimulation with IFN, the following measure would be to utilize siRNA to strike hard down YY1 and detect the effects on the cell affecting CIITA written text at the degree of messenger RNA.