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Immune system is the shield that wards off or alerts the organism of an invasion by pathogen or alien substance into the body of animals. It is able to respond to umpteen number of foreign invaders with specificity using variety of cells and molecules at its disposal. Recognition on the basis of surface molecules or chemical mediators and appropriate response to them are the two main properties required to generate an immune response. The immune system is able to identify even subtle chemical differences amongst the foreign pathogen can be picked up. The immune system also helps body to discriminate between self and non-self or sometimes altered cells. After recognition of the foreign element, immune system is able to translate the initial recognition event into a variety of effector responses, each uniquely suited for eliminating a particular type of pathogen. Later exposure to the same foreign organism induces a memory response, characterized by a more rapid and heightened immune reaction that serves to eliminate the pathogen and prevent disease.
Cells of the Immune System:-They are majorly classified into lymphoid and myeloid cells based on their lineage. The myeloid cells are classified into agranulocytes and granulocytes .Agranulocytes include the monocytes and macrophages whereas granulocytes are the eosinophils, basophils and neutrophils and the natural killer cells. Granulocytes are majorly involved in innate immune response which is more non -specific and less severe than adaptive immune response.
Lymphoid Cells:-Majorly involved in adaptive or acquired immunity. They are also agranulocytes.
The lymphocytes continually circulate in the blood and lymph and are capable of migrating into the tissue spaces and lymphoid organs, thereby integrating the immune system to a high degree.
Mature B cells display of membrane-bound immunoglobulin (antibody) molecules. Interaction between antigen and the membrane-bound antibody on a mature naive B cell; selectively induces the activation and differentiation of B-cell clones of corresponding specificity. In this process, the B cell divides repeatedly and differentiates over a 4- to 5-day period, generating a population of plasma cells and memory cells. Plasma cells, which have lower levels of membrane-bound antibody than B cells, synthesize and secrete antibody and help in elimination of antigen.
T Lymphocytes mature in thymus. These cells also have membrane bound receptor as in case of B cells. Depending upon the type of receptor they are differentiated into T Helper (TH) and T cytotoxic (Tc) cells. B cell is capable of binding soluble antigen, whereas the T cell is restricted to binding antigen displayed on self-cells. To be recognized by most T cells, this antigen must be displayed together with MHC molecules on the surface of antigen-presenting cells or on virus-infected cells, cancer cells, and grafts. The T-cell system has developed to eliminate these altered self-cells, which pose a threat to the normal functioning of the body.
MHC -is a tightly linked cluster of genes, whose products play roles in intercellular recognition and in discrimination between self and nonself. Antibodies made by plasma cells may react with antigens alone but most T cells recognize antigen only when it is combined with an MHC molecule. These MHC molecules act as antigen presenting structures on the surface of cells. The MHC determines the kind and intensity of immune response an individual will generate hence they are also critical in development of certain autoimmune diseases.
The MHC encodes three major kinds of molecules: MHC class I,MHC class II and MHC class III
Class II MHC genes encode glycoproteins expressed primarily on antigen-presenting cells (macrophages, dendritic cells, and B cells). MHC class II molecules are cell-surface glycoproteins that are of central importance to the adaptive immune system because they present peptides derived mainly from extracellular proteins to the antigen receptor of CD4+ T cells. They have two identical glycoprotein chains which are held together by non-covalent interactions. The two chains of the class II MHC molecules are encoded by DP, DQ, and DR regions in human leukocyte antigen cluster.
Regulation of MHC genes:
Genes for MHC molecules have 5' promoter sequences, which bind sequence-specific transcription factors. The promoter motifs and transcription factors that bind to these motifs have been identified for a number of MHC genes. For example MHC II transactivator, called CIITA, and another transcription factor, called RFX, both have been shown to bind to the promoter region of class II MHC genes. The expression of MHC molecules is also regulated by various cytokines. The interferons (alpha, beta, and gamma) and tumor necrosis factor have each been shown to increase expression of MHC molecules on cells. IFN gamma has been shown to be indirectly associated with regulation of MHC II genes since it activates CIITA expression. Some cytokines are also responsible for cell specific MHC expression.
CIITA and regulation of MHC II expression:
Regulation of MHC II occurs mainly at the transcription level. This regulatory module is present at 150-180 base pairs upstream of all MHC II genes and is highly conserved. This complex gene-expression profile is controlled almost exclusively by a single master regulatory factor, which is known as the class II transactivator. This molecule has several special features; an amino (N)-terminal region that is rich in acidic amino acids, three segments that are rich in proline, serine and threonine, a centrally placed GTP-binding domain, and a carboxy (C)-terminal region that consists of leucine-rich repeats (LRRs). Important region is the N terminal domain which is supposed to interact with many effector proteins that promote transcription. This is a non DNA binding activator. It has been shown to promote transcription of MHC II genes through various mechanism;
recruiting components such as transcription factor IID (TFIID) and TFIIB of the general transcription-initiation machinery
inducing phosphorylation of RNA polymerase II
interacting with the positive transcription elongation factor b (P-TEFb)
fourth, recruiting co-activators that alter chromatin accessibility by inducing histone acetylation or methylation.
recruiting the chromatin-remodelling factor BRAHMA RELATED GENE 1 (BRG1 )
This molecule has also been shown to have intrinsic Histone-acetylase activity.
Proteosome:- The proteasome is a multi protein complex consisting of two basic parts: a 20S proteolytic core complex and a 19S regulatory particle. The 19S is bound to either end of the 20S core and can be divided into a lid and a base. The base of the 19S is composed of 6 ATPases: S4, S6a, S6b, S7, Sug1 (or S8) and Sug2 (or S10b) and three non ATPase subunits which correspond, respectively, to yeast homologs Rpt1-6 . The base associates with the 20S catalytic core and regulates the access of substrates to the catalytic core.The base recognises ubiquitin bound to polyubiquitinated proteins. The 20S core has protease activity and is responsible for protein degradation.Published work from our lab has shown that 19S proteosome ATPAse Sug1and S6a play important role in regulating transcription initiation of CIITA and MHC-II genes.
Recent published work from our lab has shown that there is heavy recruitment of S6a at the CIITA pIV and and MHC II proximal promoters. So in all S6a is known to multi task as follows:
Assembly of HDAC,HAT and HMT's
CIITA promoter assembly
Assists the enhancesome assembly at MHC promoter
Recognition of Ub on CIITA
CIITA is known to be associated with kinases like CDK9 and CDK7 to phosphorylate RNApol II leading to initiation and elongation of transcription.
In yeast it has already been shown that 19S ATPase components do associate with transcription assembly and that it has crucial role in efficient elongation by RNApol II. So it would be relevant to check out whether all the 6 subunits have an interaction direct or indirect in the elongation process of transcription driven by RNA pol II and what is the specificity of interaction. Preliminary data with respect to this would include seeing whether these ATPases co-immunoprecipitate with RNA pol II.
During this semester in the the lab I majorly learnt the sterile tissue culture practices ,western blotting technique ,transfection of vector DNA into mammalian cells and finally co-immunoprecipitation technique:-
Tissue culturing for mammalian cells-HeLa Cells:- Passaging of cells or cell splitting
The attached cells on the T-175 flasks were washed with PBS(sterile).2.5 ml warm trypsin was added and the flask was incubated at 37°C for 5 minutes. This was done to detach the cells from surface. These trypsinised cells were then diluted using the DMEM media(DMEM+5mLpen/strp,37.5 FBS+5mL-Glutamine) that is used for culturing them. DMEM is known to deactivate trypsin activity so as to avoid over exposure of cells to trypsin. This cell suspension is then centrifuged at 1200rpm for 5 minutes at 4°C to get a pellet.The supernatant is discarded and the fresh 10 mL media is used to resuspend the pellet. Cells per mL are counted using a Haemocytometer. Cells are passaged twice a week.The dividing time span for HeLa being 24-30 hrs .
Transfecting mammalian cells using Fugene:-
FuGene 6 reagent[Roche] was used to transfect the adherent HeLa cells with extracellular DNA.DNA :FuGene is maintained at 1:3.FuGene 15µL was added to pre-warmed DMEM negative(without the FBS,pen/strep and L-glutamine)-500µL without touching the plastic surface of the eppendorfs.This was incubated for 5 minutes at 37°C.Vortexed DNA plasmid 5µg was added and incubatd at 37°C for 15minutes for the lipid complexes to form. After vortex and spin the entire contents of eppendorfs was added dropwise to plates with adhered cells. Proper mixing by rotating the plates was done.
To begin learning this technique, First I made 8% polyacrylamide gels using water,30%acrylamide,Tris pH 8.8,SDS,APS and TEMED in volumes as indicatd in the recipie book for various number of gels. (APS initiates the polymerisation of of acrylamide by generating free radicals followed by catalysis of decomposition of persulphate ions by TEMED ).Before this solution polymerises approximately 5 mL of it is poured into the gel forming assembly [BioRad kit].Around 1mL water i added from the top dropwise to seal the exposed gel surface thus preventing air bubbles in the gel.After polymerization the top water layer is removed by inverting and stacking gel containing water,30%acrylamide,Tris pH 6.8,sodium do-decylsulphate,APS and TEMED is made and around 2 mL is added on top of the separating(lower) gel. The stacking gel has a larger pore size(4% polyacrylamide) and allows to proteins to concentrate into sharp bands before entering the separating lower gel. This gel was then used after polymerisation to run samples.
I was given a set of lysates of some previous experiment done in the lab. These lyates were freezed and stored at -80°C. The lysates were defrosted .Then the lysate volumes were adjusted on the basis of Bradford assay and accordingly the loading dye (950µL of loading buffer or the tracker dye+50µL β-mercaptoethanol) volumes which is half of the lysate volume was added. These samples were vortexed and boiled in a water bath for 5 minutes. The β-mercaptoethanol reduces the disulphide bridges that are holding the the protein tertiary structures. Again the samples were vortexed and spun for 2 min. These samples (around 30µL each) were loaded on 8% gel as mentioned above. These samples were allowed to electrophorese in a running buffer (30gm of Tris base ,144.0 g of glycine ,add 10 g of SDS dilute to 1L of distilled water ) at 80V for first 15 minutes so that they can properly get stacked in the upper portion of the gel. Later the voltage is increased to 140V till the end of the run. The end of the run is identified when the tracker dye reaches the end of the gel. A dual colour protein marker [BioRad] loaded in the first lane(10µL) is separated out into its components as mentioned by the manufacturer. This marker is used to compare the molecular weight of the bands of the samples.
Tranferring the gels on Nitrocellulose membrane:
The gels from above were then transferred onto nitrocellulose membrane using a transfer assembly [BioRad].First the porus transfer pads were placed on the cassette, followed by filter paper, The gel was then placed on these ,followed by nitrocellulose membrane properly superimposed on it. On top of this again a filter paper is placed followed by porous filter pads.Care is taken to ensure removal of bubbles in this cassette using a roller. This cassette is then placed in the transfer buffer box and connected to 35V electric supply for overnight transfer. The nitrocellulose membrane and the filter paper are soaked in the transfer buffer ( Tris base, glycine,methanol and water) 5 minutes before use.
The nitrocellulose was then put in blocking buffer overnight (15mL)[Denville] to block the non specific sites of protein binding. The blot was then treated with a primary Ab specific for Myc tag.It was a Myc-HRP conjugate Ab.This was done because the cells from which the lysates were obtained were transfected with a Myc tagged gene segment.Transfection procedure was as mentioned above.
Figure1-Transfection of adherent cells and Western blotting
The band indicates that the transfection of Myc tagged S6a occurred because Myc-S6a is 50kDa .Since the samples were loaded as triplets similar thickness of band is expected in first three,then 2nd three and then the last three. However the last three do not appear constant.This coud be due to unequal loading.
Co-Imm unoprecipitation Experiment:-
To check whether RNA polymerase II and S6a interact with each other in a complex or indirectly in any close proximity we check if they co-immunoprecipitate
The idea behind this is that if S6a plays a role in transcription initiation RNA polymerase II which acts as a key component of the transcription machinery might get pulled out along with S6a.
RNA pol II
To check whether the antibodies pull out properly and to identify the desired band.
RNA pol II
To check the specificity of the Ab and beads used.
RNA pol II
To check whether S6a and RNA pol II immunoprecipitate
4hrs IFN-γ stimulation
RNA pol II
To check the effect of IFN stimulation on the interaction if any
750,000 HeLa cells were seeded on10cm plate containing complete DMEM media.Cells were allowed to attach onto the plates for around 4-6 hours.these cells were then transfected with 5µg Myc-S6a-7.4µL of stock--(This is the gene segment coding for S6a inserted into the vector pCMV along with an extra Myc tag).Next day morning 4th plate was stimulated with Interferon γ.After four hours of stimulatory effect ,cells were harvested. Cells were lysed using RIPA buffer(1M tris pH8,5M NaCl,10%NP40,10%SDS, DTT,dH2O).20µL PI was added to each mL of RIPA.200µL of this (RIPA+PI)is used to lyse cells for 20 minutes on ice.1%NP40(Tris pH8,KCL 100%NP40,0.5MEDTA,5M NaCl,DTT,dH2O+PI (800µL)used to dilute the effect of RIPA.This lysate was centrifuged at 13.3g for 15 to 20 minutes. 960µL of the lysate was precleared using 50µLmouse IgG beads+15µLProteinG beads to get rid of the background proteins for an hour and half.The protein levels were normalised using Bradford assay. 2 sets of lysates and one set for the immune-precipitation(IP) based on calculations of Bradford was prepared. The lysates were frozen at -80°C with the calculated amount of loading dye. The samples for IP contained Rabbit Anti-RNApolII Ab (AbCam)50µL and Anti-Mouse IgG Ab(Sigma)20µL in the first and second tubes respectively.The third and fourth tubes contained the Anti-Myc Beads 50µL.These tubes were kept overnight for IP on rotator at 4°C.Next day IgG beads were added to first and second tubes and put on rotation for two hours. This was followed by washing the beads off the nonspecific proteins using 1:5 times diluted 1%NP40.Three such washes 10 minutes each were done. 30µL loading dye was then added to the washed off bead containing IP samples.Simultaneously ,the frozen lysates were defrosted and prepared for loading on the gel.IP samples and one set of lysates were loaded onto 6% gels and the lysates to check for MYC transfection efficiency on 8% gel.Gel electrophoresis and transfer onto nitrocellulose membrane was done as mentioned earlier.
The membranes were blocked overnight [Denville's Blocking buffer] .Co-IP blot was treated with 1° Rabbit Anti-RNA pol II Ab (1:1000)-[AbCam] overnight.3 Washes 15 minutes each with 1X TBS(30mLTris pH 7.0 and 5M NaCl-10mL) with 0.5% Tween 20-1mL in one litre. This is followed by 2° HRP conjugate Anti- Rabbit Ab(1:20000)[Santa Cruz] treatment for an hour. The blot to check Myc expression is treated with Mouse HRP conjugated Anti Myc Ab(1:2000) [Santa Cruz].Blots were developed using chemiluminiscence reagent [Denville].
IB:1°Anti RNApolII Ab(1:1000)
2°-Anti rabbit HRP conjugate Ab(1;20000)
220 kDa band
1° Ab-1:1000 Rabbit Anti-RNA polII
2° Ab 1:20000 Anti rabbit HRP conjugate
Band for RNA pol II is expected at its molecular weight of 220kDa.Since we IP for S6a and on immunoblotting for RNA polII we get a band in the region of RNA polII; it seems that S6a and RNA pol II co-immunoprecipitate. However to get better blots we have been troubleshooting with the new Antibody stocks used for this experiment.
Figure 3:-Myc lysates showing Myc-S6a expressed.
IB:-Anti Myc-HRP conjugated Ab(1:2000)
Discussion and future direction:-
If S6a and RNA pol II co-immunoprecipitates then it can be hypothesised that this proteosomal subunit might have some role in the elongation process. Checking whether other subunits co-immunoprecipitate with RNA pol II could be my future line of work. This would help us understand better whether the subunits have specific function or they have a common task to achieve. Secondly studies have indicated that CIITA has a role in activating certain kinases of pTEFb complexes which is involved in the shift of transcription initiation to elongation. So the next probable question would be if the ATPases play a role in elongation do they influence in someway the pTEFb stability. Hexim which is reported as the negative transcription factor which inhibits pTEFb activity also might be involved somewhere in the S6a mediated role in elongation. So future experiments would involve checking if Sug1 one of the ATPases would co-immunoprecipitate with CDK9 or Hexim.
CIITA-Class II transactivator
DMEM-Dulbecco's Modified Eagle medium
FBS-Fetal Bovine Serum
MHC-Major Histocompatibility Complex
PAGE-Polyacryamide gel electrophoresis
Pen/strep-Penicilin and streptomycin
pTEFb-positive transcription elongation factor b
TBST:-Tris buffered saline Tween 20