Specific Aims
Activation-induced cytidine deaminase (AID) encodes a DNA-editing deaminase, which is a group of the Cytidine Deaminase family (Bello). This protein is concerned in somatic gene conversion, hypermutation, and class-switch recombination of Ig (immunoglobulin genes). Activation-induced cytidine deaminase (AID) has been expressed in B cells at germinal center and starts the events, which lead to class switch recombination of immunoglobulin genes and somatic hypermutation. Additionally to this primary responsibility in immune diversification, deviant targeting of Activation-induced cytidine deaminase activity leads to translocations and point mutations of oncogenes linked with B-cell lymphoma. Lately, AID has been related in active DNA demethylation (Sariah).
Activation-induced cytidine deaminase (AID) has been studied broadly and found to be an essential component for antibody memory, has been thoroughly studied. The main objective of the research is to determine the molecular mechanism of activation-induced cytidine deaminase regulated genetic diversity targets Ig genes (Frederick). Since activation-induced cytidine deaminase (AID) has been well understood, the study will focus how AID regulated genetic diversity targets the Ig. In order to achieve this, the study will address two objective that include determining whether FACT & H3.3 have activity at V(D)J region. In addition, it will address whether RAG targets FACT & H3.3 to those regions. The research will accomplish the broad objective of the project by determining how AID regulated genetic diversity targets the Ig (Gunnar).
Significance and Background
The molecular mechanism of activation-induced cytidine deaminase (AID), which is essential for antibody memory, has been thoroughly studied. The question that now arises is how AID regulated genetic diversity targets the Ig. Somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GC) are dependent on transcription of the target region. This leads to the idea that there are targets on the Ig genes. DNA sequences that are prone to form irregular DNA structure called non-B DNA while transcriptions are found in the Ig genes. Formation of non-B DNA structure is believed to initiate the DNA by DNA topoisomerase I. Trimethylation of Lys4 residue in histone H3 (H3K4me3) promotes SHM and CSR. In Ig genes, H3K4me3 is present on the V(D)J and Sμ regions, which are the primary targets of the AID activity. Facilitates chromatin transcription (FACT) complex accumulation is thought to increase the frequency of CSR and SHM. This suggested that the nucleosomes in the SHM target region are more rapidly exchanged, which is shown by the presence of histone variant H3.3. It is known that the V(D)J recombinase, RAG, binds to H3K4me3 (Sanjay).
Activation-induced cytidine deaminase (AID) is has been identified in multiple malignancies of mature B-cell source and adds to the progression of lymphoma in a number of mouse models. The mechanism which guides activation-induced cytidine deaminase (AID) to its genetic target point is yet unknown and might be comparatively nonspecific, as many nonimmunoglobulin genes emerge to be targeted by activation-induced cytidine deaminase (AID) in both neoplastic B cells and normal. In reality, AID (activation-induced cytidine deaminase) binds to genes on each chromosome all through the genome and be able to provoke double-stranded DNA breaks, which leads to chromosome translocations at this point.
Up-and-coming evidence supports the main role of activation-induced cytidine deaminase (AID) in lymphomagenesis during genome-wide off-target introduction of chromosome translocations and point mutations. Further work is required to additionally define the scope and repercussion of off-target AID (activation-induced cytidine deaminase) activity in person lymphoma in addition to understanding the defensive mechanisms, which break down through the progression and development of disease (Sanjay).
Since activation-induced cytidine deaminase (AID) has been liked with disease development of diseases, with understanding how it target Ig it will help to describe how it is involved in disease progression. Activation-induced cytidine deaminase (AID), which is necessary for both somatic hypermutation and class switch recombination the Ig gene, has been observed in various types of human B cell lymphoma or leukemia. AID (Activation-induced cytidine deaminase) is a powerful mutator since it is linked in DNA breakage not only of other genes but also of Ig, other genes including proto-oncogenes (Geoffrey). Current studies propose that (Activation-induced cytidine deaminase) AID is requisite for chromosomal translocation concerning Ig loci and cmyc. Conversely, it is indistinct if AID plays other responsibilities in tumorigenesis (Gunnar). It has been examined the impact of Activation-induced cytidine deaminase (AID) deficiency on the creation of facade Ig-positive B cell lymphomas in Emu-cmyc transgenic mice. Approximately all lymphomas that developed in AID (Activation-induced cytidine deaminase)-deficient transgenic rat were pre-B cell lymphomas, while control transgenic mouse had primarily B cell lymphomas, signifying that AID (Activation-induced cytidine deaminase) is requisite for the progress of B but not pre-B cell lymphomas from cmyc over expressing lump progenitors. Therefore, AID (Activation-induced cytidine deaminase) might play multiple responsibilities in B cell lymphomagenesis (Sanjay).
The study will be a progression from the previous studies since it will evaluate the molecular mechanism of activation-induced cytidine deaminase regulated genetic diversity targets Ig genes. The previous studies have examined the correlation of activation-induced cytidine deaminase with cancerous cell. Whilst l this study will go further to establish the molecular mechanism of activation-induced cytidine deaminase regulated genetic diversity targets Ig genes. In humans, activation-induced cytidine deaminase (AID) expression fallout due to irritation and this deaminase activity is also concerned in carcinogenesis. The previous study has investigated the correlation amid AID expression and the clinical categorization of the oral cancer tissues. Activation-induced cytidine deaminase (AID) is has been recognized in multiple malignancies or cancers of mature B-cell basis and adds to the series of lymphoma in numerous cases of mouse models (Sebastian).
Research Design and Procedures
It has been established that the trimethylation alteration at the Lys4 remains in histone H3 (H3K4me3) encourage or promotes CSR.5 and SHM In Ig genes, H3K4me3 is particularly enriched on the Sµ regions and V(D)J, which are the mainly targets of the Activation-induced cytidine deaminase (AID) activity. Jamming or blockingH3K4me3 lead to reserve of CSR. It is also reported that SHM and CSR occurs more regularly in the regions at the transcription elongation factors the Spt5 accumulate.2,5,6 FACT complex and as these epigenetic points is frequently established in transcriptionally active region, it was predictable that there might be extra markers which are more definite for the Ig genes (Hanitsch).
Facilitates chromatin transcription abbreviated as, (FACT) is a heterodimeric protein multifaceted comprised of Spt16 and SSRP1 and was initially recognized from its biochemical activity to assist RNAPII transcription to lengthen on the chromatin template. It is projected that FACT (facilitates chromatin transcription) interrupts the nucleosome in front of the lengthening RNAPII and reverts it following sequence of the polymerase. It has been formerly identified, that FACT is obligatory for CSR.5 FACT knockdown radically reduced CSR effectiveness as well as the histone alteration H3K4me3, which was consequently found to be requisite for CSR (Philipsen).
Recently it was established that FACT (Facilitates chromatin transcription) also promotes SHM and accumulates particularly on the SHM target point on the Ig genes. From candidate screening for the SHM- supporting transcription lengthening factors, it is been found that the reduction of FACT (Facilitates chromatin transcription) most powerfully condensed the mutation on the SHM reporter transgene in individuals BL2 lymphoma cell. Significantly, it is observed strong FACT occupancy on the igh (immunoglobulin heavy chain) loci amid the Eµ intronic enhancer and the V(D)J region, and the 5′ surface of the Sµ switch region, together of which accrue SHM upon AID expression. In the meantime, FACT (Facilitates chromatin transcription) occupancy was only feeble at extremely transcribed non-Ig genes, which bear high Spt5, RNAPII, and H3K4me3 occupancies, revealing that the FACT (Facilitates chromatin transcription) accumulation on Igh is a gene- definite episode rather than a universal trait of transcription. Accumulations of FACT (Facilitates chromatin transcription) proposed that the nucleosomes in the SHM target point are more swiftly exchanged compared to those in other points or regions. Certainly, accretion of the histone variant H3.3, a characteristic of replication- autonomous histone turnover, concurs with the FACT (Facilitates chromatin transcription) enrichment. This double enrichment of FACT and H3.3 were observed not only in the kappa light chain locus, but also Igh in mouse spleen B cells (Rosen).
It may be interesting to test if FACT and H3.3 is concerned in the V(D)J recombination also. It is widely known and understood that the V(D)J recombinase RAG attaches to H3K4me3. Furthermore, current studies propose that the transcription-coupled commotion of nucleosome permits RAG to assail the recombination signal series. The current study will clarify the molecular methods of how FACT and H3.3 accrue on the Ig genes. Cooperatively, the finding will shed light on the significance of histone switch dynamics in the SHM-targeting method (Philipsen).
RAG targets FACT and H3.3 to those regions
V(D)J recombination is instigated by the lymphoid definite proteins RAG2 and RAG1, which jointly comprise the V(D)J recombinase. Nevertheless, the RAG1 and RAG2 complex may also act as transposes, interleaving the kaput DNA molecules produced through V (D) J recombination into an unconnected piece of DNA. This procedure, referred RAG transposition, can potentially lead chromosomal translocations, insertional mutagenesis, and genomic instability. It is well identified that the V(D)J recombinase RAG attaches to H3K4me3. H3K4me3 plays a serious responsibility in the activation-induced cytidine deaminase (AID) induced DNA cleavage of switch points in the IgH (immunoglobulin heavy chain locus) during class switch recombination (CSR). Facilitates chromatin transcription (FACT) is responsible for outlining H3K4me3 at AID loci. It is found that H3K4me3 loss is connected or correlate with defects in AID tempted DNA breakage and condensed mutation frequencies in immunoglobulin heavy chain loci in both the S and variable points and in non- immunoglobulin heavy chain loci for instance metastasis-linked to lung adenocarcinoma transcript 1 and miniature nucleolar RNA human gene 3 (SNHG3). Universal gene expression analysis exposed that Spt6 be able to act as both a negative and positive transcriptional controller in B cells, upsetting about 5% of the genes that comprise suppressor of AID and Ty4 (Spt4) (Sanjay). Captivatingly, Spt6 regulate AID and CSR expression during two distinctive histone alteration pathways, H3K36me3 and H3K4me3, correspondingly. RAG transposition actions are potentially injurious. Therefore, it is vitally significant which RAG transposition be concealed in rising T cells and B cells (Marta).
Methods
HSC-2 and HSC-3 cells will be extracted from lymph node tumors, which originate in patients having oral squamous cell carcinoma. Cells will be then cultured or treated in Dulbecco’s Modified Eagle Medium (GIBCO-BRL, Grand Island, NY) complemented with ten percent fetal bovine serum (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin in culture flask in humidified 5% CO2 at 37°C. Human germinal center B cell line (BL2 cells) will be cultured in RPMI1640 medium complemented with ten percent FBS, 100 mg/mL streptomycin, 100 U/mL penicillin, and 0.004% 2-mercaptoethanol in culture flask that will be humidified in 5% CO2 at 37°C (Sanjay).
Immuno staining of AID
For immunohistochemical analysis or scrutiny, formalin fixed, paraffin entrenched blocks will be attained from key tumors. Uninterrupted 4-µm sections will be consequently cut from every tumor block. The monoclonal antibody against AID to be incorporated EK2 5G9 and mAID2. Immunohistochemical staining will be executed with the Vectastain Elite ABC kit. The sections will be color-developed with substrate or chromogen diaminobenzidine (DAKO) and consequently counterstained with methyl green (Marta).
Evaluation of AID Immunohistochemical Staining
Two authors independently will examine the stained sections. In every case, two differently randomly chosen microscopic fields (200×) comprising more than 200 growth cells will be evaluated. After including the immune reactive cells and the sum number of cancer cells, the mean percentages of immune reactive cells will be calculated exclusive of knowledge of the clinical facts, and specimens will be classified into two clusters: negative (<5%) and positive (≥5%) (Marta).
Dual Fluorescent Immunostaining
All oral squamous cell carcinoma samples that showed AID on solitary immune staining will be used for double fluorescent immune staining. Double fluorescent staining for cytokeratins (CK) (AE1/AE3; DAKO) and AID (EK2 5G9) will be performed to determine the position of cancer cells exposing AID in every specimen. Deparaffinized division will be microwaved in the citrate buffer (pH 6.0) for twenty minute and stored in Protein Block Serum-Free for over twenty minutes (Philipsen). The specimens will be stored during the night at 4°C with key antibodies to CK and AID. The response or reaction product will be visualized by fluorescent goat anti-mouse. Samples will be counterstained with 4 inches; 6-diamidino-2-phenylindole and to be observed under a light microscope. No positive staining will be observed when the main antibodies to be replaced or omitted with usual serum in the negative controls through the staining process (Epeldegui).
The approach selected is aimed at establishing on the molecular mechanism of activation-induced cytidine deaminase regulated genetic diversity targets Ig genes. The approach can also attest if FACT and H3.3 is concerned in the V(D)J recombination also. It is widely known and understood that the V (D) J recombinase RAG attaches to H3K4me3. The result will enable one understand how Activation-induced cytidine deaminase (AID) has been identified in multiple malignancies of mature B-cell source and adds to the progression of lymphoma in a number of mouse models (Philipsen).
Work cited
Allen, Sariah J. “How phosphorylation affects the biochemical regulation and targeting of activation induced cytidine deaminase (AID).” Los Angeles, California: University of Southern California, 2012. Print.
Alt, Frederick W. “AID for Immunoglobulin Diversity.” San Diego, CA: Elsevier Science & Technology, 2007. Print.
Coico, Richard, and Geoffrey Sunshine. “Immunology: a short course.” 6th ed. Hoboken, N.J.: John Wiley, 2008. Print.
Alt, Frederick W. “AID for immunoglobulin diversity.” Amsterdam: Academic Press, 2007. Print.
Epeldegui, Marta. “The role of virus induced activation induced cytidine deaminase (AID) expression in the pathogenesis of AIDS-associated non-Hodgkin's lymphoma.” New York: klin, 2007. Print.
Fugmann, Sebastian. “DNA Deamination and the immune system aid in health and disease.” London: Imperial College Press ;, 2011. Print.
Geha, Raif S., and Fred S. Rosen. “Case studies in immunology: a clinical companion.” 5th ed. New York, N.Y.: Garland Science, Taylor and Francis Group, 2008. Print.
Hanitsch, Leif Gunnar. “Molecular mechanisms of class switch recombination the role of Activation-induced cytidine deaminase (AID) and the histone variant H2AX.” Newcastle upon Tyne: L. Erlbaum Associates, 2006. Print.
Philipsen, Antje. “Expression von AID-mRNA in menschlichen B-Lymphozyten und Non-Hodgkin-Lymphomen.” New York: New York University Press, 2007. Print.
Ranjit, Sanjay. “The C Terminus of Activation Induced Cytidine Deaminase (AID) Recruits Proteins Important for Class Switch Recombination to the IG Locus: a dissertation.” Kathmandu: Kluwer Academic Publishers, 2010. Print.
