Gotowa bibliografia na temat „Recombination activating genes”

Abstract It has been established that meiotic recombination and chromosome segregation are inhibited when meiotic DNA replication is blocked. Here we demonstrate that early meiotic gene (EMG) expression is also inhibited by a block in replication. Since early meiotic genes are required to promote meiotic recombination and DNA division, the low expression of these genes may contribute to the block in meiotic progression. We have identified three Hur– (HU reduced recombination) mutants that fail to couple meiotic recombination and gene expression with replication. One of these mutations is in RPD3, a gene required to maintain meiotic gene repression in mitotic cells. Complete deletions of RPD3 and the repression adapter SIN3 permitted recombination and early meiotic gene expression when replication was inhibited with hydroxyurea (HU). Biochemical analysis showed that the Rpd3p-Sin3p-Ume6p repression complex does exist in meiotic cells. These observations suggest that repression of early meiotic genes by SIN3 and RPD3 is critical for the normal response to inhibited replication. A second response to inhibited replication has also been discovered. HU-inhibited replication reduced the accumulation of phospho-Ume6p in meiotic cells. Phosphorylation of Ume6p normally promotes interaction with the meiotic activator Ime1p, thereby activating EMG expression. Thus, inhibited replication may also reduce the Ume6p-dependent activation of EMGs. Taken together, our data suggest that both active repression and reduced activation combine to inhibit EMG expression when replication is inhibited.

Assembly of T cell receptor (TCR)α/β genes by variable/diversity/joining (V[D]J) rearrangement is an ordered process beginning with recombination activating gene (RAG) expression and TCRβ recombination in CD4−CD8−CD25+ thymocytes. In these cells, TCRβ expression leads to clonal expansion, RAG downregulation, and TCRβ allelic exclusion. At the subsequent CD4+CD8+ stage, RAG expression is reinduced and V(D)J recombination is initiated at the TCRα locus. This second wave of RAG expression is terminated upon expression of a positively selected α/β TCR. To examine the physiologic role of the second wave of RAG expression, we analyzed mice that cannot reinduce RAG expression in CD4+CD8+ T cells because the transgenic locus that directs RAG1 and RAG2 expression in these mice is missing a distal regulatory element essential for reinduction. In the absence of RAG reinduction we find normal numbers of CD4+CD8+ cells but a 50–70% reduction in the number of mature CD4+CD8− and CD4−CD8+ thymocytes. TCRα rearrangement is restricted to the 5′ end of the Jα cluster and there is little apparent secondary TCRα recombination. Comparison of the TCRα genes expressed in wild-type or mutant mice shows that 65% of all α/β T cells carry receptors that are normally assembled by secondary TCRα rearrangement. We conclude that RAG reinduction in CD4+CD8+ thymocytes is not required for initial TCRα recombination but is essential for secondary TCRα recombination and that the majority of TCRα chains expressed in mature T cells are products of secondary recombination.

A recent paper from the Alt laboratory shows that recombination activating genes (RAGs) are not responsible for double-strand DNA breaks associated with some chromosomal translocations in pre–T-cell lymphomas.

Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.

T lymphocytes regulate the initiation, duration, and magnitude of adaptive immune responses and function as effector cells in cell mediated immunity. To become immunologically competent they must generate functional antigen receptors. This process takes place in the thymus and requires somatic recombination of T cell receptor (TCR) genes. It is mediated by the endonucleases recombination activating gene-1 (RAG1) and RAG2. Although the thymus regresses at puberty, T cells are present throughout life implying that other tissues must provide the proper milieu for T cell development. This thesis describes extrathymic T cell maturation in man. RAG1, RAG2, and the preTα-chain (pTα), which is exclusively utilized in developing T cells, were used as markers for TCR gene rearrangement. Two new exons (1A and 1B) encoding sequences in the 5’ untranslated region (5’UTR) of mRNA were discovered in the human RAG1 gene. The previously described 5’UTR exon (renamed 1C) was located between the new exons and exon 2, the latter containing the entire coding sequence. We found that small intestinal lymphocytes of the T cell lineage expressed the new exons in three different splice forms. RAG1 mRNA containing the 1C exon was not expressed in small intestinal lymphocytes. In contrast, splice forms containing the 1A exon were not expressed in thymocytes. RAG1 and pTα mRNA expressing lymphocytes were seen both within the epithelium and in lamina propria. Thymocyte-like CD2⁺CD7⁺CD3^-, CD4⁺CD8⁺, CD1a⁺, and IL7-R+ lymphocytes were identified in the small intestinal mucosa. CD2⁺CD7⁺CD3^- cells had the highest expression levels of mRNA for RAG1 and pTα, suggesting that the small intestinal mucosa is indeed a site for T cell maturation. Small intestinal T lymphocytes were also shown to kill via the Fas/FasL pathway in a TCR/CD3 independent manner and via the perforin/granzyme pathway in a TCR/CD3 dependent manner. The Fas/FasL-mediated cytotoxicity may reflect an ongoing selection process of extrathymically maturated T cells.

The nasopharyngeal tonsil is the major inductive site for immune reactions against inhaled antigens. Previous demonstration of RAG1 expression in tonsillar B cells was interpreted as antigen driven receptor revision. The present study confirms the expression of RAG1 in B cells. We also found that RAG1, RAG2, and pTa mRNAs were expressed in lymphocytes of the T cell lineage. A small population of cells with the immature phenotype CD2+CD7+CD3- was demonstrated. This population had the highest expression levels of mRNA for RAG1, RAG2, pTα and terminal deoxynucleotidyl transferase. All four splice-forms of RAG1 mRNA were expressed. RAG1 and pTα mRNA expressing cells were mainly located in the proximity of the surface epithelium and in the outer rim of the follicles. These results suggest that the nasopharyngeal tonsil is a site where extrathymic T cell development and antigen driven TCR revision are occurring in parallel.

Celiac disease (CD) is a small intestinal enteropathy characterized by permanent intolerance to gluten. Gluten reactive intestinal T cells are central in the pathogenesis and CD can be regarded as a failure to maintain tolerance to this food antigen. Expression of the RAG1 1A/2 splice form was significantly decreased in small intestinal T cell subsets of CD patients suggesting that impaired TCR gene rearrangement could contribute to failure of maintain tolerance in CD.

Together, these findings show that both small intestinal and nasopharyngeal tonsillar lymphocytes of T cell lineage have the molecular machinery for antigen receptor rearrangement and that thymocyte-like lymphocytes are present in both tissues. Thus these organs are likely sites of T lymphocyte ontogeny as well as for secondary T cell receptor rearrangement in man.

RAGs (Recombination Activating Genes) are responsible for generation of antigen receptor diversity in case of B-cells and T-cells, through the process of combinatorial joining of different V (variable), D (diversity) and J (joining) gene segments. Each of these segments are flanked by recombination signal sequences (RSS), which consist of a conserved heptamer and nonamer separated by a less conserved spacer of 12 or 23 bp. RAGs recognize and cleave at the 5’ end of heptamer, leading to the formation of hairpin coding ends and blunt signal ends. The coding ends are joined through the process of no homologous DNA end joining (NHEJ), leading to the rearrangement of variable region of antigen receptors. Apart from its physiological property, RAGs can also act as a structure-specific nuclease. Previously, it has been shown that inadvertent action of RAGs on cryptic RSS and non B-DNA structures can lead to the generation of genomic instability and cancer. A very coordinated expression of RAGs has been observed in pro- and pre-B cells of the lymphoid system, which overlaps with the window of productive rearrangement during V(D)J recombination. Besides, studies by us and others have shown that RAG cleavage at altered DNA structures and cryptic RSS leads to chromosomal translocations resulting into cancer. However, several questions related to regulation of RAG expression and its activity in lymphoid cells remains to be answered. Previous studies have suggested regulation of RAG expression at different levels, such as methylation, ubiquitination, phosphorylation and by coordinate action of various transcription factors. In the present study, we evaluate the potential role of miRNAs in the regulation of RAG expression and its function in lymphoid cells. miRNAs are small, single-stranded non-coding RNAs, which play an important role in the regulation of gene expression. They play a critical role in the regulation of different cellular functions. Although there are miRNAs identified to play critical role during development of immune system, several key questions such as its role in the regulation of RAGs is yet to be addressed. In the current study, we have used bioinformatics approach to extract potential miRNAs that bind to 3’UTR of RAG1 and RAG2. miRNA expression datasets were downloaded from NCBI SRA database and extensive evaluation was done using various bioinformatics tools such as Bowtie, Sam tools, Bam tools, Bed tools and R package. We screened the miRNA expression profile across different stages of B-cell development (pro, pre, immature and mature B-cells), which overlap with the narrow window of RAG expression. The shortlisted miRNAs were further analyzed using miRNA databases such as miRBase, Targetscan and EMBL. Results showed that 33 miRNAs were specific to RAG1, among that one (miRNA1) followed RAG expression profile in B-cells. Besides miRNA2, which is a novel miRNA, was selected only on the basis of RAGs expression profile in a stage specific manner and the complementarity of the seed sequence of miRNA2 to the 3’UTR of RAG1 was checked manually. Interestingly, we observed that RAG1 expression was significantly down regulated in the presence of these miRNAs. However, there was no significant difference in the levels of other genes analysed. Further, semi-quantitative RT-PCR analysis confirmed the endogenous processing of pre-miRNA into mature miRNA using the cellular machinery. Besides, enrichment of 3’UTR of seed region of these miRNAs, enhanced the expression level of RAG1. Importantly, the enhancement in RAG1 expression level was limited in case of mature B-cells, where RAG expression is normally not observed. Further, transfection of lymphoid cells with miRNA inhibitors, specific to the miRNAs under study, showed the enhancement in RAG1 expression in lymphoid cells. In addition to this, specificity of selected miRNAs was confirmed by performing 3’UTR reporter assays, where enhanced luciferase expression was observed in case of mutant 3’UTR, while it was minimal in case of wild type constructs. Endogenous expression levels of selected miRNAs were evaluated in both lymphoid and nonlymphoid normal tissues and cancer cells using RT-PCR. Interestingly, we observed inverse correlation of expression levels of miRNA and RAG expression in all the cells tested. Besides, miRNA expression levels were less in pre-B cells and T-cells, owing to the increased expression of RAGs. Apart from this, recombinogenic potential of candidate miRNAs was assessed using episomal based V(D)J recombination assays. Interestingly we observed significant decrease (2-4 fold) in the V(D)J recombination efficiency when miRNA1 or 2 constructs were transfected in Nalm6 cells, as compared to that of controls, where no miRNAs were used. However, in case of Reh cells upon transfection with miRNA1construct, the decrease in recombination potential was upto 9 fold. Hence, we identify two miRNAs that can play an important role in the regulation of RAG1 expression and its physiological activity. Further, studies are being carried out to confirm their role in the regulation of RAG1 during different developmental stages of lymphoid cells in mice. As stated above, in addition to the sequence-specific activity, RAG possesses structure-specific nuclease activity as well. It has been shown that RAGs can cleave different types of altered DNA structures. Studies from our laboratory showed that even when RAGs act as a structure-specific nuclease there is a sequence bias. Presence of cytosine and thymine at the single-stranded region of heteroduplex DNA is important for RAG nicking and double-strand break (DSB) formation. In addition, proximity of a nonamer to bubble structures can enhance RAG cleavage. However, the role of immediate flanking sequences in the RAG mediated cleavage at heteroduplex regions is not understood. We investigated the role of flanking double-stranded DNA sequences in the regulation of RAG cleavage on non-B DNA structures. We found that RAG binding and cleavage on heteroduplex DNA is dependent on the length of double-stranded flanking region. Besides, immediate flanking regions of the heteroduplex DNA affected the RAG binding and cleavage in a sequence dependent manner. Interestingly, we also observed that the cleavage efficiency of RAGs at heteroduplex region was influenced by the phasing of DNA. Thus, our results suggest that sequence, length and phase positions of the DNA can affect the efficiency of RAG cleavage when it acts as a structure-specific nuclease. These findings provide novel insights into regulation of the pathological action of RAGs. Previous studies have shown that in addition to formation of coding and signal joints during V(D)J recombination, nonstandard V(D)J recombination products known as hybrid joints and open-shut joints may be formed, particularly in certain aberrant conditions such as defective NHEJ machinery. Interestingly, the hybrid and open-shut joints closely resemble the transposition mechanisms associated with transposons oretroviruses. Studies have also shown that RAGs possess structural similarity with integrases in domain organization. Both the proteins have Zinc Finger Binding domain (ZFB) which helps in multimerization of the protein, a central catalytic core domain comprising three acidic amino acids D, D and E essential for enzymatic activity and C-terminal domain (CTD) responsible for nonspecific binding to the DNA. Previous studies from our laboratory showed that, Elvitegravir, an inhibitor of integrase could interfere with the biochemical functions of the RAGs in vitro. Specifically, it inhibited the RAG binding and cleavage at RSS, hairpin formation, post-cleavage complex formation involving 12RSS and 23RSS. Using the episomal assay system that mimics signal joints (pGG49) and coding joints (pGG51), we show that Elvitegravir can inhibit V(D)J recombination inside cells. Interestingly we observed 3-6 fold decrease in the recombination frequency in signal ends joining, when treated with increasing concentrations (100, 500 and 1000 nM) of Elvitegravir. A 5-8 fold decrease in coding joints formation was also observed upon treatment with the inhibitor. The presence of recombination was confirmed by restriction digestion followed by sequencing analysis. Further analysis of recombination junctions revealed extensive deletion before joining in the case of Elvitegravir treated samples. Insertions or substitutions near to the recombination junctions were also prominent in treated samples. In depth analysis of sequenced junctions showed the presence of sequence having the features to form hairpins both upstream and downstream to the RSS sequences and was the site of cleavage in cases were higher deletion was observed. The analyzed recombinants did not show any signal joints or coding joints formation in treated samples. This suggests that Elvitegravir affects the physiological function, the V(D)J recombination of RAGs inside the cells. Thus, in the present study, we show that RAGs can be regulated by specific miRNAs. We have identified two potential miRNAs, which can regulate the RAG expression as well as its function in different stages of B- and T-cell development. Further, we also identify a novel regulatory mechanism for the structure-specific activity of the RAG complex. In addition to this, we find that integrase inhibitor, Elvitegravir, affects V(D)J recombination within B-cells, indicating its potential deleterious impact in HIV patients, which needs to be further evaluated.

碩士
國立成功大學
生物學系碩博士班
97
Orange-spotted grouper (Epinephelus coioides) is a fish species with a high economic importance in the aquaculture industry in Taiwan. The high mortalities observed throughout early development such as viral nervous necrosis (VNN) causes the highest mortalities up to 100% always occur among 1-month-old larvae with total body lengths of 2.0 cm. Teleost is the oldest species has the adaptive immune system. However, there is a risk of inducing immunological tolerance if fish that are immunised at a very early age before they are immunocompetent. Thus, it is important to establish the earliest time that grouper can be vaccinated or give immunopotentiating agent. Recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination. During B lymphocyte development, the variable region of Ig gene is assembled by the recombination of multiple V, D, J segments, which can generate a vast array of immunoglobulin M (IgM) to against numerous antigen. IgM produced by B lymphocytes is also an important gene that can be used in the study of the ontogenesis of the immune system, as it is the first formed antibody of the primary humoral component of the acquired immune system in fish species. These genes are expressed together in this study in order to understand the expression profile of them. The genes, rag1 and rag2, of E. coioides were cloned and sequenced the open reading frames. The full-length cDNA of rag1 and rag2 were 3653 and 1875 base pairs (bp) long respectively. The lengths of rag1 and rag2 open reading frame were 3216 and 1602 bp encoding 1071 and 533 amino acids with the molecular weight of putative protein were about 117 and 58 kDa. Subsequently, the rag1, rag2 and IgM mRNA expression level in ontogeny of fish and different organs was evaluated by reverse transcriptase PCR (RT-PCR). The result showed the expression level of rag1, rag2 and IgM mRNA raised after 13, 13 and 22 dpf of fries respectively. This data was suggested that orange-spotted grouper at this stage might possess mature immunity and is able to produce immunoglobulin. The expression of rag1 was observed in thymus, head kidney and trunk kidney. The expression of rag2 was observed in thymus and head kidney. The expression of IgM was observed in thymus, head kidney, trunk kidney, spleen, intestines and pancreas. This data forms the basis for a proposal that the thymus and head kidney of teleost species play an essential developmental role in lymphopoiesis and thus can be regarded as a primary lymphoid organ.

Recombination Activating Genes (RAGs) orchestrate the process called V (D) J recombination, which enables the vertebrate adaptive immune system to specifically recognize millions of antigens. During this recombination process, V (variable), D (diversity) and J (joining) gene segments of antibody (B cell receptor) and TCR (T cell receptor) join by different possible combinations to generate antigen receptor diversity. This unique site specific recombination process is actuated by lymphoid specific proteins called RAG1 and RAG2 (RAGs or RAG complex). RAGs recognize a conserved sequence motif flanking the above subexons called Recombination Signal Sequence (RSS). There are two types of RSS known as 12-RSS and 23-RSS, where a conserved heptamer sequence and nonameric sequence is separated by 12 or 23 bp, respectively. RAGs specifically bind to RSS by RAG1 Nonamer Binding Domain (NBD) and generate nicks which are converted to DSBs via a hairpin intermediate and finally repaired by Non-Homologous DNA End Joining (NHEJ), a major DSB repair pathway in eukaryotes. Thus, RAGs act as a sequence specific endonuclease, and is unique to higher eukaryotes. Therefore, reduced or loss of RAG activity could result in immune deficiency syndromes like Omenn Syndrome (OS) and Severe Combined Immunodeficiency (SCID). Apart from acting as a sequence specific nuclease, RAGs have been shown to cleave on altered DNA structures like mismatches (bubbles), hairpins, flaps, gaps, triplexes and 3’ overhangs. This structure specific nuclease activity is implicated in causing genomic instability in B and T cells, particularly leading to generation of chromosomal translocations in certain lymphoid cancers. However, unlike the sequence- specific cleavage activity, this novel property of RAGs is poorly understood. Structure-specific nuclease activity of RAGs was characterized by using heteroduplex DNA substrate containing bubble region. RAG proteins were overexpressed and purified from human cell line and used for the assay. Results showed that RAGs cleave different bubble substrates with different efficiency. The role of DNA sequence at single-stranded region of heteroduplex DNA on RAG cleavage was investigated by synthesizing the substrate DNA having either adenineguanine/ thymine/ cytosine in the bubble sequence. Interestingly, efficient RAG cleavage was observed only when cytosines were present at single-stranded region, while thymine bubbles were cleaved with much lower efficiency. Adenine and guanine containing bubbles were not cleaved by RAGs. This was the first observation showing sequence specificity during structure-specific nuclease activity of RAGs. Besides, it was observed that RAG cleavage on bubbles with cytosines resulted in DSB formation, which is essential for generation of chromosomal translocations. Further, such specificity and cytosine preference was observed, even when RAGs acted on other altered DNA substrates like hairpin loops, 3’ overhangs and gaps. When the role of flanking duplex region on RAG cleavage was tested, only the 5’ duplex nucleotide was critical for RAG cleavage reaction and cytosine was the most preferred nucleotide. By systematic mutation of bubble region, it was observed that the two cytosines present at the double strand-single strand junction are critical for RAG cleavage. A single nucleotide bubble (mismatch) with cytosines was cleaved by RAGs with low but detectable efficiency. A bubble with at least 2 nt length possessing cytosine was cleaved with higher efficiency resulting in both single-stranded nicks and DSBs. Based on these studies, “C(d)C(s)C(s)” was proposed as a novel recognition motif for RAG cleavage, on altered DNA structures, where“d” and “s” represent double- and single-strand region, respectively. To be targeted by RAGs in vivo, the altered DNA substrates have to compete with RSS, the physiological substrate. It is not known whether such structures will be cleaved by RAGs, when present along with RSS. Besides, the regulation of the both structure and sequence specific nuclease activities are not studied. To address the above questions, RAG cleavage on bubble substrates was performed in presence of RSS either in cis or trans configuration. Results showed that both bubble substrates and RSS were cleaved with similar efficiency by RAGs. In fact, they can compete out each other in a concentration dependent manner. When kinetics of RSS and bubble cleavage were performed, RSS cleavage reaction was faster and saturated within 10-15 min, while bubbles cleavage started slow and went on increasing with time. This difference in kinetics persisted when both substrates were present together. This could be a regulatory mechanism for targeting RAGs to RSS sites and limiting bubble cleavage which can be deleterious to cells. HMGB1, a DNA binding protein which is shown to enhance RSS binding and synapsis, does not affect RAG action on bubble substrates. RAG postcleavage complex is formed during V(D)J recombination process where RAGs remain bound to cleaved RSS after cleavage, which limits further RAG action on other sites. Such cleavage complex was not detected on bubble substrates, which suggests that after cleavage RAGs were not associated to DSBs of bubble cleavage. Finally, the nonamer binding domain of RAG1 involved in RSS binding in V(D)J recombination, was found to be dispensable for the structure-specific nuclease activity and it appears that RAGs bind to bubble substrates using a different domain. In summary, in this study, the structure-specific nuclease activity of RAGs was characterized. A novel sequence motif that can regulate this activity of RAGs was identified. Though altered structures can be equally favored substrates as RSS, differences in reaction kinetics, cleavage complex formation and separate DNA binding domains regulate RAG cleavage, when it acts as a structure-specific nuclease. Thus, this study may help in the better understanding of RAG induced genomic instabilities in lymphoid tissues.

The lymphoid tissues consist of distinct cell subpopulations of B and T cell lineages and possess complex signaling pathways that are controlled by a myriad of molecular interactions. During the fine-tuned developmental process of the lymphoid system, inappropriate activation of oncogenes and loss of tumor suppressor gene activity can push lymphocytes into uncontrolled clonal expansion, causing several lymphoid malignancies. V(D)J recombination is one such essential process, important for the proper development of the mammalian immune system. However, mistakes in normal V(D)J recombination can lead to deletion of tumor suppressor genes or activation of proto-oncogenes. In the first part of the study, the physiological and pathological roles of DNA binding domain of RAG1 have been characterized. RAG (Recombination Activating Gene) complex consisting of RAG1 and RAG2, is a site specific endonuclease responsible for the generation of antigen receptor diversity. It cleaves a specific DNA sequence termed as recombination signal sequence (RSS), comprising of a conserved heptamer and nonamer. Recent studies have shown that RAGs can also act as a structure-specific nuclease by cleaving flaps, heterologous loops, bubbles, hairpins etc. Nonamer binding domain (NBD) of RAG1 plays a central role in the recognition of RSS during its sequence specific activity. To investigate its DNA binding properties, NBD of murine RAG1 was cloned, overexpressed and purified from E. coli. Electrophoretic mobility shift assays showed that NBD binds with high affinity to nonamer in the context of 12/23 RSS. However, it did not bind to heteroduplex DNA, irrespective of the sequence of the single-stranded region. Interestingly, when a nonamer was present next to a heteroduplex DNA, NBD exhibited robust binding. NBD binding was specific to thymines when single stranded DNA containing poly A, C, G and T were used. Biolayer interferometry studies showed that the observed poly T binding to NBD was robust with a binding constant of 0.45±0.16 µM. >23 nt was essential for NBD binding at homothymidine stretches. On a double-stranded DNA, NBD could bind to A:T stretches, but not G:C stretches or random sequences. Although NBD is indispensable for sequence-specific activity of RAGs, external supplementation of purified nonamer binding domain to NBD deleted cRAG1/cRAG2 did not restore the sequence specific activity, suggesting that the overall domain architecture of RAG1 is important for maintaining its properties. Therefore, we define the sequence requirements of NBD binding to double- and single-stranded DNA, which will have implications in generation of chromosomal rearrangement and genomic instability in lymphoid cells. Genetic alterations are one of the hallmarks of lymphoid malignancies. Many genes involved in chromosomal abnormalities are known to play central roles in the development of normal lymphocytes. In the second part of the study, molecular mechanism associated with fragility of the transcription factor, B cell leukemia 11B (BCL11B) that drives malignant transformation of T-cells has been studied. BCL11B is a zinc finger protein transcription factor with multiple functions. It plays a key role in both development and subsequent maintenance of T-cells. BCL11B gene alterations are implicated in a number of diseases including T-cell malignancies. It acts as a haplo-insufficient tumor suppressor and loss of BCL11B allele leads to susceptibility to mouse thymic lymphoma and human T-ALL. Recent studies reveal heterozygous BCL11B mutations and deletions across each of the major molecular subtypes of T-ALL (15% of patients). Most of the BCL11B missense mutations identified so far affected the residues within BCL11B zinc finger domains of the exon 4. However, mechanism of generation of such specific mutations leading to altered functions of BCL11B remains to be explored. In the present study, we address the potential mechanism of fragility of BCL11B gene during leukemia genesis. Firstly, we have evaluated different regions of BCL11B gene for presence of non-B DNA sequence motifs. Studies using non-B DB database reveal clustering of several non-B DNA forming motifs at the region spanning exon 4 of BCL11B gene. In order to biochemically evaluate the potential of non-B DNA structure formation, two different regions of exon 4 were PCR amplified and cloned. Using bisulfite modification assay we demonstrate that, single strandedness exists at both region I and II of BCL11B exon 4, when the region is present on a plasmid DNA. Bisulfite reactivity on chromosomal DNA confirmed existence of such altered DNA structures in the context of human genome. In vitro gel shift assays showed formation of both intra and intermolecular G-quadruplexes. Primer extension studies revealed that non-B DNA structures could block polymerization during replication on a plasmid, leading to DNA replication arrest. Extrachromosomal assays showed that non-B DNA structure motifs, in contrast to its mutants, blocked transcription leading to reduced expression of green fluorescent protein (GFP) within cells. Many non-B DNA-forming sequences have been mapped to regions of common chromosomal breakpoints in human tumors, known as “hotspots”, which are associated with leukemia, lymphomas and genomic disorders. Thus, alternative DNA conformations are believed to contribute to mutations, deletions and other genetic instability, leading to the deregulation of cancer-related genes in malignant diseases such as leukemia and lymphoma. Activation induced cytidine deaminase (AID), is an essential enzyme involved in antibody diversification of immunoglobulin genes. However, aberrant AID expression in B- cell and non-B cell background is reported in various cancers including leukemia and lymphoma. AID activity requires single stranded DNA (ssDNA) as a substrate. Since activation induced cytidine deaminase (AID) deaminates cytosines when present on a single stranded DNA and its expression is deregulated in many cancers, we investigated the role of AID in BCL11B gene mutagenesis. We observed substantial AID expression in many T-cell leukemic cell lines. Thus, we hypothesize that AID might be targeted to single stranded DNA present at BCL11B exon 4 due to formation of non-B DNA structures such as G-quadruplexes causing AID mediated deamination, further leading to nucleotide alterations and the mutational signature observed at BCL11B exon 4 resulting in T-ALL. Based on our findings, we propose that single strandedness resulted due to formation of non-B DNA structures such as G-quadruplex DNA, triplex DNA or cruciform DNA during physiological processes like DNA replication and transcription at exon 4 of BCL11B, can act as the target for AID. Thus, our findings uncover a new possible link between non-B DNA structure motifs and AID expression in causing mutations at BCL11B exon 4 which could lead to T cell leukemia genesis. BCL11B is a bifunctional transcriptional regulator that can act as a repressor and transactivator, and is known to differentially control the expression of specific genes in a context-dependent manner. In order to understand the transcriptional network involving BCL11B, it was cloned, overexpressed and purified from E. coli. To investigate the DNA binding properties of BCL11B protein, electrophoretic mobility shift assays were performed. Our results lead to identification of a specific sequence motif that is responsible for DNA binding. Competition experiments in presence of specific and nonspecific oligomers further confirmed the binding specificity. Thus, in the present study, we have characterized the binding properties of nonamer binding domain of RAG1, emphasizing its pathological relevance in causing genomic instability in lymphoid cells. The study may help in better understanding of RAG induced genomic instability in lymphoid tissues and role of aberrant AID expression in inducing mutations at BCL11B Zinc finger domain, leading to its deregulation and culminating into T-cell leukemia

La protéine AID (déaminase induite par l’activation) joue un rôle central dans la réponse immunitaire adaptative. En désaminant des désoxycytidines en désoxyuridines au niveau des gènes immunoglobulines, elle initie l’hypermutation somatique (SHM), la conversion génique (iGC) et la commutation isotypique (CSR). Elle est essentielle à une réponse humorale efficace en contribuant à la maturation de l’affinité des anticorps et au changement de classe isotypique. Cependant, son activité mutagénique peut être oncogénique et causer une instabilité génomique propice au développement de cancers et de maladies autoimmunes. Il est donc critique de réguler AID, en particulier ses niveaux protéiques, pour générer une réponse immunitaire efficace tout en minimisant les risques de cancer et d’autoimmunité. Un élément de régulation est le fait qu’AID transite du cytoplasme vers le noyau mais reste majoritairement cytoplasmique à l’équilibre. AID est par ailleurs plus stable dans le cytoplasme que dans le noyau, ce qui contribue à réduire sa présence à proximité de l’ADN. Le but de cette thèse était d’identifier de nouveaux partenaires et déterminants d’AID régulant sa stabilité et ses fonctions biologiques. Dans un premier temps, nous avons identifié AID comme une nouvelle protéine cliente d’HSP90. Nous avons montré qu’HSP90 interagit avec AID dans le cytoplasme, ce qui empêche la poly-ubiquitination d’AID et sa dégradation par le protéasome. En conséquence, l’inhibition d’HSP90 résulte en une diminution significative des niveaux endogènes d’AID et corrèle avec une réduction proportionnelle de ses fonctions biologiques dans la diversification des anticorps mais aussi dans l’introduction de mutations aberrantes. Dans un second temps, nous avons montré que l’étape initiale dans la stabilisation d’AID par la voie de chaperonnage d’HSP90 dépend d’HSP40 et d’HSP70. En particulier, la protéine DnaJa1, qui fait partie de la famille des protéines HSP40s, limite la stabilisation d’AID dans le cytoplasme. La farnésylation de DnaJa1 est importante pour l’interaction entre DnaJa1 et AID et moduler les niveaux de DnaJa1 ou son état de farnésylation impacte à la fois les niveaux endogènes d’AID mais aussi la diversification des anticorps. Les souris DNAJA1-/- présentent une réponse immunitaire compromise en cas d’immunisation, qui est dûe à des niveaux réduits d’AID et un défaut de commutation de classe. Dans un troisième temps, nous avons montré que la protéine AID est intrinsèquement plus instable que sesprotéines paralogues APOBEC. Nous avons identifié l’acide aspartique en seconde position d’AID ainsi qu’un motif semblable au PEST comme des modulateurs de la stabilité d’AID. La modification de ces motifs augmente la stabilité d’AID et résulte en une diversification des anticorps plus efficace. En conclusion, l’instabilité intrinsèque d’AID est un élément de régulation de la diversification des anticorps. Cette instabilité est en partie compensée dans le cytoplasme par l’action protective de la voie de chaperonnage DnaJa1-HSP90. Par ailleurs, l’utilisation d’inhibiteurs d’HSP90 ou de farnésyltransférases pourrait être un outil intéressant pour la modulation indirecte des niveaux d’AID et le traitement de lymphomes/leucémies et de maladies auto-immunes causés par AID.
Activation induced deaminase (AID) plays a central role in adaptive immunity. AID deaminates deoxycytidine to deoxyuridine in defined regions of the immunoglobulin (Ig) genes and initiates somatic hypermutation (SHM), gene conversion (iGC) and class switch recombination (CSR). While being essential for an effective immune response by underpinning antibody affinity maturation and isotype switching, the mutagenic activity of AID can also be oncogenic and causes genomic instability leading to the development of cancer, or exacerbate autoimmune diseases. Therefore, AID regulation, including the control of its protein level, is central to balancing effective immunity with cancer/autoimmunity. Notably, AID shuttles between the cytoplasm and the nucleus but is predominantly cytoplasmic at steady-state, with cytoplasmic AID being much more stable than nuclear AID. These regulatory steps contribute to limit the exposure of the genome to AID but their mechanisms are unknown. This thesis aimed at identifying AID partners and intrinsic determinants regulating its stability and modulating its biological functions. Firstly, we identified AID as a novel HSP90 client protein. We demonstrated that HSP90 interacts with AID in the cytoplasm and prevents its polyubiquitination and subsequent proteasomal degradation. Consequently, HSP90 inhibition results in a significant reduction of endogenous AID levels and correlates with a proportional reduction in both AID-mediated antibody diversification and off-target mutations. Secondly, we showed that the first step in the HSP90 molecular chaperoning pathway and stabilization is the interaction of AID with the HSP40 and HSP70 system. In fact, a specific HSP40 protein, DnaJa1, is the limiting step in cytoplasmic AID stabilization. DnaJa1 farnesylation is required for DnaJa1-AID interaction and modulation of DnaJa1 levels or its farnesylation impacts endogenous AID levels and antibody diversification. In vivo, DnaJa1- deficient mice display compromized response to immunization, resulting from reduced AID protein levels and isotype switching. Thirdly, we found that AID is intrinsically less stable than its APOBEC paralogs. We identified the AID N-terminal aspartic acid residue at position two and an internal PEST-like motif as destabilizing modulators of AID protein turnover. Disruption of these motifs increases AID protein stability and antibody diversification.We conclude that AID’s intrinsic instability directly contributes to regulating antibody diversification. This intrinsic instability is at least partially compensated for in the cytoplasm by the protective action of the DnaJa1-HSP90 molecular chaperoning pathway. Pharmacologically targeting AID in an indirect way, by using HSP90 or farnesyltransferase inhibitors, could be relevant for treating some AID-associated lymphomas/leukemias and/or autoimmune diseases.

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

The innate and the adaptive immune system efficiently cooperate to protect us from infections. The ancient innate immune system, dating back to the first multicellular organisms, utilizes phagocytic cells, soluble antimicrobial peptides, and the complement system for an immediate line of defence against pathogens. Using a limited number of germline-encoded pattern recognition receptors including the Toll-like, RIG-1-like, and NOD-like receptors, the innate immune system recognizes so-called pathogen-associated molecular patterns (PAMPs). PAMPs are specific for groups of related microorganisms and represent highly conserved, mostly non-protein molecules essential for the pathogens' life cycles. Hence, escape mutants strongly reduce the pathogen's fitness. An important task of the innate immune system is to distinguish between harmless antigens and potentially dangerous pathogens. Ideally, innate immune cells should activate the adaptive immune cells only in the case of invading pathogens. The evolutionarily rather new adaptive immune system, which can be found in jawed fish and higher vertebrates, needs several days to mount an efficient response upon its first encounter with a certain pathogen. As soon as antigen-specific lymphocyte clones have been expanded, they powerfully fight the pathogen. Importantly, memory lymphocytes can often protect us from reinfections. During the development of T and B lymphocytes, many millions of different receptors are generated by somatic recombination and hypermutation of gene segments making up the antigen receptors. This process carries the inherent risk of autoimmunity, causing most inflammatory rheumatic diseases. In contrast, inadequate activation of the innate immune system, especially activation of the inflammasomes, may cause autoinflammatory syndromes.

AbstractMicrococcal nuclease (MNase) originating from Staphylococcus aureus is a calcium dependent ribo- and desoxyribonuclease which has endo- and exonucleolytic activity of low sequence preference. MNase is widely used to analyze nucleosome positions in chromatin by probing the enzyme’s DNA accessibility in limited digestion reactions. Probing reactions can be performed in a global way by addition of exogenous MNase, or locally by “chromatin endogenous cleavage” (ChEC) reactions using MNasefusion proteins. The latter approach has recently been adopted for the analysis of local RNA environments of MNasefusion proteins which are incorporated in vivo at specific sites of ribonucleoprotein (RNP) complexes. In this case, ex vivo activation of MNase by addition of calcium leads to RNA cleavages in proximity to the tethered anchor protein thus providing information about the folding state of its RNA environment.Here, we describe a set of plasmids that can be used as template for PCR-based MNase tagging of genes by homologous recombination in S. cerevisiae. The templates enable both N- and C-terminal tagging with MNase in combination with linker regions of different lengths and properties. In addition, an affinity tag is included in the recombination cassettes which can be used for purification of the particle of interest before or after induction of MNase cleavages in the surrounding RNA or DNA. A step-by-step protocol is provided for tagging of a gene of interest, followed by affinity purification of the resulting fusion protein together with associated RNA and subsequent induction of local MNase cleavages.

The innate and the adaptive immune system efficiently cooperate to protect us from infections. The ancient innate immune system, dating back to the first multicellular organisms, utilizes phagocytic cells, soluble antimicrobial peptides, and the complement system for an immediate line of defence against pathogens. Using a limited number of germline-encoded pattern recognition receptors including the Toll-like, RIG-1-like, and NOD-like receptors, the innate immune system recognizes so-called pathogen-associated molecular patterns (PAMPs). PAMPs are specific for groups of related microorganisms and represent highly conserved, mostly non-protein molecules essential for the pathogens’ life cycles. Hence, escape mutants strongly reduce the pathogen’s fitness. An important task of the innate immune system is to distinguish between harmless antigens and potentially dangerous pathogens. Ideally, innate immune cells should activate the adaptive immune cells only in the case of invading pathogens. The evolutionarily rather new adaptive immune system, which can be found in jawed fish and higher vertebrates, needs several days to mount an efficient response upon its first encounter with a certain pathogen. As soon as antigen-specific lymphocyte clones have been expanded, they powerfully fight the pathogen. Importantly, memory lymphocytes can often protect us from reinfections. During the development of T and B lymphocytes, many millions of different receptors are generated by somatic recombination and hypermutation of gene segments making up the antigen receptors. This process carries the inherent risk of autoimmunity, causing most inflammatory rheumatic diseases. In contrast, inadequate activation of the innate immune system, especially activation of the inflammasomes, may cause autoinflammatory syndromes.

Research activities in both our laboratories were directed toward development of control of the FLP/frt recombination system for plants. As described in the text of the research proposal, the US lab has been engaged in developing regulatory strategies such as tissue-specific promoters and the steroid-inducible activation of the FLP enzyme while the main research activities in Israel have been directed toward the development and testing of a copper-regulated expression of flp recombinase in tobacco (this is an example of a promoter activation by metal ions). The Israeli lab hat additionally completed experiments of previous studies regarding factors affecting the efficiency of recombinase activity using both a gain-of-function assay (excisional-activation of a gusA marker) and loss of function assay (excision of a rolC marker) in tobacco. Site-specific recombinase systems, in particular the FLP/frt and R/RS systems of yeast and the Cre/lox system of bacteriophage P1, have become an essential component of targeted genetic transformation procedures both in animal and plant organisms. To provide more flexibility in transgene excisions by the recombinase systems as well as gene targeting, and to widen possible applications, the development of controlled or regulated recombination systems is highly desirable and was therefore the subject of this research proposal. There are a few possible mechanisms to regulate expression of a recombinase system. They include: 1) control of the recombination system by having the target sites (e.g. frt) in one plant and the flp recombinase gene in another, and bringing the two together by cross fertilization. 2) regulation of promoter activities by external stimuli such as temperature, chemicals, metal ions, etc. 3) regulation of promoter activities by internal signals, i.e. cell- or tissue-specific, or developmental regulation. 4) regulation of enzyme activity by providing cofactors essential for biochemical reactions to take place such as steroid molecules in conjunction with a steroid ligand-binding protein (domains). During the course of this research our major emphasis have been focused toward studying the feasibility of hybrid seed production in Arabidopsis, using FLP/frt. Male-sterility was induced using the antisence of a pollen- and tapetum-specific gene, bcp1, isolated from Arabidopsis. The sterility inducing gene was flanked by frt sites. Upon cross pollination of flowers of male-sterile plants with pollen from FLP-containing plants, viable seeds were produced, and the progeny hybrid plants developed normally. The major achievement from this work is the first demonstration of using a site-specific recombinase to restore fertility in male-sterile plants (see attached paper, Luo et al., Plant J 2000; 23:423-430). The implication from this finding is that site-specific recombination systems can be applied in crop plants as a useful alternative method for hybrid seed production.

Objectives: The original objectives of this project were to: 1) evaluate inducible promoters for the expression of recombinase in apple (USDA-ARS); 2) develop alternative selectable markers for use in apple to facilitate the positive selection of gene excision by recombinase (Cornell University); 3) compare the activity of three different recombinase systems (Cre/lox, FLP/FRT, and R/RS)in apple using a rapid transient assay (ARO); and 4) evaluate the use of recombinase systems in apple using the best promoters, selectable markers and recombinase systems identified in 1, 2 and 3 above (Collaboratively). Objective 2 was revised from the development alternative selectable markers, to the development of a marker-free selection system for apple. This change in approach was taken due to the inefficiency of the alternative markers initially evaluated in apple, phosphomannose-isomerase and 2-deoxyglucose-6-phosphate phosphatase, and the regulatory advantages of a marker-free system. Objective 3 was revised to focus primarily on the FLP/FRT recombinase system, due to the initial success obtained with this recombinase system. Based upon cooperation between researchers (see Achievements below), research to evaluate the use of the FLP recombinase system under light-inducible expression in apple was then conducted at the ARO (Objective 4). Background: Genomic research and genetic engineering have tremendous potential to enhance crop performance, improve food quality and increase farm profits. However, implementing the knowledge of genomics through genetically engineered fruit crops has many hurdles to be overcome before it can become a reality in the orchard. Among the most important hurdles are consumer concerns regarding the safety of transgenics and the impact this may have on marketing. The goal of this project was to develop plant transformation technologies to mitigate these concerns. Major achievements: Our results indicate activity of the FLP\FRTsite-specific recombination system for the first time in apple, and additionally, we show light- inducible activation of the recombinase in trees. Initial selection of apple transformation events is conducted under dark conditions, and tissue cultures are then moved to light conditions to promote marker excision and plant development. As trees are perennial and - cross-fertilization is not practical, the light-induced FLP-mediated recombination approach shown here provides an alternative to previously reported chemically induced recombinase approaches. In addition, a method was developed to transform apple without the use of herbicide or antibiotic resistance marker genes (marker free). Both light and chemically inducible promoters were developed to allow controlled gene expression in fruit crops. Implications: The research supported by this grant has demonstrated the feasibility of "marker excision" and "marker free" transformation technologies in apple. The use of these safer technologies for the genetic enhancement of apple varieties and rootstocks for various traits will serve to mitigate many of the consumer and environmental concerns facing the commercialization of these improved varieties.