Tag Archives: Rabbit Polyclonal to B-Raf.

Ig class change recombination (CSR) requires expression of activation-induced cytidine deaminase

Ig class change recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Antigen stimulation of B lymphocytes induces Cyclosporin H the expression of activation-induced cytidine deaminase (AID) which is responsible for generation of antibody memory (1 2 Somatic hypermutation and class switch recombination (CSR) are two genetic events that engrave antibody memory into the Ig heavy-chain (H) locus of the B cell genome. CSR takes place between two switch (S) regions located upstream of the individual H constant regions (CH) and converts the isotype from IgM to another class by bringing the specific CH region close to the H variable region (VH) exons and looping out the intervening DNA segment (3). Gene-targeting experiments in the IgH locus have shown that active transcription through the S regions is an essential requirement of CSR (4 5 This transcription initiates from the I promoter located upstream of each S region and proceeds through the I exon the intronic S region the CH exons and the CH introns. The mature transcripts designated as germline transcripts (GLTs) are generated by splicing out the S region and CH intronic sequences (3). However it is not well understood whether the transcription itself the transcription products or both are important for CSR. The original chromatin-opening hypothesis suggested that transcription of the S region causes its chromatin structure to be relatively open which increases its accessibility to a putative recombinase (6 7 In fact the migration of the transcription machinery accumulates positive and negative supercoil in its front and rear respectively. During this process R-loop formation was detected in the DNA from switching B cells by the bisulfite sensitivity assay (8). The R-loop formation was considered to support the DNA deamination hypothesis proposed for the function of AID as the single-strand DNA can serve as an efficient substrate of cytidine deamination by AID as exhibited in vitro (9). This hypothesis postulates that dU generated by AID deamination is recognized as a dU/dG mismatch and excised by uracil DNA glycosylase (10). The abasic sites thus formed is usually then cleaved by apyrimidinic/apurinic endonuclease. It has been also proposed that dU/dG mismatches are recognized and cleaved by mismatch repair proteins such as Msh2 and Msh6. On the other hand AID was recently shown to reduce the translation of Topoisomerase 1 (Top1) mRNA and thus decrease its protein level (11). The decrease in Top1 causes inefficient recovery of the excessive unfavorable supercoil of the transcribed S region because Top1 removes the excessive supercoil by nicking and transient covalent binding to DNA followed by rotation and religation. It was postulated that this resultant prolongation of the unfavorable supercoil can induce the formation of non-B form DNA in the S region (12). Top1 can cleave non-B form DNA but not rotate efficiently because Cyclosporin H of its aberrant structure resulting in irreversible single-stranded cleavage. According to this model Top1 is the enzyme that cleaves the S region during CSR. Despite these studies however the transcription requirement for CSR has not been fully elucidated partly because in vivo transcription occurs on a chromatin template in which the DNA is usually wrapped around core histone octamers (H2A H2B H3 and H4) (13). The transcriptional migration of RNA polymerase II (Pol II) along the chromatin Cyclosporin H template requires the reorganization of nucleosomes and numerous histone posttranslational modifications (PTMs) that include H2B ubiquitination and H3 methylation in Rabbit Polyclonal to B-Raf. transcribed regions (14). Such nucleosomal reorganization and modifications require the orchestrated contribution of numerous accessory factors. One of the most important of these components is the facilitates chromatin transcription (FACT) complex which has been proposed to facilitate the passage of Cyclosporin H Pol II through the chromatin (15). An in vitro chromatin transcription assay exhibited that human FACT acts as a histone chaperone that can displace H2A and H2B (16) from a nucleosome in front of Pol II and can replace them again behind.