Histone3 lysine4 trimethylation regulated by the facilitates chromatin transcription complex is critical for DNA cleavage in class switch recombination

Abstract

Ig class switch recombination (CSR) requires expression of activation-induced cytidine deaminase (AID) and transcription through target switch (S) regions. Here we show that knockdown of the histone chaperone facilitates chromatin transcription (FACT) completely inhibited S region cleavage and CSR in IgA-switch-inducible CH12F3-2A B cells. FACT knockdown did not reduce AID or S region transcripts but did decrease histone3 lysine4 trimethylation (H3K4me3) at both the Sμ and Sα regions. Because knockdown of FACT or H3K4 methyltransferase cofactors inhibited DNA cleavage in H3K4me3-depleted S regions, H3K4me3 may serve as a mark for recruiting CSR recombinase. These findings revealed an unexpected evolutionary conservation between CSR and meiotic recombination.A ntigen stimulation of B lymphocytes induces 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 heavychain (H) locus of the B cell genome. CSR takes place between two switch (S) regions, located upstream of the individual H constant regions (C H ) and converts the isotype from IgM to another class by bringing the specific C H region close to the H variable region (V H ) 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 C H exons, and the C H introns. The mature transcripts, designated as germline transcripts (GLTs), are generated by splicing out the S region and C H 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 demonstrated 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 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...

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

“…As shown in Fig. 4A, the SSRP1 knockdown did not alter the H3.3 deposition levels in the V(D)J and Sμ regions, but it reduced the H3K4me3 occupancy, in agreement with our previous findings in SSRP1-depleted murine B lymphoma cells (16), indicating that FACT is not required for H3.3 deposition into Igh chromatin.…”

“…We previously reported that H3K4me3 serves as a marker for SHM and CSR (5,16). In CSR, FACT contributes to the DNA break by promoting the trimethylation of H3K4 (16). Regarding SHM, we reported that H3K4me3 is enriched in SHM target regions (5).…”

“…Therefore, we speculate that the more frequently DNA is unwound from the nucleosome, the higher the chance of the DNA being hypermutated. We previously reported that H3K4me3 serves as a marker for SHM and CSR (5,16). In CSR, FACT contributes to the DNA break by promoting the trimethylation of H3K4 (16).…”

“…FACT is proposed to evict nucleosomal histones and deposit them at the site of transcription by RNAPII, so that the polymerase can proceed beyond the nucleosomes (15). We previously discovered that FACT is required to induce CSR (16). FACT knockdown blocks CSR at the DNA-cleavage step without affecting the level of the Ig heavy chain gene (Igh) transcription, and FACT is important for inducing trimethylation on histone H3K4 (H3K4me3), which seems to be recognized by a protein complex with DNAcleaving activity.…”

Elucidation of IgH 3′ region regulatory role during class switch recombination via germline deletion

“…Location of breakpoints in S 1 implicated in the targeting of the CSR machinery (including AID and RNA Pol II) to S regions primed for CSR [25][26][27][28] . Altogether with DNA transcription, this remodelling of chromatin in S regions constitutes a hallmark of the accessibility to CSR factors.…”