The DSIF Subunits Spt4 and Spt5 Have Distinct Roles at Various Phases of Immunoglobulin Class Switch Recombination

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription of S regions and AID, expression array analysis suggests that Spt4 and Spt5 regulate a distinct subset of transcripts in CH12F3-2A cells. Curiously, Spt4 is critically important in suppressing cryptic transcription initiating from the intronic Sμ region. Depletion of Spt5 reduced the H3K4me3 level and DNA cleavage at the Sα region, whereas Spt4 knockdown did not perturb the H3K4me3 status and S region cleavage. H3K4me3 modification level thus correlated well with the DNA breakage efficiency. Therefore we conclude that Spt5 plays a role similar to the histone chaperone FACT complex that regulates H3K4me3 modification and DNA cleavage in CSR. Since Spt4 is not involved in the DNA cleavage step, we suspected that Spt4 might be required for DNA repair in CSR. We examined whether Spt4 or Spt5 is essential in non-homologous end joining (NHEJ) and homologous recombination (HR) as CSR utilizes general repair pathways. Both Spt4 and Spt5 are required for NHEJ and HR as determined by assay systems using synthetic repair substrates that are actively transcribed even in the absence of Spt4 and Spt5. Taken together, Spt4 and Spt5 can function independently in multiple transcription-coupled steps of CSR

Polo kinase recruitment via the constitutive centromere-associated network at the kinetochore elevates centromeric RNA

The kinetochore, a multi-protein complex assembled on centromeres, is essential to segregate chromosomes during cell division. Deficiencies in kinetochore function can lead to chromosomal instability and aneuploidy-a hallmark of cancer cells. Kinetochore function is controlled by recruitment of regulatory proteins, many of which have been documented, however their function often remains uncharacterized and many are yet to be identified. To identify candidates of kinetochore regulation we used a proteome-wide protein association strategy in budding yeast and detected many proteins that are involved in post-translational modifications such as kinases, phosphatases and histone modifiers. We focused on the Polo-like kinase, Cdc5, and interrogated which cellular components were sensitive to constitutive Cdc5 localization. The kinetochore is particularly sensitive to constitutive Cdc5 kinase activity. Targeting Cdc5 to different kinetochore subcomplexes produced diverse phenotypes, consistent with multiple distinct functions at the kinetochore. We show that targeting Cdc5 to the inner kinetochore, the constitutive centromere-associated network (CCAN), increases the levels of centromeric RNA via an SPT4 dependent mechanism

Spt4 and Spt5 regulate small yet distinct sets of transcripts.

<p>(A) A Venn diagram showing the number of up- or down-regulated ranscripts by at least 2-fold in the absence of either Spt4 or Spt5, in CIT(+) treated CH12F3-2A cells. (B) Differential expressions of selected genes identified by microarray by either Spt4 or Spt5 knockdown stimulated for 24 hours with CIT; results are presented relative to control, which was set as 1. A complete list is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002675#pgen.1002675.s006" target="_blank">Table S1</a>. (C) Top: A schematic diagram of the positions of primers (triangles) used to quantify the cryptic S region transcripts. Bottom: after introducing the RNAi oligonucleotides indicated, the cryptic Sμ and Sα transcripts were quantified by RT-qPCR normalized to HPRT. SD values were determined from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P<0.03; **, P<0.004; ***, P<0.0002).</p

The DSIF complex is required for efficient NHEJ.

<p>(A) ChIP assays of the various knockdown and control samples indicated, using anti-Ku80 antibody. Background values from controls with no antibody were subtracted. Values were normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P<0.01). (B) Schematic diagram of the <i>I-Sce</i>I-induced NHEJ repair substrate. (C) Percentage of EGFP-positive cells, assessed by FACS analysis 48 hours after co-transfection of <i>I-Sce</i>I expression plasmids and the indicated RNAi oligonucleotides into H1299dA3-1 cells. (D) Relative EGFP-positive cells with respect to to <i>I-Sce</i>I-treated control cells. SD values were derived from three independent experiments. A.U: arbitrary unit. (E) Knockdown efficiency of the indicated genes was quantified by RT-qPCR. (F) <i>I-Sce</i>I(+)-treated control cells from (D) were divided and transfected with the indicated RNAi oligonucleotides (indicated by dotted line); the percent EGFP-positive population was quantified and compared to control samples. SD values were derived from three independent experiments. A.U: arbitrary unit. (G) PCR of genomic DNA products; uncut and repaired fragments derived from control and knockdown samples are indicated by arrowheads. (H) PCR products of repaired genomic DNA fragments derived from the indicated knockdown samples. Arrowheads represent insertions.</p

Spt4 and Spt5 differentially control S region DNA cleavage and H3K4me3 status.

<p>(A) DNA break assay by γH2AX ChIP using anti-γH2AX antibody were performed in Spt4- or Spt5-knockdown or control samples. Pulled-down DNA was subjected to Sμ- and Sα-specific detection by RT-PCR, normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. (B) DNA break assay performed using biotin-dUTP end labeling method derived from Spt4- or Spt5-knockdown and control samples. Pulled-down DNA was subjected to Sμ- and Sα-specific detection by RT-PCR, normalized to the input DNA. SD values were derived from three independent experiments. (C) Top: schematic diagram of the position of the ChIP assay PCR products. Bottom: the knockdown and control samples indicated were assayed by ChIP, using anti-H3K4me3 and anti-H3 antibodies. Background values from controls with no antibody were subtracted. Values were normalized to the input DNA signals. The maximum value in each data set was set as 100%. SD values were derived from three independent experiments. The p-values were calculated using the unpaired two-tailed Student's <i>t</i> test (*, P = 0.05; **, P<0.03; ***, P<0.02; ****, P<0.005). (D) Immunoblotting of histone H3K4me3, H3, and tubulin derived from the indicated knockdown samples.</p

Spt4 and Spt5 are critical for CSR.

<p>(A) Flow cytometry (FACS) profile of the percent IgA-switching population, indicated by the number in the box, after introducing the indicated RNAi oligonucleotide under CIT(−) or (+) conditions. (B) Top: Summary of the percent IgA-switching population data derived from the indicated gene knockdown samples. Bottom: Percent of dead cells as determined by PI staining. SD values were determined from three independent experiments. Knockdown efficiency for each gene indicated was analyzed by (C) RT-qPCR and (D) immunoblotting. (E) Immunoblotting of Spt4 and Spt5 derived from the indicated knockdown samples. (F) Various transcripts quantified by RT-qPCR and normalized to HPRT, after introduction of the indicated RNAi oligonucleotide, under the CIT(+) condition. SD values were determined from three independent experiments. (G) After introducing Spt4 or Spt5 RNAi oligonucleotides, γ3GLT was quantified by RT-qPCR normalized to HPRT. SD values were determined from three independent experiments. (H) Summary of IgG3-switching population data derived from the indicated gene-knockdown samples. SD values were determined from three independent experiments. The p-values were calculated using the unpaired two-tailed. Student's <i>t</i> test (*, P<0.03; **, P<0.007; ***, P<0.001). (I) FACS profile of the IgG3-switching population after introducing the indicated RNAi oligonucleotide under CIT(−) or (+) conditions.</p

Further evidence for involvement of a noncanonical function of uracil DNA glycosylase in class switch recombination

Activation-induced cytidine deaminase (AID) introduces DNA cleavage in the Ig gene locus to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells. The DNA deamination model assumes that AID deaminates cytidine (C) on DNA and generates uridine (U), resulting in DNA cleavage after removal of U by uracil DNA glycosylase (UNG). Although UNG deficiency reduces CSR efficiency to one tenth, we reported that catalytically inactive mutants of UNG were fully proficient in CSR and that several mutants at noncatalytic sites lost CSR activity, indicating that enzymatic activity of UNG is not required for CSR. In this report we show that CSR activity by many UNG mutants critically depends on its N-terminal domain, irrespective of their enzymatic activities. Dissociation of the catalytic and CSR activity was also found in another UNG family member, SMUG1, and its mutants. We also show that Ugi, a specific peptide inhibitor of UNG, inhibits CSR without reducing DNA cleavage of the S (switch) region, confirming dispensability of UNG in DNA cleavage in CSR. It is therefore likely that UNG is involved in a repair step after DNA cleavage in CSR. Furthermore, requirement of the N terminus but not enzymatic activity of UNG mutants for CSR indicates that the UNG protein structure is critical. The present findings support our earlier proposal that CSR depends on a noncanonical function of the UNG protein (e.g., as a scaffold for repair enzymes) that might be required for the recombination reaction after DNA cleavage