Acetyltransferases GCN5 and PCAF Are Required for B Lymphocyte Maturation in Mice

B lymphocyte development has two DNA recombination processes: V(D)J recombination of the immunoglobulin (Igh) gene variable region, and class switching of the Igh constant regions from IgM to IgG, IgA, or IgE. V(D)J recombination is required for the successful maturation of B cells from pro-B to pre-B to immature-B and then to mature B cells in the bone marrow. CSR occurs outside of the bone marrow when mature B cells migrate to peripheral lymphoid organs, such as spleen and lymph nodes. Both V(D)J recombination and CSR depend on an open chromatin state that makes DNA accessible to specific enzymes, recombination activating gene (RAG), and activation-induced cytidine deaminase (AID). Acetyltransferases GCN5 and PCAF possess redundant functions acetylating histone H3 lysine 9 (H3K9). Here, we generated a mouse model that lacked both GCN5 and PCAF in B cells. Double-deficient mice possessed low levels of mature B cells in the bone marrow and peripheral organs, an accumulation of pro-B cells in bone marrow, and reduced CSR levels. We concluded that both GCN5 and PCAF are required for B-cell development in vivo.

Then, mature B cells initiate another DNA recombination process to change the constant regions of immunoglobulin genes, referred to as class switch recombination (CSR). During the CSR in mice, IgM is replaced by IgG3, IgG1, IgG2a, IgG2b, IgE, or IgA [2]. The CSR is initiated by nonproductive transcription known as a germline transcription (GLT), which is needed to separate two DNA strands. Single-stranded DNA is then targeted by activation-induced cytidine deaminase (AID), a B lymphocyte-specific enzyme deaminating cytosine to uracil (C to U). Then, uracil DNA N-glycosylase (UNG) removes uracil from

Generation of Mice Lacking GCN5 and PCAF in B Cells
The combined inactivation of Gcn5 and Pcaf genes in mice results in embryonic lethality [32]. To overcome this challenge, we designed a complex genetic model when the floxed Gcn5 gene is conditionally inactivated in B-cell lineages by the CRE enzyme under the Cd19 promoter (Cd19 Cre+ ) [34]. To sort out cells with activated CRE, we used a model with knocked-in yellow fluorescent protein gene (YFP) into the ROSA-26 locus. YFP is inactive until CRE removes the "STOP" signal (Rosa-26-YFP+) [35]. Thus, we obtained Gcn5 f/f Pcaf −/− Cd19 +/Cre YFP + mice and simpler controls. Further in the text, we skip Cd19 +/Cre and YFP+ for simplicity in most cases, and refer to mice on the basis of the status of Gcn5 and Pcaf genes, i.e., as Gcn5 f/f Pcaf −/− , Gcn5 f/f , Pcaf −/− , and WT. When the CRE is active and describing sorted B cells, we indicate Gcn5 −/− , a knockout status of the gene. The lack of GCN5 and PCAF, and H3K9 acetylation, were validated using Western blot ( Figure S1).

Mice Lacking GCN5 and PCAF in B Cells Possess Small Spleens
We obtained mice with the germline inactivation of Pcaf gene and conditional inactivation of Gcn5 in B cells under the Cd19 promoter ( Figure 1). The germline inactivation of Pcaf gene alone had no detectable effect on mouse development, in line with a previous observation [32]. The conditional inactivation of the Gcn5 gene in B cells had no visible Biomolecules 2022, 12, 61 4 of 10 effect on sizes of WT and Pcaf -deficient mice, which were 15 to 19 g on average (p > 0.1433) ( Figure 1A). However, the inactivation of Gcn5 resulted in smaller spleens in mice (Gcn5 −/− , 54 mg) when compared to WT (69 mg) and Pcaf −/− (72 mg) mice. The combined inactivation of Pcaf and Gcn5 in B cells resulted in even smaller spleens (Gcn5 −/− Pcaf −/− , 29 mg, p < 0.0001). The spleens of mice without CRE activity with the Gcn5 gene being floxed and functional (Gcn5 f/f Pcaf −/− , 67 mg) were comparable in size to the ones of WT mice ( Figure 1B,C).
describing sorted B cells, we indicate Gcn5 , a knockout status of the gene. The lack of GCN5 and PCAF, and H3K9 acetylation, were validated using Western blot ( Figure S1).

Mice Lacking GCN5 and PCAF in B Cells Possess Small Spleens
We obtained mice with the germline inactivation of Pcaf gene and conditional inactivation of Gcn5 in B cells under the Cd19 promoter ( Figure 1). The germline inactivation of Pcaf gene alone had no detectable effect on mouse development, in line with a previous observation [32]. The conditional inactivation of the Gcn5 gene in B cells had no visible effect on sizes of WT and Pcaf-deficient mice, which were 15 to 19 g on average (p > 0.1433) ( Figure 1A). However, the inactivation of Gcn5 resulted in smaller spleens in mice (Gcn5 −/− , 54 mg) when compared to WT (69 mg) and Pcaf −/− (72 mg) mice. The combined inactivation of Pcaf and Gcn5 in B cells resulted in even smaller spleens (Gcn5 −/− Pcaf −/− , 29 mg, p < 0.0001). The spleens of mice without CRE activity with the Gcn5 gene being floxed and functional (Gcn5 f/f Pcaf −/− , 67 mg) were comparable in size to the ones of WT mice ( Figure  1B,C).

Mice Lacking GCN5 and PCAF in B Cells Possess Delayed B Lymphocyte Development
To detect mature Gcn5 −/− Pcaf −/− B cells, we identified B220+IgM+ cells in the spleen using flow cytometry (Figure 2A

Mice Lacking GCN5 and PCAF in B Cells Possess Delayed B Lymphocyte Development
To detect mature Gcn5 −/− Pcaf −/− B cells, we identified B220+IgM+ cells in the spleen using flow cytometry (Figure 2A

Inactivation of Gcn5 and Pcaf Results in a Reduced Proportion of B Cells in the Blood
To detect mature B cells in the blood, we used B220 markers ( Figure 2C,D). The inactivation of Pcaf alone resulted in 62% of B cells after red blood cells had been lysed, which was comparable to WT mice with 65% of B cells in the blood (p = 0.92). The inactivation of the Gcn5 gene alone resulted in a modest reduction of B-cell proportion to 56% (p = 0.12), while the combined inactivation of Gcn5 and Pcaf led to even lower B-cell levels in the blood (22%, p < 0.0001). The levels of B cells in the blood of control mice without CRE recombinase expression when the Gcn5 gene was functional (Gcn5 f/f Pcaf −/− ) were comparable to those of WT mice (78%). GCN5 and PCAF are thus both required and functionally redundant for B-cell development in mice.

Inactivation of Gcn5 and Pcaf Results in a Reduced Proportion of B Cells in the Blood
To detect mature B cells in the blood, we used B220 markers ( Figure 2C,D). The inactivation of Pcaf alone resulted in 62% of B cells after red blood cells had been lysed, which was comparable to WT mice with 65% of B cells in the blood (p = 0.92). The inactivation of the Gcn5 gene alone resulted in a modest reduction of B-cell proportion to 56% (p = 0.12), while the combined inactivation of Gcn5 and Pcaf led to even lower B-cell levels in the blood (22%, p < 0.0001). The levels of B cells in the blood of control mice without CRE recombinase expression when the Gcn5 gene was functional (Gcn5 f/f Pcaf −/− ) were comparable to those of WT mice (78%). GCN5 and PCAF are thus both required and functionally redundant for B-cell development in mice.
One reason for the low Gcn5 −/− Pcaf −/− B-cell count in spleen and blood in mice could be cell death following the normal development of B cells in bone marrow and migration to the periphery. Another option could be the blocked or delayed maturation of B cells in bone marrow during the earlier developmental stages. To test the latter possibility, we analyzed B cells in the bone marrow of the mice (Figure 3).

Inactivation of Gcn5 and Pcaf Results in Accumulation of Pro-B Cells in Bone Marrow
To characterize B-cell maturation in bone marrow, we followed the expression of B220 (B220+IgM−) and IgM (B220+IgM+) on the lymphocyte surface. The inactivation of Gcn5 or Pcaf resulted in an insignificant decline in the B220+IgM+ population (26%, 5-6 One reason for the low Gcn5 −/− Pcaf −/− B-cell count in spleen and blood in mice could be cell death following the normal development of B cells in bone marrow and migration to the periphery. Another option could be the blocked or delayed maturation of B cells in bone marrow during the earlier developmental stages. To test the latter possibility, we analyzed B cells in the bone marrow of the mice (Figure 3).

Inactivation of Gcn5 and Pcaf Results in Accumulation of Pro-B Cells in Bone Marrow
To characterize B-cell maturation in bone marrow, we followed the expression of B220 (B220+IgM−) and IgM (B220+IgM+) on the lymphocyte surface. The inactivation of Gcn5 or Pcaf resulted in an insignificant decline in the B220+IgM+ population (26%, 5-6 million) when compared to WT (32%, 8 million, p = 0.44) (Figure 3 A,B). The combined inactivation of Gcn5 and Pcaf resulted in an additional reduction in mature B cells in bone marrow (3 million, 16%) ( Figure 3A,B).
We concluded that GCN5 and PCAF are required for the maturation of B cells from the pro-B to the pre-B cell stage, and later to mature B cells.

GCN5 Is Required for Robust Class Switch Recombination
The CSR relies on the ATM-dependent DDR [2,4,5,27]. Because H3K9 acetylation works downstream of H2AX phosphorylation, and GCN5/PCAF might work downstream of ATM/ATR/DNA-PKcs, we tested if the CSR depends on GCN5 and PCAF (Figure 4). We purified B splenocytes from 8-to 12-week-old mice and stimulated the CSR  Figure S3 and 3B).
We concluded that GCN5 and PCAF are required for the maturation of B cells from the pro-B to the pre-B cell stage, and later to mature B cells.

GCN5 Is Required for Robust Class Switch Recombination
The CSR relies on the ATM-dependent DDR [2,4,5,27]. Because H3K9 acetylation works downstream of H2AX phosphorylation, and GCN5/PCAF might work downstream of ATM/ATR/DNA-PKcs, we tested if the CSR depends on GCN5 and PCAF (Figure 4). We purified B splenocytes from 8-to 12-week-old mice and stimulated the CSR from IgM to IgG3 using established protocols [40,42]. We focused on matched pairs of Gcn5 f/f (the functional equivalent of WT cells) and Gcn5 −/− , as well as Gcn5 f/f Pcaf −/− (the functional equivalent of Pcaf −/− ) and Gcn5 −/− Pcaf −/− cells. We used Aid −/− cells as a CSR-deficient control to detect an experimental background (Figure 4). The inactivation of Pcaf alone had no effect on CSR levels (WT vs. Gcn5 f/f Pcaf −/− , p > 0.96). On the other hand, the inactivation of Gcn5 resulted in a reduction in CSR from about 14% in WT and Gcn5 f/f cells to 6% in Gcn5 −/− cells, * p < 0.0008 (Figure 4). The combined deletion of Pcaf and Gcn5 resulted in a similar reduction from 12% in Gcn5 f/f Pcaf −/− cells to 6% in Gcn5 −/− Pcaf −/− cells, ** p = 0.0080. We concluded that GCN5 is required for robust CSR to IgG3 because the additional inactivation of Pcaf did not affect CSR levels when compared to Gcn5 −/− and Gcn5 −/− Pcaf −/− B cells, p > 0.9999 (Figure 4).
Biomolecules 2022, 12, x FOR PEER REVIEW 7 of 11 from IgM to IgG3 using established protocols [40,42]. We focused on matched pairs of Gcn5 f/f (the functional equivalent of WT cells) and Gcn5 −/− , as well as Gcn5 f/f Pcaf −/− (the functional equivalent of Pcaf −/− ) and Gcn5 −/− Pcaf −/− cells. We used Aid −/− cells as a CSR-deficient control to detect an experimental background (Figure 4). The inactivation of Pcaf alone had no effect on CSR levels (WT vs. Gcn5 f/f Pcaf −/− , p > 0.96). On the other hand, the inactivation of Gcn5 resulted in a reduction in CSR from about 14% in WT and Gcn5 f/f cells to 6% in Gcn5 −/− cells, * p < 0.0008 (Figure 4). The combined deletion of Pcaf and Gcn5 resulted in a similar reduction from 12% in Gcn5 f/f Pcaf −/− cells to 6% in Gcn5 −/− Pcaf −/− cells, ** p = 0.0080. We concluded that GCN5 is required for robust CSR to IgG3 because the additional inactivation of Pcaf did not affect CSR levels when compared to Gcn5 −/− and Gcn5 −/− Pcaf −/− B cells, p > 0.9999 (Figure 4).

Discussion
Both GCN5 and PCAF are involved in chromatin modification and DDR response, which made them relevant candidates to facilitate lymphocyte development [13,14]. One challenge was the lack of a relevant in vivo model because GCN5 and PCAF have certain redundant functions in acetylating H3K9, and because the germline inactivation of Gcn5 results in early embryonic lethality in mice [32]. Here, we generated and analyzed a complex mouse model that allowed for studying of double-deficient Gcn5 −/− Pcaf −/− B cells development in vivo and ex vivo. We used a germline knockout of Pcaf [32], a conditional knockout of Gcn5 f/f , a knockin of CRE recombinase expressed under the B-cell-specific Cd19 promoter [34], and a knockin of YFP to track the activity of CRE recombinase [35].
For such a complex mouse model (Gcn5 f/f Pcaf −/− Cd19 +/cre Rosa-26-YFP + ), multiple controls were used. In one line of the controls, mice lacking PCAF and having a floxed

Discussion
Both GCN5 and PCAF are involved in chromatin modification and DDR response, which made them relevant candidates to facilitate lymphocyte development [13,14]. One challenge was the lack of a relevant in vivo model because GCN5 and PCAF have certain redundant functions in acetylating H3K9, and because the germline inactivation of Gcn5 results in early embryonic lethality in mice [32]. Here, we generated and analyzed a complex mouse model that allowed for studying of double-deficient Gcn5 −/− Pcaf −/− B cells development in vivo and ex vivo. We used a germline knockout of Pcaf [32], a conditional knockout of Gcn5 f/f , a knockin of CRE recombinase expressed under the B-cell-specific Cd19 promoter [34], and a knockin of YFP to track the activity of CRE recombinase [35].
For such a complex mouse model (Gcn5 f/f Pcaf −/− Cd19 +/cre Rosa-26-YFP + ), multiple controls were used. In one line of the controls, mice lacking PCAF and having a floxed Gcn5 gene but expressing no CRE recombinase were considered (Gcn5 f/f Pcaf −/− Rosa-26-YFP + , Figure S2). The GCN5-deficient and GCN5/PCAF double-deficient B cells possessed developmental delay, with lower levels of mature B cells in thee spleen and blood, and the accumulation of progenitor B cells in bone marrow (Figures 1-4). Control mice without CRE expression, on the other hand, demonstrated WT levels of B-cell development in all groups, i.e., WT levels of B220+IgM+ mature B cells in the spleen ( Figure S2A,B) and blood ( Figure S2C,D). In addition, these mice possessed high levels of B220+IgM+ cells ( Figure S2E,F), and stable and low levels of pro-B cells (B220+IgM−CD43+) in bone marrow ( Figure S2G,H).
Mice lacking PCAF and with conditional knockout of Gcn5 in B cells were alive and resembled WT littermates ( Figure 1A). One clear feature that the Gcn5 f/f Pcaf −/− Cd19 +/cre Rosa-26-YFP + mice had was a small spleen ( Figure 1B,C), which was also the case in mice lacking only Gcn5 in B cells. The small spleen could indicate a defect in B-cell development, and we indeed found low numbers of mature B cells in the spleen, blood, and bone marrow. One could propose that mature B cells lacking GCN5 or both GCN5 and PCAF possess low proliferation speed or tend to trigger apoptosis. Alternatively, GCN5 and PCAF might be required for the V(D)J recombination. This option could be tested by, for example, using vAbl pre-B cell lines, as we and others did before [4,15,18,23,[27][28][29][30][31]. Another intriguing question is whether the physical presence or enzymatic activity of GCN5 and PCAF are required for the observed phenotypes, i.e., abrogated B-cell maturation and reduced levels of CSR. To investigate this question, one could use specific inhibitors of GCN5 and PCAF enzymes, or enzyme-dead mutations introduced to the Gcn5 and Pcaf genes.
The inactivation of Gcn5 in murine B cells also resulted in reduced lymphomagenesis in mice overexpressing MYC oncoprotein [44]. Our findings further highlight this observation, suggesting that GCN5 and potentially also PCAF enzymes are attractive targets for cancer therapy [44].
CSR levels were reduced in B cells lacking GCN5 (Figure 4). The challenge in this set of experiments was that mice of the Gcn5 f/f Pcaf −/− Cd19 +/cre Rosa-26-YFP + genotype were rather rare and possessed a very low number of suitable B splenocytes (Figures 1 and 2). Although our data on IgG3 are sufficient, one could extend the study by generating knockout cell lines lacking GCN5 and PCAF, and suitable for CSR. One possible model system is CH12F3 cells capable of supporting CSR to IgA [45], which were used for this kind of experiment [24,37,42]. The CSR itself is a complex multistage process. Generating relevant cell lines also provides tools to determine specific stages of CSR affected in GCN5-deficient mice, i.e., germline transcription, AID recruitment, generation of DSBs, or DNA repair.

Conclusions
Acetyltransferases GCN5 and PCAF possess redundant functions in B-cell maturation. GCN5 is required for robust class switch recombination ex vivo.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biom12010061/s1, Figure S1: Detection of GCN5, PCAF and histones; Figure S2: Detection of developing B cells in mice of indicated genotypes using flow cytometry; Figure S3