FUNCTIONAL DISSECTION OF HISTONE LYSINE METHYLATION IN B CELL SPECIFICATION

Methylation on lysine 4 of histone H3 (H3K4) by the histone methyltransferases (HMTs) of the trithorax group is associated with the activation and maintenance of transcriptional programs, but virtually nothing is known about the function of these key epigenetic regulators in differentiating cell compartments of adult animals. Here I present the first characterization of the role of the H3K4 HMT Mll2 (Mixed lineage leukemia), also known as Mll4 or Wbp7 in adult tissues focusing on the ontogeny of B cells as the best characterized system for the dissection of lineage commitment and terminal specification. Expression analysis in sorted populations revealed that both Mll2 and its closest homolog Mll1 are expressed throughout B cell maturation, and the hypothesis I set out to test was whether they form a redundant regulatory circuit or whether their selective ablation would uncover functional requirements for specific B cell subsets. To this end I used a conditional knock out mouse line in which the Mll2 gene is ablated according to the ‘knock-out-first’ approach, (Glaser et al., 2006; Testa et al., 2004; Testa et al., 2003), an innovative engineering strategy that allows to knock out the gene by transcription trapping, restore it using FLP-mediated recombination and knock it out again using Cre-mediated recombination. In order to knock out Mll2 specifically in mature B cells I selected the CD21-Cre3A mouse line that expresses Cre recombinase under the CD21 promoter (Kraus et al., 2004). In this line Cre-mediated deletion follows the expression of CD21, which takes place at the immature B to mature B cell transition (Kraus et al., 2004) (Takahashi et al., 1997). Flow cytometric analysis of B cell subsets in compound mutants performed on over 50 pairs of mice (homozygous versus heterozygous Mll2 flox mice carrying the CD21-Cre transgene) revealed that upon Mll2 deletion, size of marginal zone (MZ) (CD19high - CD21high - CD38high - CD1dhigh - CD23low) B cell compartment on average was reduced by about 50% with respect to the control animals. This was confirmed by histological analysis of the spleen of control and mutant mice. Other B mature cell compartments, including follicular, B1 and germinal center B cells from different lymphopoietic organs were not affected in mutant animals, indicating a specific requirement for Mll2 in the establishment and/or maintenance of the MZ B cell lineage. A gene expression profiling did not reveal aberrant expression of key regulators of the homeostasis of MZ B cell compartment, nor remarkable differences between transcriptomes of Mll2 deficient and proficient cells. However in vitro analysis of the dynamics demonstrated higher, by about 75%, rate of proliferation of mutant MZ B cell compartment. Similarly significant increase in the proliferating fraction was also observed in mutant follicular and immature/transitional B cells. This higher turnover of splenic B cell populations upon loss of Mll2 may reflect the presence of a stimulus promoting cell proliferation as part of a feed back loop triggered by the decrease in MZ B cells. Moreover functional analysis of MZ B cells revealed that upon loss of Mll2 cells are not capable to switch class of immunoglobulin in a response to NP-Ficoll immunization indicating a crucial role of Mll2 in the establishment of a new transcriptional program. These findings are in agreement with previously published results demonstrating requirement of Mll2 for the activation and timing of lineage-specific transcriptional programs in both sperm and oocyte germ layers (Andreu-Vieyra et al., 2010; Glaser et al., 2009). Together, present results could suggest that Mll2 plays a role also in B lymphophoiesis and therefore we consider a candidacy of Mll2 as a putative lymphopoietic regulating factor.