A Critical Role of the Thy28-MYH9 Axis in B Cell-Specific Expression of the Pax5 Gene in Chicken B Cells

Accumulating evidence suggests that Pax5 plays essential roles in B cell lineage commitment. However, molecular mechanisms of B cell-specific expression of Pax5 are not fully understood. Here, we applied insertional chromatin immunoprecipitation (iChIP) combined with stable isotope labeling using amino acids in cell culture (SILAC) (iChIP-SILAC) to direct identification of proteins interacting with the promoter region of the endogenous single-copy chicken Pax5 gene. By comparing B cells with macrophage-like cells trans-differentiated by ectopic expression of C/EBPβ, iChIP-SILAC detected B cell-specific interaction of a nuclear protein, Thy28/Thyn1, with the Pax5 1A promoter. Trans-differentiation of B cells into macrophage-like cells caused down-regulation of Thy28 expression. Loss-of-function of Thy28 induced decrease in Pax5 expression and recruitment of myosin-9 (MYH9), one of Thy28-interacting proteins, to the Pax5 1A promoter. Loss-of-function of MYH9 also induced decrease in Pax5 expression. Thus, our analysis revealed that Thy28 is functionally required for B cell-specific expression of Pax5 via recruitment of MYH9 to the Pax5 locus in chicken B cells.     

Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.     

BSAP／Pax—5在B淋巴细胞发育、增殖、分化中的作用

BSAP,一个B细胞特异性激活蛋白,由Pax-5转录的核蛋白。作为核转录因子,其在B细胞的发育、增殖和分化中起重要作用。同时也影响B细胞分化晚期的Ig的分泌。     

Stepwise specification of lymphocyte developmental lineages

B and T lymphocytes differentiate from multipotent precursors through distinct specification and commitment steps. New findings on the unique role of Pax5 in B-lineage commitment, dichotomous action of Notch signaling in B versus T cell development, and the gene expression changes comprising T-lineage specification and commitment now illuminate this process.     

Reduced EBF expression underlies loss of B‐cell potential of hematopoietic progenitors with age

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age‐related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2–5‐fold, while myeloid potential remains unchanged. This age‐related decrease in B‐cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B‐lineage specifying factors, EBF and Pax5. Notably, retrovirus‐mediated expression of EBF complemented the age‐related loss of B‐cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B‐cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B‐cell fate, without altering their potential to generate myeloid cells.     

Regulation of DNA replication within the immunoglobulin heavy-chain locus during B cell commitment

The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh C(H)-3'RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.     

Restricted STAT5 activation dictates appropriate thymic B versus T cell lineage commitment

The molecular mechanisms regulating lymphocyte lineage commitment remain poorly characterized. To explore the role of the IL7R in this process, we generated transgenic mice that express a constitutively active form of STAT5 (STAT5b-CA), a key downstream IL7R effector, throughout lymphocyte development. STAT5b-CA mice exhibit a 40-fold increase in pro-B cells in the thymus. As documented by BrdU labeling studies, this increase is not due to enhanced B cell proliferation. Thymic pro-B cells in STAT5b-CA mice show a modest increase in cell survival ( approximately 4-fold), which correlates with bcl-x(L) expression. However, bcl-x(L) transgenic mice do not show increases in thymic B cell numbers. Thus, STAT5-dependent bcl-x(L) up-regulation and enhanced B cell survival are not sufficient to drive the thymic B cell development observed in STAT5b-CA mice. Importantly, thymic pro-B cells in STAT5b-CA mice are derived from early T cell progenitors (ETPs), suggesting that STAT5 acts by altering ETP lineage commitment. Supporting this hypothesis, STAT5 binds to the pax5 promoter in ETPs from STAT5b-CA mice and induces pax5, a master regulator of B cell development. Conversely, STAT5b-CA mice exhibit a decrease in the DN1b subset of ETPs, demonstrating that STAT5 activation inhibits early T cell differentiation or lineage commitment. On the basis of these findings, we propose that the observed expression of the IL-7R on common lymphoid progenitors, but not ETPs, results in differential STAT5 signaling within these distinct progenitor populations and thus helps ensure appropriate development of B cells and T cells in the bone marrow and thymic environments, respectively.