Sox4 B-lymphocyte progenitors

In this issue of Blood, Mallampati et al provide mechanistic insight into the functions of the transcription factor Sox4 in pro-B lymphocytes using both gain-of-function and loss-of-function approaches combined with global gene expression and genome-wide transcription factor binding analysis.¹ 

Lymphocyte development is regulated by transcriptional regulatory networks that include cell type–specific, as well as broadly expressed transcriptional regulators. These networks guide the activation of lineage-appropriate genes, a process called lineage specification, and they repress genes for alternative lineages, thereby enforcing commitment to the specified fate.²  The networks controlling B-lymphocyte development are among the best studied and have provided many of the paradigms that shape our thinking on gene regulatory circuits and lymphocyte development. Compelling evidence also indicates that lineage-specifying transcriptional networks form the basis for growth regulatory and differentiation control throughout lymphocyte development because key members are frequently mutated in immature and mature B-cell malignanies.³  Nonetheless, multiple transcription factors whose functions remain enigmatic have an impact on B-lymphocyte development, and their activities are not easily placed into the known regulatory networks.

One enigmatic transcription factor is Sox4, a high-mobility group family transcription factor that is required for early B-cell development and whose expression is a poor prognostic factor in B-cell leukemia.⁴  In 1996, Sox4 was reported to be essential for B-cell development at the stage of interleukin-7–dependent expansion, and at least a portion of the function of Sox4 involves promoting the survival of pro-B lymphocytes.^5,6  However, Sox4 is not a B-lymphocyte–specific transcription factor; it plays critical roles in T-lymphocyte development as well as in nonhematopoietic cells, and therefore, its functions are likely influenced by the context in which it is expressed.⁷  The essential targets of Sox4 in B lymphopoiesis have been difficult to identify because pro-B lymphocytes are absent in Sox4^−/− mice. However, the development of a conditional allele for Sox4 has opened the door for investigation into its functions in vivo and in vitro.⁸  Mallampati et al¹  created gain- and loss-of-function models in vitro to study the requirements for Sox4 in pro-B lymphocytes. By using a self-excising Cre-producing retrovirus, they deleted conditional alleles of Sox4 in primary and Bcr-Abl–expressing pro-B lymphocytes and subsequently complemented the Sox4 deficiency with Sox4-producing retrovirus. Combining a careful analysis of the developmental requirements for Sox4 with microarray analysis of messenger RNA (mRNA) expression and chromatin immunoprecipitation of biotin-labeled Sox4 followed by high-throughput sequencing (Sox4 chromatin immunoprecipitation sequencing), they revealed some of the critical targets of Sox4 in pro-B lymphocytes. Of note, approximately 35% of the identified binding sites contained the canonical Sox4 binding sequence; however, Sox4 also appeared to bind DNA through potential GABPA sites, and signature motifs for additional transcription factors were also enriched. Reporter assays revealed that Sox4 can activate transcription via GABPA consensus sites. Therefore, Sox4 may regulate a substantial number of genes through unanticipated interactions, either with unique DNA binding motifs or through interactions with the factors that bind to those motifs. Future experiments will be needed to clarify the mechanisms by which Sox4 regulates gene expression through DNA binding motifs that are not the Sox4 consensus. However, these data raise the intriguing hypothesis that Sox4 function may be highly context dependent because of an ability to regulate genes through interactions with other sequence-specific transcription factors.

The combined analysis performed in the study by Mallampati et al revealed a direct role for Sox4 in expression of the recombinase-activating genes Rag1 and Rag2, with a consequent effect on recombination of the B-cell receptor gene loci. In addition, an unexpected effect on the targets of the Wnt signaling pathway was also identified. Sox4 was bound to the promoter of the Csnk1e gene, encoding casein kinase 1 ε (CK1ε), a known kinase in the GSK3β-containing complex that controls the stability of β-catenin and consequently the function of the Tcf1/Lef1 family of transcription factors.⁹  Sox proteins and Tcf1/Lef1 proteins can interact and have been implicated as antagonistic regulators of the development of subsets of γδ T cells.¹⁰  The findings in this study raise the possibility that Sox4 may also antagonize Lef1 (Tcf1 is not expressed in pro-B lymphocytes) function by controlling the stability of β-catenin in pro-B lymphocytes. Csnk1e mRNA-directed short hairpin RNA resulted in a decreased number of BP1⁺ pro-B lymphocytes and increased the activity of a Tcf1/Lef1 reporter, implicating CK1ε-dependent destruction of β-catenin and inhibition of the Wnt pathway in pro-B lymphocyte survival or expansion. However, the kinase activity of CK1ε targets many proteins, and the current experiments do not rule out a role for these other proteins in pro-B lymphocytes. Nonetheless, this study provides a foundation for further understanding of how Sox4 controls B-cell development and how its functions integrate with other critical regulators of B lymphocyte development and transformation.