The main finding of this study is the identification of 26 genes influenced by SOX11 in MCL. SOX11 has critical roles in embryonic neurogenesis and tissue remodeling 
. It is also required for neuron survival and neurite growth 
but no defined role in hematopoiesis has yet been shown.
The SOX11 protein belongs to the HMG box super family of DNA-binding proteins which are highly conserved between animals. They are involved in the regulation of such diverse development processes as specification of early embryonic germ layers 
, organ development and neurogenesis 
. SOX11, together with two other SOX family members SOX4 and SOX12, belong to the same subgroup, SOXC. SOX4 is so far the only gene in this family that has been found to have an important role in lymphopoiesis. SOX4–null hematopoietic cells grafted into wild type mice remain blocked at the pro-B cell stage 
. Although SOX11 is highly homologous to SOX4, its role in lymphopoiesis remains unclear.
Recently, SOX11 was found to be aberrantly expressed in MCL 
. Importantly, approximately 10% of cyclin D1 positive MCL lack nuclear expression of SOX11 
. SOX11 is also expressed in subsets of Burkitt lymphomas, lymphoblastic leukemias and hairy cell leukemias 
. Thus, expression of SOX11 in lymphoma seems independent of the t(11;14) cyclin D1 translocation.
The possible role of the aberrant SOX11 expression in lymphoma is unknown. Others have shown that SOX11 is needed for the expression of pan-neuronal genes, including the Class-Ш β-tubulin gene TUBB3 (also named Tuj1). TUBB3 was the first gene to be identified as a potential direct target of SOXC proteins. In this study, we showed that SOX11 can modify the expression of TUBB3 also in MCL.
By gene expression analysis using a very stringent threshold, we identified 26 genes, all significantly downregulated, after SOX11 knockdown in MCL cells. Three of these genes were also found to be significantly correlated to SOX11 expression levels in 16 SOX11 positive primary MCL samples from various tissues. During the course of our study, Fernandez et al. published results on MCL with leukemic, non-nodal presentation and a very indolent clinical course 
. Many of these tumors had in fact been misdiagnosed as other types of lymphomas. 15 of the 26 SOX11siRNA downregulated genes in Granta 519 were found to be significantly correlated to SOX11 expression in the Fernandez cohort, confirming our observations.
Prior to this study, Tubb3 and Tead2 were the only genes proven to be directly regulated by Sox11 
. By chromatin immunoprecipitation we showed direct binding of the SOX11 protein to the promoter regions of the DBN1, SETMAR and HIG2 genes in MCL cells.
DBN1 and three other genes downregulated by SOX11 silencing, TMSB15A, TMSB15B and C5orf13 are actin binding and involved in cell shape and motility. DBN1 encodes for the actin binding protein drebrin 1 (d
), cloned in 1988 
. DBN1 was later shown to be is involved in neurite formation and synaptic signalling (reviewed in 
) and to be downregulated in brains of Alzheimer patients 
. Drebrin is also expressed in epithelial cells of the stomach, kidney, colon and in cell lines from fibroblasts and astrocytoma 
. Interestingly, drebrin is cleaved by caspase-6 
, encoded by another of the SOX11siRNA downregulated genes.TMSB15A and TMSB15B encode for different isoforms of the actin binding protein thymosin beta 15. Thymosin beta 15 promotes cell motility and was discovered as highly upregulated in human breast, prostate and colon cancer 
. According to NCBI, C5orf13 is coding for the protein P311, highly expressed in glioma and involved in glioma cell migration through the reorganization of the actin cytoskeleton 
. Thus a number of genes involved in cell shape and motility were downregulated by SOX11 siRNA treatment of Granta 519 cells. We could, however, not detect any significant change in shape or growth pattern of the cells after siRNA treatment (data not shown).
SETMAR, HIG2 and HMGB3 could potentially influence cell division and response to cytostatic treatment. SETMAR, also called METNASE, is cooperating with topoisomerase II alpha in decoiling of chromosomes during mitosis. Recent evidence in acute myeloid leukemia suggests that SETMAR may confer resistance to the topoisomerase II alpha inhibitor VP-16 
. The HMGB3 protein belongs to the high mobility group of proteins and may, as shown for other HMG proteins, play a role in DNA replication and transcription. Hmgb3 is highly expressed in hematopoietic stem cells 
. Recent experiment in mice indicate that Hmgb3 downregulation is associated with increased Wnt-signalling, more rapid renewal of stem cells and fewer lymphoid and myeloid progenitor cells 
. We therefore investigated a possible influence on cell proliferation or cell survival in vitro
after SOX11 knock-down by siRNA. In these short term experiments, we did however not detect any significant changes in cell proliferation or viability after SOX11 downregulation (manuscript in preparation), in contrast to what has been reported for neural cells 
. However, since the effect on gene expression in siRNA treated cells is transient, we cannot exclude possible effects of SOX11 expression on cell survival or proliferation in other experimental settings.
SOX11 is aberrantly expressed in MCL but the molecular mechanism(s) responsible for its upregulation in lymphoid and hematopoietic cells are not yet defined, in contrast to other cell types. In chicken neural cells overexpression of Ngn2 and Ascl1 upregulates Sox11 while the transcriptional repressors Id1 and REST/NRSF downregulates its expression 
. In the mouse embryonal neuronal plate Sox11 was upregulated by FoxD5 and Notch signalling 
. SOX11 mRNA expression was also rapidly (within 3 hours) upregulated after exposure of retinal microglia to 17beta-estradiol 
. Even though SOX11 is highly expressed in most MCL, expression of SOX11 seems not to be dependent on the t(11;14) since it is a feature of both cyclin D1 positive and cyclin D1 negative cases 
. Furthermore, SOX11 may be expressed in other aggressive B cell lymphomas that lack cyclin D1.
In summary, we have used selective siRNA targeting and downregulation of SOX11 to identify a set of SOX11 responsive genes. Our results have been validated in primary MCL tumors and freshly isolated lymphoma cells. Three of the identified genes, DBN1, SETMAR and HIG2, were found to be directly targeted by the SOX11 protein in MCL.