Sox4 Downregulates Pu.1 Gene Expression by Binding to An Upper Regulatory Element of Pu.1, a Mechanism Contributing to Leukemogenesis.
Authors: Aue Georg , Du Yang , Cleveland Susan , Smith Stephen , Dave Utpal P. , Jenkins Nancy , Copeland Neal G. , Dunbar Cynthia 
Published: October 2019

Abstract
Abstract 3979
Poster Board III-915
Mice that express 20% the normal levels of the Ets transcription factor Pu.1 develop AML, unlike mice that express 50% to 90% the normal levels, indicating that Pu.1 is a dosage-sensitive tumor suppressor gene. Furthermore, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one Pu.1 allele, suggesting that downregulation of Pu.1 may cooperate with Sox4 in AML induction. Since the other Pu.1 allele remains intact in these AMLs and a 50% decrease in Pu.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate Pu.1 expression in these AMLs. To test this hypothesis, we transfected HL60 promyelocytes with an expression vector carrying both GFP and Sox4 cDNAs or a GFP vector control. Transfected GFP+ cells were purified by flow cytometry and Pu.1 mRNA levels were analyzed by real-time RT-PCR. Pu.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, while Beta-actin mRNA levels were maintained constant, confirming that overexpression of Sox4 downregulates Pu.1 expression in myeloid cells. The decrease of Pu.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly repress the Pu.1 promoter in myeloid cells. Consistent with this, analysis of a published microarray databases comprising 285 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with Pu.1 expression (r= -0.337, p-value<0.001). In order to confirm that downregulation of Pu.1 cooperates with Sox4 in AML induction, we infected Pu.1 heterozygous knockout or wild type bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. An increased penetrance of myeloid leukemia was observed in mice transplanted with Sox4-infected Pu.1 +/- bone marrow (95%) compared to mice receiving Sox4-infected wild type marrow (60%, p<0.001). Myeloid leukemia was confirmed by histology in all animals (100%) of the Sox4-infected Pu.1 +/ cohort. A Southern blot with a Sox4 probe confirmed clonal integrations. Consistent with our hypothesis, integration site analysis of the Sox4-infected Pu.1 +/- cohort tumor spleen DNA could not detect a Pu.1 integration site. Binding motif analysis found a Sox4 binding site in an upper regulatory element (URE) 14 kb upstream of the Pu.1 gene. Chromatin immunohybridization (ChIP) with a Sox4 antibody performed in 32D clone 3 lymphoblasts confirmed binding in a highly conserved area of the Pu.1 upstream control region. An electromobility shift assay (EMSA) is currently pursued. In summary, these results elucidate how the transcription factor Pu.1 is regulated by Sox4 though an upper regulatory element and can play a role in leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.