BackgroundThe embryonic stem cell (ESC) factor, SALL4, plays an essential role in both development and leukemogenesis. It is a unique gene that is involved in self-renewal in ESC and leukemic stem cell (LSC).Methodology/Principal FindingsTo understand the mechanism(s) of SALL4 function(s), we sought to identify SALL4-associated proteins by tandem mass spectrometry. Components of a transcription repressor Mi-2/Nucleosome Remodeling and Deacetylase (NuRD) complex were found in the SALL4-immunocomplexes with histone deacetylase (HDAC) activity in ESCs with endogenous SALL4 expression and 293T cells overexpressing SALL4. The SALL4-mediated transcriptional regulation was tested on two potential target genes: PTEN and SALL1. Both genes were confirmed as SALL4 downstream targets by chromatin-immunoprecipitation, and their expression levels, when tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR), were decreased in 293T cells overexpressing SALL4. Moreover, SALL4 binding sites at the promoter regions of PTEN and SALL1 were co-occupied by NuRD components, suggesting that SALL4 represses the transcriptions of PTEN and SALL1 through its interactions with the Mi-2/NuRD complex. The in vivo repressive effect(s) of SALL4 were evaluated in SALL4 transgenic mice, where decreased expressions of PTEN and SALL1 were associated with myeloid leukemia and cystic kidneys, respectively.Conclusions/SignificanceIn summary, we are the first to demonstrate that stem cell protein SALL4 represses its target genes, PTEN and SALL1, through the epigenetic repressor Mi-2/NuRD complex. Our novel finding provides insight into the mechanism(s) of SALL4 functions in kidney development and leukemogenesis.

Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2–4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

The mixed-lineage leukemia (MLL) H3K4 methyltransferase protein, and the heterodimeric RUNX1/CBF␤ transcription factor complex, are critical for definitive and adult hematopoiesis, and both are frequently targeted in human acute leukemia. We identified a physical and functional interaction between RUNX1 (AML1) and MLL and show that both are required to maintain the histone lysine 4 trimethyl mark (H3K4me3) at 2 critical regulatory regions of the AML1 target gene PU. IntroductionThe transcriptional regulation of hematopoiesis requires coordinate changes in gene expression to control the processes of stem cell self-renewal, differentiation, and maturation. The primary importance of transcriptional regulation in hematopoiesis is exemplified by human acute myelogenous leukemia, where recurrent chromosomal translocations are found that affect transcriptional regulators including transcription factors and histone modifying enzymes, such as AML1, CBF␤, RAR␣, TEL, MLL, MOZ, CBP, and p300. 1,2 Acute leukemias accompanied by MLL translocations have traditionally been thought of being distinct from the CBF leukemias (those associated with AML1 or CBF␤ translocations) at least in part because of their different gene expression profile, different prognosis, and different frequency of occurrence in de novo versus secondary AML. Yet mutations and translocations involving the mixed-lineage leukemia and AML1 genes are found in both AML and ALL, and it has been postulated that MLL and AML1/CBF␤ may both participate in regulating some common target genes. 3 The MLL gene was isolated as a common target of chromosomal translocations involving 11q23. [4][5][6] These translocations, which are observed in adult, childhood, and infant acute leukemias, fuse MLL with more than 60 different partner proteins. However, the most common translocations generate the MLL/AF6, MLL/ AF9, MLL/ENL(s), MLL/AF10, and the MLL/AF17 fusion proteins in AML, and the MLL/AF4, MLL/LAF4, MLL/5q31, and MLL/ENL(l) fusion proteins in B-ALL. 7 MLL is a functional ortholog of the Drosophila trithorax (trx) protein, 8 which is involved in maintaining epigenetic transcriptional memory at homeobox (Hox) gene loci. 9 The SET domain of MLL has histone methyltransferase activity 10 and MLL forms a multicomponent complex that specifically methylates lysine 4 on histone H3 (H3-K4), a modification typically associated with transcriptionally active regions of chromatin. The best-studied downstream targets of MLL (and trx) are the Hox genes, which control segment pecificity and cell fate in the developing embryo. 11 Targeted homozygous disruption of MLL in mice was embryonic lethal at day 10.5-14, 12 and similar to loss of trx function in flies, Hox gene expression was initiated but not maintained in these mice. 13 Another group, who targeted exons 12-14 of Mll, obtained a slight delay in lethality, 14 but both groups reported that Mll-deficient mice have defective yolk sac and fetal liver hematopoiesis. 15 Animals carrying a single normal Mll allele are phenotypically abnor...