In vitro hematopoietic differentiation of mouse embryonic stem cells requires the tumor suppressor menin and is mediated by Hoxa9

Inactivating mutations in the tumor suppressor gene MEN1 cause the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1). The ubiquitously expressed MEN1 encoded protein, menin, interacts with MLL (mixed-lineage leukemia protein), and together they are essential components of a multiprotein complex with histone methyl transferase activity. MLL is also essential for hematopoiesis, and plays a critical role in leukemogenesis via epigenetic regulation of Hoxa9 expression that also requires menin. Therefore we chose to explore the role of menin in hematopoiesis. We generated Men1^−/− embryonic stem (ES) cell lines, and induced them to differentiate in vitro. While these cells were able to form embryoid bodies (EBs) expressing the early markers Flk-1 and c-Kit, their ability to further differentiate into hematopoietic colonies was compromised. The Men1^−/− ES cells show reduced expression of Hoxa9 that can be recovered by reexpression of Menin. We demonstrate that the block in differentiation of Men1^−/− ES cell lines can be rescued not only by the expression of menin but also that of Hoxa9. These results suggest that, similar to MLL, menin is required for hematopoiesis, and this requirement may be mediated through regulation of Hoxa9 expression.     

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8

Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.

Structured summary

MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007)MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007)MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)     

MLL3 suppresses tumorigenesis through regulating TNS3 enhancer activity

MLL3 is a histone H3K4 methyltransferase that is frequently mutated in cancer, but the underlying molecular mechanisms remain elusive. Here, we found that MLL3 depletion by CRISPR/sgRNA significantly enhanced cell migration, but did not elevate the proliferation rate of cancer cells. Through RNA-Seq and ChIP-Seq approaches, we identified TNS3 as the potential target gene for MLL3. MLL3 depletion caused downregulation of H3K4me1 and H3K27ac on an enhancer ~ 7 kb ahead of TNS3. 3C assay indicated the identified enhancer interacts with TNS3 promoter and repression of enhancer activity by dCas9-KRAB system impaired TNS3 expression. Exogenous expression of TNS3 in MLL3 deficient cells completely blocked the enhanced cell migration phenotype. Taken together, our study revealed a novel mechanism for MLL3 in suppressing cancer, which may provide novel targets for diagnosis or drug development.Subject terms: Tumour-suppressor proteins, Chromosomes     

HOXA9 regulates miR-155 in hematopoietic cells

HOXA9-mediated up-regulation of miR-155 was noted during an array-based analysis of microRNA expression in Hoxa9^−/−bone marrow (BM) cells. HOXA9 induction of miR-155 was confirmed in these samples, as well as in wild-type versus Hoxa9-deficient marrow, using northern analysis and qRT–PCR. Infection of wild-type BM with HOXA9 expressing or GFP⁺ control virus further confirmed HOXA9-mediated regulation of miR-155. miR-155 expression paralleled Hoxa9 mRNA expression in fractionated BM progenitors, being highest in the stem cell enriched pools. HOXA9 capacity to induce myeloid colony formation was blunted in miR-155-deficient BM cells, indicating that miR-155 is a downstream mediator of HOXA9 function in blood cells. Pu.1, an important regulator of myelopoiesis, was identified as a putative down stream target for miR-155. Although miR-155 was shown to down-regulate the Pu.1 protein, HOXA9 did not appear to modulate Pu.1 expression in murine BM cells.     

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

The majority of eukaryotic pre-mRNAs are processed by 3′-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3′-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the ∼ 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 Å resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3′-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.     

The Human Mixed Lineage Leukemia 5 (MLL5), a Sequentially and Structurally Divergent SET Domain-Containing Protein with No Intrinsic Catalytic Activity

Mixed Lineage Leukemia 5 (MLL5) plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. Chromatin binding is ensured by its plant homeodomain (PHD) through a direct interaction with the N-terminus of histone H3 (H3). In addition, MLL5 contains a Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, a protein module that usually displays histone lysine methyltransferase activity. We report here the crystal structure of the unliganded SET domain of human MLL5 at 2.1 Å resolution. Although it shows most of the canonical features of other SET domains, both the lack of key residues and the presence in the SET-I subdomain of an unusually large loop preclude the interaction of MLL5 SET with its cofactor and substrate. Accordingly, we show that MLL5 is devoid of any in vitro methyltransferase activity on full-length histones and histone H3 peptides. Hence, the three dimensional structure of MLL5 SET domain unveils the structural basis for its lack of methyltransferase activity and suggests a new regulatory mechanism.     

MicroRNA-142-3p inhibits cell proliferation in human acute lymphoblastic leukemia by targeting the MLL-AF4 oncogene

The mixed-lineage leukemia (MLL)-AF4 fusion protein encoded by the chromosomal translocation t(4;11) predicts a poorer prognosis in acute lymphoblastic leukemia (ALL) than in other MLL-associated leukemias. However, the detailed mechanism underlying regulation of MLL-AF4 expression remains largely unknown. In this study, we showed that microRNA (miR)-142-3p was significantly downregulated in ALL patients expressing MLL-AF4. Upregulation of miR-142-3p decreased MLL-AF4 expression in the RS4;11 leukemic cell line, which suggests that MLL-AF4 is a direct target of miR-142-3p. Ectopic expression of miR-142-3p remarkably suppressed cell proliferation and induced apoptosis in RS4;11 cells expressing the MLL-AF4 fusion protein. We also found that exogenous expression of miR-142-3p strongly reduced the expression of MLL-AF4 target genes such as homeobox A (HOXA)9, HOXA7, and HOXA10 in RS4;11 cells. Taken together, our results indicate that miR-142-3p functions as a growth suppressor in MLL-AF4⁺ ALL, and its suppressive effects are mediated primarily through repression of MLL-AF4 expression.     

MicroRNA-126 regulates HOXA9 by binding to the homeobox

The PicTar program predicted that microRNA-126 (miR-126), miR-145, and let-7s target highly conserved sites within the Hoxa9 homeobox. There are increased nucleotide constraints in the three microRNA seed sites among Hoxa9 genes beyond that required to maintain protein identity, suggesting additional functional conservation. In preliminary experiments, forced expression of these microRNAs in Hoxa9-immortalized bone marrow cells downregulated the HOXA9 protein and caused loss of biological activity. The microRNAs were shown to target their predicted sites within the homeobox. miR-126 and Hoxa9 mRNA are coexpressed in hematopoietic stem cells and downregulated in parallel during progenitor cell differentiation; however, miR-145 is barely detectable in hematopoietic cells, and let-7s are highly expressed in bone marrow progenitors, suggesting that miR-126 may function in normal hematopoietic cells to modulate HOXA9 protein. In support of this hypothesis, expression of miR-126 alone in MLL-ENL-immortalized bone marrow cells decreased endogenous HOXA9 protein, while inhibition of endogenous miR-126 increased expression of HOXA9 in F9 cells.     

Learning from mouse models of MLL fusion gene-driven acute leukemia

5–10% of human acute leukemias carry chromosomal translocations involving the mixed lineage leukemia (MLL) gene that result in the expression of chimeric protein fusing MLL to >80 different partners of which AF4, ENL and AF9 are the most prevalent. In contrast to many other leukemia-associated mutations, several MLL-fusions are powerful oncogenes that transform hematopoietic stem cells but also more committed progenitor cells. Here, I review different approaches that were used to express MLL fusions in the murine hematopoietic system which often, but not always, resulted in highly penetrant and transplantable leukemias that closely phenocopied the human disease. Due to its simple and reliable nature, reconstitution of irradiated mice with bone marrow cells retrovirally expressing the MLL-AF9 fusion became the most frequently in vivo model to study the biology of acute myeloid leukemia (AML). I review some of the most influential studies that used this model to dissect critical protein interactions, the impact of epigenetic regulators, microRNAs and microenvironment-dependent signals for MLL fusion-driven leukemia. In addition, I highlight studies that used this model for shRNA- or genome editing-based screens for cellular vulnerabilities that allowed to identify novel therapeutic targets of which some entered clinical trials. Finally, I discuss some inherent characteristics of the widely used mouse model based on retroviral expression of the MLL-AF9 fusion that can limit general conclusions for the biology of AML. This article is part of a Special Issue entitled: The MLL family of proteins in normal development and disease edited by Thomas A Milne.