RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element.

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

C/EBPα is required for development of dendritic cell progenitors.

Dendritic cells (DCs) are master regulators of the immune system, but molecular regulation of early DC differentiation has been poorly understood. Here, we report that the transcription factor C/EBPα coordinates the development of progenitor cells required for production of multiple categories of DCs. C/EBPα was needed for differentiation from stem/progenitor cells to common DC progenitors (CDPs), but not for transition of CDP to mature DCs. C/EBPα deletion in mature DCs did not affect their numbers or function, suggesting that this transcription factor is not needed for maintenance of DCs in lymphoid tissues. ChIP-seq and microarrays were used to identify candidate genes regulated by C/EBPα and required for DC formation. Genes previously shown to be critical for DC formation were bound by C/EBPα, and their expression was decreased in the earliest hematopoietic compartments in the absence of C/EBPα. These data indicate that C/EBPα is important for the earliest stages of steady-state DC differentiation.

Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling.

Activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain determine responsiveness to EGFR tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer (NSCLC). The modulation of transcriptional pathways by mutant EGFR signaling is not fully understood. Previously, we and others identified a single base pair change leading to a threonine to methionine (T790M) amino acid alteration in the ATP-binding pocket of the EGFR as a common mechanism of acquired resistance. The gefitinib-resistant, T790M-mutant H1975 NSCLC cell line undergoes prominent growth arrest and apoptosis when treated with the irreversible EGFR inhibitor, CL-387,785. We did a transcriptional profiling study of mutant EGFR target genes that are differentially expressed in the "resistant" gefitinib-treated and the "sensitive" CL387,785-treated H1975 cells to identify the pivotal transcriptional changes in NSCLC with EGFR-activating mutations. We identified a small subset of early gene changes, including significant reduction of cyclin D1 as a result of EGFR inhibition by CL-387,785 but not by gefitinib. The reduction in cyclin D1 transcription was associated with subsequent suppression of E2F-responsive genes, consistent with proliferation arrest. Furthermore, cyclin D1 expression was higher in EGFR-mutant lung cancer cells compared with cells with wild-type EGFR. EGFR-mutant cells were routinely sensitive to the cyclin-dependent kinase inhibitor flavopiridol, confirming the functional relevance of the cyclin D axis. These studies suggest that cyclin D1 may contribute to the emergence of EGFR-driven tumorigenesis and can be an alternative target of therapy.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression.

Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non-small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1.

Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.

C/EBPbeta is required for 'emergency' granulopoiesis.

During 'emergency' situations such as infections, host defense requires rapid mobilization of bone marrow granulocyte progenitors. 'Steady-state' granulopoiesis is absolutely dependent on the C/EBPalpha transcription factor, but the transcriptional mechanisms underlying emergency granulopoiesis remain unclear. Here we show that large numbers of granulocytes were generated from C/EBPalpha-deficient progenitors after cytokine stimulation in vivo. Cytokine treatment or fungal infection induced upregulation of C/EBPbeta but not C/EBPalpha or C/EBPepsilon transcripts in granulocyte progenitors, and C/EBPbeta-deficient progenitors showed decreased emergency-induced granulopoiesis in vitro and in vivo. C/EBPbeta inhibited proliferation less severely than did C/EBPalpha. These data suggest a critical function for C/EBPbeta in emergency granulopoiesis, which demands both differentiation and proliferation of granulocyte precursors.

Dysregulation of protein modification by ISG15 results in brain cell injury.

UBP43 (USP18) is a protease that removes the ubiquitin-like modifier ISG15 from conjugated proteins. Here we present the first report of dysregulation of protein ISG15 modification by the generation of UBP43 knockout mice. In the absence of UBP43, brain tissue showed an elevated level of ISG15 conjugates, and cellular necrosis was evident in the ependyma. Such disruption of the blood-brain barrier resulted in severe neurologic disorders. These results demonstrate that UBP43 plays a critical role in maintaining the homeostatic balance of ISG15-conjugated protein, and that regulation of cellular levels of ISG15 protein modification is essential for brain cell function.