Person:

Tenen, Daniel

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a (Runx1^{−/−}) embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPβ and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.

Background: C/EBPα is a transcription factor essential for terminal differentiation of several cell types. It has not known if C/EBPα protein is expressed and functions in the prostate gland. Methods: The presence of C/EBPα in normal and cancerous prostate epithelium was examined by immunochemistry. Over expression of C/EBPα in LNCaP cells was conducted with retrovirus-mediated transduction. PSA expression was examined by RT-PCR and western blot and PSA promoter activity by luciferase reporter assay. Results: In normal prostate C/EBPα was expressed in the basal layer of the epithelium. In prostate cancer C/EBPα was detected at low levels throughout the cancers and in advanced prostate cancer C/EBPα expression was associated with decreased expression of AR and PSA. Overexpression of C/EBPα inhibited epigenetically PSA expression and was accompanied by the loss of expression of AR. Transient increase of C/EBPα inhibited the PSA promoter/enhancer activity independently of expression of AR. Conclusion: In LNCaP cells C/EBPα over expression inhibits expression of PSA by AR -dependent and independent mechanisms and by extinguishing AR expression provides a model for hormonal independent cell growth.

The t(8;21) translocation fuses the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO protein. The resultant RUNX1/ETO fusion protein is a leukemia-initiating transcription factor that interferes with RUNX1 function. The result of this interference is a block in differentiation and, finally, the development of acute myeloid leukemia (AML). To obtain insights into RUNX1/ETO-dependant alterations of the epigenetic landscape, we measured genome-wide RUNX1- and RUNX1/ETO-bound regions in t(8;21) cells and assessed to what extent the effects of RUNX1/ETO on the epigenome depend on its continued expression in established leukemic cells. To this end, we determined dynamic alterations of histone acetylation, RNA Polymerase II binding and RUNX1 occupancy in the presence or absence of RUNX1/ETO using a knockdown approach. Combined global assessments of chromatin accessibility and kinetic gene expression data show that RUNX1/ETO controls the expression of important regulators of hematopoietic differentiation and self-renewal. We show that selective removal of RUNX1/ETO leads to a widespread reversal of epigenetic reprogramming and a genome-wide redistribution of RUNX1 binding, resulting in the inhibition of leukemic proliferation and self-renewal, and the induction of differentiation. This demonstrates that RUNX1/ETO represents a pivotal therapeutic target in AML.

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPβ. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPβ-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPβ were involved in the positive regulation of C/EBPβ transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPβ mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPβ transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPβ upregulation.

Summary In blood, transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver (FL) hematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa deficient adult HSCs underwent a pronounced expansion with enhanced proliferation, characteristics resembling FL HSCs. Consistently, transcription profiling of C/EBPa deficient HSCs revealed a gene expression programme similar to FL HSCs. Moreover we observed that age-specific C/EBPa expression correlated with its inhibitory effect on HSC cell cycle. Mechanistically we identified N-Myc as C/EBPa downstream target, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.

An exciting recent approach to targeting transcription factors in cancer is to block formation of oncogenic complexes. We investigated whether interfering with the interaction of the transcription factor SALL4, which is critical for leukemic cell survival, and its epigenetic partner complex represents a novel therapeutic approach. The mechanism of SALL4 in promoting leukemogenesis is at least in part mediated by its repression of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through its interaction with a histone deacetylase (HDAC) complex. In this study, we demonstrate that a peptide can compete with SALL4 in interacting with the HDAC complex and reverse its effect on PTEN repression. Treating SALL4-expressing malignant cells with this peptide leads to cell death that can be rescued by a PTEN inhibitor. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells using an RNAi approach. In summary, our results demonstrate a novel peptide that can block the specific interaction between SALL4 and its epigenetic HDAC complex in regulating its target gene, PTEN. Furthermore, targeting SALL4 with this approach could be an innovative approach in treating leukemia.

Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease.

Objective: Hepatocellular carcinoma (HCC) is a heterogeneous tumour displaying a complex variety of genetic and epigenetic changes. In human cancers, aberrant post-transcriptional modifications, such as alternative splicing and RNA editing, may lead to tumour specific transcriptome diversity. Design: By utilising large scale transcriptome sequencing of three paired HCC clinical specimens and their adjacent non-tumour (NT) tissue counterparts at depth, we discovered an average of 20 007 inferred A to I (adenosine to inosine) RNA editing events in transcripts. The roles of the double stranded RNA specific ADAR (Adenosine DeAminase that act on RNA) family members (ADARs) and the altered gene specific editing patterns were investigated in clinical specimens, cell models and mice. Results: HCC displays a severely disrupted A to I RNA editing balance. ADAR1 and ADAR2 manipulate the A to I imbalance of HCC via their differential expression in HCC compared with NT liver tissues. Patients with ADAR1 overexpression and ADAR2 downregulation in tumours demonstrated an increased risk of liver cirrhosis and postoperative recurrence and had poor prognoses. Due to the differentially expressed ADAR1 and ADAR2 in tumours, the altered gene specific editing activities, which was reflected by the hyper-editing of FLNB (filamin B, β) and the hypo-editing of COPA (coatomer protein complex, subunit α), are closely associated with HCC pathogenesis. In vitro and in vivo functional assays prove that ADAR1 functions as an oncogene while ADAR2 has tumour suppressive ability in HCC. Conclusions: These findings highlight the fact that the differentially expressed ADARs in tumours, which are responsible for an A to I editing imbalance, has great prognostic value and diagnostic potential for HCC.