In liver, most genes are expressed with a porto-central gradient. The transcription factor hepatic nuclear-factor4␣ (HNF4␣) is associated with 12% of the genes in adult liver, but its involvement in zonation of gene expression has not been investigated. A putative HNF4␣-response element in the upstream enhancer of glutamine synthetase (GS), an exclusively pericentral enzyme, was protected against DNase-I and interacted with a protein that is recognized by HNF4␣-specific antiserum. Chromatin-immunoprecipitation assays of HNF4␣-deficient (H4LivKO) and control (H4Flox) livers with HNF4␣ antiserum precipitated the GS upstream enhancer DNA only from H4Flox liver. Identical results were obtained with a histone-deacetylase1 (HDAC1) antibody, but antibodies against HDAC3, SMRT and SHP did not precipitate the GS upstream enhancer. In H4Flox liver, GS, ornithine aminotransferase (OAT) and thyroid hormone-receptor 1 ( T he development and maintenance of liver architecture and function is regulated by liver-enriched transcription factors. 1 One of these, hepatic nuclear factor 4␣ (HNF4␣; NR2A1) is expressed at high levels in liver, kidney, intestine, and pancreas 2,3 and binds to the promoter of 12% of genes that are expressed in adult liver. 4 HNF4␣ is an orphan member of the nuclear-receptor superfamily. 2 Depending on chain length and degree of saturation, 5 fatty acyl-coenzyme A thioesters may act as agonistic or antagonistic factors, but whether or not these thioesters function as ligands remains unsettled. 2,[6][7][8] Transcriptional regulation by HNF4␣ is accomplished by interactions with coactivator or corepressor mediators (e.g., GRIP1, SRC-1, CBP/p300, SMRT). 6,7,9,10 The resulting coactivator or corepressor complexes have intrinsic histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity, respectively. Histone modifications play an important role in the regulation of the
LMO2 is a bridging factor within a DNA binding complex and is required for definitive haematopoiesis to occur. The developmental stage of the block in haematopoietic specification is not known. We show that Lmo2−/− mouse embryonic stem cells differentiated to Flk-1+ haemangioblasts, but less efficiently to haemogenic endothelium, which only produced primitive haematopoietic progenitors. Genome-wide approaches indicated that LMO2 is required at the haemangioblast stage to position the TAL1/LMO2/LDB1 complex to regulatory elements that are important for the establishment of the haematopoietic developmental program. In the absence of LMO2, the target site recognition of TAL1 is impaired. The lack of LMO2 resulted in altered gene expression levels already at the haemangioblast stage, with transcription factor genes accounting for ∼15% of affected genes. Comparison of Lmo2−/− with Tal1−/− Flk-1+ cells further showed that TAL1 was required to initiate or sustain Lmo2 expression.
T-cell acute lymphoblastic leukaemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients relapse and die from the disease. To improve patient outcome new drugs are needed. With the aim to identify new therapeutic targets, we integrated transcriptomics and metabolomics data, including live-cell NMR-spectroscopy, of cell lines and patient samples. We found that T-ALL cells have limited energy availability, resulting in active AMPK-signalling and reduced autophagy. Despite this, mTOR kinase remains active and essential for glutamine-uptake that fuels rapid proliferation.Glutamine fuels aspartate synthesis and together they supply three nitrogen atoms in purines and all atoms but one carbon in pyrimidine rings. We show that EAAT1, a glutamateaspartate transporter normally only expressed in the CNS, is crucial for glutamine conversion to nucleotides and that T-ALL cell proliferation depends on EAAT1 function, identifying it as a target for T-ALL treatment. Finally, we performed an in silico screen and identified a novel EAAT1-specific allosteric inhibitor.
The transcriptional mediator LIM domain only 2 (LMO2) forms a large multi-protein complex together with TAL1/LYL1, HEB/E2A, LDB1 and GATA. This complex regulates transcription from the onset of haematopoietic development and during differentiation. Chromosomal re-arrangements involving LMO and other members of the complex are causative for T-cell lymphoblastic leukaemia (T-ALL). We have identified Plant Homeodomain (PHD)-like Finger 6 (PHF6) as a new LMO2 interacting factor. Somatic mutations in PHF6 have been found to occur in several types of leukaemia. We show that PHF6 interacts with LMO2 during the initial stages of the haematopoietic development, myeloid differentiation and in T-ALL. The LMO2/PHF6 complex binds the DNA and regulates linage-specific gene expression. Additionally, a loss or reduction of LMO2 and PHF6 leads to chromosomal instability. PHF6 and LMO2 are required for maintaining levels of γH2AX and 53BP1, where PHF6 is important for γH2AX accumulation and LMO2 has a role in recruiting 53BP1 to γH2AX foci.
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