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1.
Sci Rep ; 9(1): 12342, 2019 08 26.
Article in English | MEDLINE | ID: mdl-31451756

ABSTRACT

Indole derivatives are a structurally diverse group of compounds found in food, toxins, medicines, and produced by commensal microbiota. On contact with acidic stomach conditions, indoles undergo condensation to generate metabolites that vary in solubility, activity and toxicity as they move through the gut. Here, using halogenated ions, we map promising chemo-preventative indoles, i) 6-bromoisatin (6Br), ii) the mixed indole natural extract (NE) 6Br is found in, and iii) the highly insoluble metabolites formed in vivo using desorption/ionisation on porous silicon-mass spectrometry imaging (DIOS-MSI). The functionalised porous silicon architecture allowed insoluble metabolites to be detected that would otherwise evade most analytical platforms, providing direct evidence for identifying the therapeutic component, 6Br, from the mixed indole NE. As a therapeutic lead, 0.025 mg/g 6Br acts as a chemo-preventative compound in a 12 week genotoxic mouse model; at this dose 6Br significantly reduces epithelial cell proliferation, tumour precursors (aberrant crypt foci; ACF); and tumour numbers while having minimal effects on liver, blood biochemistry and weight parameters compared to controls. The same could not be said for the NE where 6Br originates, which significantly increased liver damage markers. DIOS-MSI revealed a large range of previously unknown insoluble metabolites that could contribute to reduced efficacy and increased toxicity.


Subject(s)
Colorectal Neoplasms/metabolism , Gastrointestinal Tract/metabolism , Imaging, Three-Dimensional , Indoles/metabolism , Metabolome , Silicon/chemistry , Animals , Male , Mice, Inbred C57BL , Porosity , Solubility , Xenobiotics/metabolism
2.
Blood Cells Mol Dis ; 44(4): 287-90, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20194037

ABSTRACT

The transcription factor RUNX1 is essential for definitive hematopoiesis and is required for the expression of a number of important hematopoietic regulator genes. It was recently shown that RUNX1 acts within a narrow developmental window during which it cannot be replaced by other members of the RUNX transcription factor family. Studies of the molecular basis of this phenomenon revealed that RUNX1 is required for the opening of chromatin of important hematopoietic regulator genes and for the formation, but not the maintenance of stable transcription factor complexes on these genes. However, the chromatin opening activity of RUNX1 is context dependent, indicating that it cooperates with alternate transcription factors at different stages of hematopoietic development. This review summarizes recent results on the regulation of chromatin structure by RUNX1 in developing hematopoietic cells.


Subject(s)
Chromatin/metabolism , Core Binding Factor Alpha 2 Subunit/physiology , Gene Expression Regulation, Developmental/physiology , Hematopoiesis/genetics , Animals , Cell Lineage , Chromatin Assembly and Disassembly , Core Binding Factor beta Subunit/physiology , Endothelium/cytology , Epigenesis, Genetic/genetics , Hematopoietic Stem Cells/metabolism , Humans , Mice , Multiprotein Complexes , Proto-Oncogene Proteins/physiology , Receptor, Macrophage Colony-Stimulating Factor/physiology , Trans-Activators/physiology , Transcription Factors/metabolism
3.
Blood ; 114(2): 299-309, 2009 Jul 09.
Article in English | MEDLINE | ID: mdl-19339695

ABSTRACT

At the cellular level, development progresses through successive regulatory states, each characterized by their specific gene expression profile. However, the molecular mechanisms regulating first the priming and then maintenance of gene expression within one developmental pathway are essentially unknown. The hematopoietic system represents a powerful experimental model to address these questions and here we have focused on a regulatory circuit playing a central role in myelopoiesis: the transcription factor PU.1, its target gene colony-stimulating-factor 1 receptor (Csf1r), and key upstream regulators such as RUNX1. We find that during ontogeny, chromatin unfolding precedes the establishment of active histone marks and the formation of stable transcription factor complexes at the Pu.1 locus and we show that chromatin remodeling is mediated by the transient binding of RUNX1 to Pu.1 cis-elements. By contrast, chromatin reorganization of Csf1r requires prior expression of PU.1 together with RUNX1 binding. Once the full hematopoietic program is established, stable transcription factor complexes and active chromatin can be maintained without RUNX1. Our experiments therefore demonstrate how individual transcription factors function in a differentiation stage-specific manner to differentially affect the initiation versus maintenance of a developmental program.


Subject(s)
Blood Cells/metabolism , Chromatin/genetics , Chromatin/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression Regulation , Animals , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/deficiency , Core Binding Factor Alpha 2 Subunit/genetics , DNA Methylation , Mice , Promoter Regions, Genetic/genetics , Protein Binding , RNA, Messenger/genetics , Time Factors
4.
Semin Immunol ; 20(4): 257-63, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18579409

ABSTRACT

Hematopoietic stem cells exhibit a multi-lineage gene expression program, and this expression program is either maintained when these cells self-renew, or re-programmed when they differentiate. Both processes require the regulated expression of sequence-specific transcription factors and their interaction with the epigenetic regulatory machinery which programs the chromatin of hematopoietic genes in a cell type specific fashion. This article describes recent findings on the complexity of these molecular interactions and their consequences with respect to the regulation of cell fate decisions. We also describe recent findings from studies of genes expressed in the myeloid lineage (Pu.1 and csf1r) which highlight some of the molecular principles governing cell fate decisions at the epigenetic level.


Subject(s)
Cell Differentiation/physiology , Chromatin/physiology , Gene Expression Regulation/physiology , Myeloid Cells/physiology , Transcription Factors/physiology , Animals , Cell Lineage , Gene Silencing/physiology , Humans , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor, Macrophage Colony-Stimulating Factor/genetics , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism
5.
Mol Cell Biol ; 27(21): 7425-38, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17785440

ABSTRACT

The Ets family transcription factor PU.1 is crucial for the regulation of hematopoietic development. Pu.1 is activated in hematopoietic stem cells and is expressed in mast cells, B cells, granulocytes, and macrophages but is switched off in T cells. Many of the transcription factors regulating Pu.1 have been identified, but little is known about how they organize Pu.1 chromatin in development. We analyzed the Pu.1 promoter and the upstream regulatory element (URE) using in vivo footprinting and chromatin immunoprecipitation assays. In B cells, Pu.1 was bound by a set of transcription factors different from that in myeloid cells and adopted alternative chromatin architectures. In T cells, Pu.1 chromatin at the URE was open and the same transcription factor binding sites were occupied as in B cells. The transcription factor RUNX1 was bound to the URE in precursor cells, but binding was down-regulated in maturing cells. In PU.1 knockout precursor cells, the Ets factor Fli-1 compensated for the lack of PU.1, and both proteins could occupy a subset of Pu.1 cis elements in PU.1-expressing cells. In addition, we identified novel URE-derived noncoding transcripts subject to tissue-specific regulation. Our results provide important insights into how overlapping, but different, sets of transcription factors program tissue-specific chromatin structures in the hematopoietic system.


Subject(s)
Chromatin/chemistry , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Proto-Oncogene Proteins/genetics , RNA, Untranslated/genetics , Trans-Activators/genetics , Transcription, Genetic , Animals , B-Lymphocytes/enzymology , B-Lymphocytes/metabolism , Base Sequence , Cell Differentiation , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/metabolism , Macrophages/enzymology , Macrophages/metabolism , Mice , Molecular Sequence Data , Myeloid Cells/cytology , Myeloid Cells/metabolism , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Binding , RNA Polymerase II/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/metabolism , Transcription Factors/metabolism
6.
Mol Cell Biol ; 27(3): 878-87, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17116688

ABSTRACT

Hematopoietic stem cells and multipotent progenitors exhibit low-level transcription and partial chromatin reorganization of myeloid cell-specific genes including the c-fms (csf1R) locus. Expression of the c-fms gene is dependent on the Ets family transcription factor PU.1 and is upregulated during myeloid differentiation, enabling committed macrophage precursors to respond to colony-stimulating factor 1. To analyze molecular mechanisms underlying the transcriptional priming and developmental upregulation of the c-fms gene, we have utilized myeloid progenitors lacking the transcription factor PU.1. PU.1 can bind to sites in both the c-fms promoter and the c-fms intronic regulatory element (FIRE enhancer). Unlike wild-type progenitors, the PU.1(-/-) cells are unable to express c-fms or initiate macrophage differentiation. When PU.1 was reexpressed in mutant progenitors, the chromatin structure of the c-fms promoter was rapidly reorganized. In contrast, assembly of transcription factors at FIRE, acquisition of active histone marks, and high levels of c-fms transcription occurred with significantly slower kinetics. We demonstrate that the reason for this differential activation was that PU.1 was required to promote induction and binding of a secondary transcription factor, Egr-2, which is important for FIRE enhancer activity. These data suggest that the c-fms promoter is maintained in a primed state by PU.1 in progenitor cells and that at FIRE PU.1 functions with another transcription factor to direct full activation of the c-fms locus in differentiated myeloid cells. The two-step mechanism of developmental gene activation that we describe here may be utilized to regulate gene activity in a variety of developmental pathways.


Subject(s)
Chromatin Assembly and Disassembly , Gene Expression Regulation, Developmental , Genes, fms/genetics , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription, Genetic/genetics , Animals , Base Sequence , Chromatin Assembly and Disassembly/genetics , Deoxyribonuclease I/metabolism , Early Growth Response Protein 2/metabolism , Enhancer Elements, Genetic , Histones/metabolism , Kinetics , Methylation , Mice , Models, Genetic , Molecular Sequence Data , NIH 3T3 Cells , Promoter Regions, Genetic/genetics , Protein Binding , Proto-Oncogene Proteins/deficiency , RNA Polymerase II/metabolism , TATA-Box Binding Protein/metabolism , Trans-Activators/deficiency , Transcription Factors/metabolism , Transcriptional Activation
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