Publications by authors named "Siebe Loontiens"

6 Publications

  • Page 1 of 1

PHF6 Expression Levels Impact Human Hematopoietic Stem Cell Differentiation.

Front Cell Dev Biol 2020 4;8:599472. Epub 2020 Nov 4.

Department of Diagnostic Sciences, Ghent University, Ghent, Belgium.

Transcriptional control of hematopoiesis involves complex regulatory networks and functional perturbations in one of these components often results in malignancies. Loss-of-function mutations in , encoding a presumed epigenetic regulator, have been primarily described in T cell acute lymphoblastic leukemia (T-ALL) and the first insights into its function in normal hematopoiesis only recently emerged from mouse modeling experiments. Here, we investigated the role of PHF6 in human blood cell development by performing knockdown studies in cord blood and thymus-derived hematopoietic precursors to evaluate the impact on lineage differentiation in well-established models. Our findings reveal that levels differentially impact the differentiation of human hematopoietic progenitor cells into various blood cell lineages, with prominent effects on lymphoid and erythroid differentiation. We show that loss of PHF6 results in accelerated human T cell development through reduced expression of and its downstream target genes. This functional interaction in developing thymocytes was confirmed using a -deficient zebrafish model that also displayed accelerated developmental kinetics upon reduced or notch1 activation. In summary, our work reveals that appropriate control of expression is important for normal human hematopoiesis and provides clues towards the role of in T-ALL development.
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http://dx.doi.org/10.3389/fcell.2020.599472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672048PMC
November 2020

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes.

BMC Genomics 2019 Mar 20;20(1):228. Epub 2019 Mar 20.

Department of Biomolecular Medicine & Center for Medical Genetics, Ghent University, 9000, Ghent, Belgium.

Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.

Results: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.

Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.
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http://dx.doi.org/10.1186/s12864-019-5608-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425699PMC
March 2019

ALK positively regulates MYCN activity through repression of HBP1 expression.

Oncogene 2019 04 11;38(15):2690-2705. Epub 2018 Dec 11.

Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium.

ALK mutations occur in 10% of primary neuroblastomas and represent a major target for precision treatment. In combination with MYCN amplification, ALK mutations infer an ultra-high-risk phenotype resulting in very poor patient prognosis. To open up opportunities for future precision drugging, a deeper understanding of the molecular consequences of constitutive ALK signaling and its relationship to MYCN activity in this aggressive pediatric tumor entity will be essential. We show that mutant ALK downregulates the 'HMG-box transcription factor 1' (HBP1) through the PIK-AKT-FOXO3a signaling axis. HBP1 inhibits both the transcriptional activating and repressing activity of MYCN, the latter being mediated through PRC2 activity. HBP1 itself is under negative control of MYCN through miR-17~92. Combined targeting of HBP1 by PIK antagonists and MYCN signaling by BET- or HDAC-inhibitors blocks MYCN activity and significantly reduces tumor growth, suggesting a novel targeted therapy option for high-risk neuroblastoma.
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http://dx.doi.org/10.1038/s41388-018-0595-3DOI Listing
April 2019

TBX2 is a neuroblastoma core regulatory circuitry component enhancing MYCN/FOXM1 reactivation of DREAM targets.

Nat Commun 2018 11 19;9(1):4866. Epub 2018 Nov 19.

Center for Medical Genetics, Ghent University, Ghent, 9000, Belgium.

Chromosome 17q gains are almost invariably present in high-risk neuroblastoma cases. Here, we perform an integrative epigenomics search for dosage-sensitive transcription factors on 17q marked by H3K27ac defined super-enhancers and identify TBX2 as top candidate gene. We show that TBX2 is a constituent of the recently established core regulatory circuitry in neuroblastoma with features of a cell identity transcription factor, driving proliferation through activation of p21-DREAM repressed FOXM1 target genes. Combined MYCN/TBX2 knockdown enforces cell growth arrest suggesting that TBX2 enhances MYCN sustained activation of FOXM1 targets. Targeting transcriptional addiction by combined CDK7 and BET bromodomain inhibition shows synergistic effects on cell viability with strong repressive effects on CRC gene expression and p53 pathway response as well as several genes implicated in transcriptional regulation. In conclusion, we provide insight into the role of the TBX2 CRC gene in transcriptional dependency of neuroblastoma cells warranting clinical trials using BET and CDK7 inhibitors.
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http://dx.doi.org/10.1038/s41467-018-06699-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6242972PMC
November 2018

Cell of origin dictates aggression and stem cell number in acute lymphoblastic leukemia.

Leukemia 2018 08 18;32(8):1860-1865. Epub 2018 Apr 18.

Department of Pathology, Massachusetts General Hospital Research Institute, Boston, MA, 02114, USA.

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http://dx.doi.org/10.1038/s41375-018-0130-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620784PMC
August 2018