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Nfix expression critically modulates early B lymphopoiesis and myelopoiesis.

O'Connor C, Campos J, Osinski JM, Gronostajski RM, Michie AM, Keeshan K - PLoS ONE (2015)

Bottom Line: We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells.The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes.These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland.

ABSTRACT
The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification.

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Loss of Nfix perturbs myelopoiesis while driving B cell differentiation.Total BM from WT or Nfix deficient mice was plated onto OP9 cells and analysed by flow cytometry on day 4 (A) and day 6 (B). Bar chart shows the mean percentages of B220+ cells from 3 independent experiments +/- SD. (C) Representative flow cytometric analysis of cells expressing B220 at day 6. (D) Total BM from WT or Nfix deficient mice was plated in methylcellulose (M3434). Colonies were counted after 11 days. Bar chart represents the mean number of colonies from 2 independent experiments +/- SD. (E) Bar chart shows the mean percentages of Gr1 + and F4/80+ cells from 2 independent experiments +/- SD. (F) Representative flow cytometric analysis of cells from colony assay which were stained with Gr-1 and F4/80. (G) Graphical presentation of relative mRNA expression of indicated genes assessed using high-throughput qPCR on the 48.48 Dynamic Array IFC system (Fluidigm). Bars represent the average of 3 biological Nfix-/- replicates normalized to WT control, and error bars denote +/- SD.
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pone.0120102.g006: Loss of Nfix perturbs myelopoiesis while driving B cell differentiation.Total BM from WT or Nfix deficient mice was plated onto OP9 cells and analysed by flow cytometry on day 4 (A) and day 6 (B). Bar chart shows the mean percentages of B220+ cells from 3 independent experiments +/- SD. (C) Representative flow cytometric analysis of cells expressing B220 at day 6. (D) Total BM from WT or Nfix deficient mice was plated in methylcellulose (M3434). Colonies were counted after 11 days. Bar chart represents the mean number of colonies from 2 independent experiments +/- SD. (E) Bar chart shows the mean percentages of Gr1 + and F4/80+ cells from 2 independent experiments +/- SD. (F) Representative flow cytometric analysis of cells from colony assay which were stained with Gr-1 and F4/80. (G) Graphical presentation of relative mRNA expression of indicated genes assessed using high-throughput qPCR on the 48.48 Dynamic Array IFC system (Fluidigm). Bars represent the average of 3 biological Nfix-/- replicates normalized to WT control, and error bars denote +/- SD.

Mentions: To investigate the physiological relevance of Nfix in myeloid and lymphoid lineage specification, we isolated BM from Nfix-/- neonate mice and cultured it on OP9 cells. There was a marked increase in cells expressing B220 after 4 and 6 days in culture compared with WT littermate control cells (Fig. 6A-C). Myeloid differentiation of Nfix deficient cells was assessed in methylcellulose colony assays. Nfix deficient BM cells formed significantly fewer colonies than their WT counterparts (Fig. 6D), and expressed significantly lower percentages of mature myeloid markers CD11b, Gr1 and F4/80 (Fig. 6D-F). These data indicate that loss of Nfix perturbs normal myeloid differentiation. Gene expression analysis by qRT-PCR reveals that Nfix deficient BM cells have a marked disruption in the expression of key transcription factors involved in lineage fate determination (Fig. 6G). Of particular interest is the downregulation of Id genes and MMP9 (the inverse of our overexpression data), and an upregulation of CD19 and Pu.1 in Nfix deficient cells, supporting a role of Nfix in cell lineage fate. Taken together, these loss of function data show that loss of Nfix in the BM leads to altered hematopoiesis by enhancing B cell development while disrupting myelopoiesis, and that this effect is mediated by changes in key transcription factors associated with lineage fate determination.


Nfix expression critically modulates early B lymphopoiesis and myelopoiesis.

O'Connor C, Campos J, Osinski JM, Gronostajski RM, Michie AM, Keeshan K - PLoS ONE (2015)

Loss of Nfix perturbs myelopoiesis while driving B cell differentiation.Total BM from WT or Nfix deficient mice was plated onto OP9 cells and analysed by flow cytometry on day 4 (A) and day 6 (B). Bar chart shows the mean percentages of B220+ cells from 3 independent experiments +/- SD. (C) Representative flow cytometric analysis of cells expressing B220 at day 6. (D) Total BM from WT or Nfix deficient mice was plated in methylcellulose (M3434). Colonies were counted after 11 days. Bar chart represents the mean number of colonies from 2 independent experiments +/- SD. (E) Bar chart shows the mean percentages of Gr1 + and F4/80+ cells from 2 independent experiments +/- SD. (F) Representative flow cytometric analysis of cells from colony assay which were stained with Gr-1 and F4/80. (G) Graphical presentation of relative mRNA expression of indicated genes assessed using high-throughput qPCR on the 48.48 Dynamic Array IFC system (Fluidigm). Bars represent the average of 3 biological Nfix-/- replicates normalized to WT control, and error bars denote +/- SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363787&req=5

pone.0120102.g006: Loss of Nfix perturbs myelopoiesis while driving B cell differentiation.Total BM from WT or Nfix deficient mice was plated onto OP9 cells and analysed by flow cytometry on day 4 (A) and day 6 (B). Bar chart shows the mean percentages of B220+ cells from 3 independent experiments +/- SD. (C) Representative flow cytometric analysis of cells expressing B220 at day 6. (D) Total BM from WT or Nfix deficient mice was plated in methylcellulose (M3434). Colonies were counted after 11 days. Bar chart represents the mean number of colonies from 2 independent experiments +/- SD. (E) Bar chart shows the mean percentages of Gr1 + and F4/80+ cells from 2 independent experiments +/- SD. (F) Representative flow cytometric analysis of cells from colony assay which were stained with Gr-1 and F4/80. (G) Graphical presentation of relative mRNA expression of indicated genes assessed using high-throughput qPCR on the 48.48 Dynamic Array IFC system (Fluidigm). Bars represent the average of 3 biological Nfix-/- replicates normalized to WT control, and error bars denote +/- SD.
Mentions: To investigate the physiological relevance of Nfix in myeloid and lymphoid lineage specification, we isolated BM from Nfix-/- neonate mice and cultured it on OP9 cells. There was a marked increase in cells expressing B220 after 4 and 6 days in culture compared with WT littermate control cells (Fig. 6A-C). Myeloid differentiation of Nfix deficient cells was assessed in methylcellulose colony assays. Nfix deficient BM cells formed significantly fewer colonies than their WT counterparts (Fig. 6D), and expressed significantly lower percentages of mature myeloid markers CD11b, Gr1 and F4/80 (Fig. 6D-F). These data indicate that loss of Nfix perturbs normal myeloid differentiation. Gene expression analysis by qRT-PCR reveals that Nfix deficient BM cells have a marked disruption in the expression of key transcription factors involved in lineage fate determination (Fig. 6G). Of particular interest is the downregulation of Id genes and MMP9 (the inverse of our overexpression data), and an upregulation of CD19 and Pu.1 in Nfix deficient cells, supporting a role of Nfix in cell lineage fate. Taken together, these loss of function data show that loss of Nfix in the BM leads to altered hematopoiesis by enhancing B cell development while disrupting myelopoiesis, and that this effect is mediated by changes in key transcription factors associated with lineage fate determination.

Bottom Line: We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells.The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes.These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification.

View Article: PubMed Central - PubMed

Affiliation: Paul O'Gorman Leukaemia Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland.

ABSTRACT
The commitment of stem and progenitor cells toward specific hematopoietic lineages is tightly controlled by a number of transcription factors that regulate differentiation programs via the expression of lineage restricting genes. Nuclear factor one (NFI) transcription factors are important in regulating hematopoiesis and here we report an important physiological role of NFIX in B- and myeloid lineage commitment and differentiation. We demonstrate that NFIX acts as a regulator of lineage specification in the haematopoietic system and the expression of Nfix was transcriptionally downregulated as B cells commit and differentiate, whilst maintained in myeloid progenitor cells. Ectopic Nfix expression in vivo blocked early B cell development stage, coincident with the stage of its downregulation. Furthermore, loss of Nfix resulted in the perturbation of myeloid and lymphoid cell differentiation, and a skewing of gene expression involved in lineage fate determination. Nfix was able to promote myeloid differentiation of total bone marrow cells under B cell specific culture conditions but not when expressed in the hematopoietic stem cell (HSPC), consistent with its role in HSPC survival. The lineage choice determined by Nfix correlated with transcriptional changes in a number of genes, such as E2A, C/EBP, and Id genes. These data highlight a novel and critical role for NFIX transcription factor in hematopoiesis and in lineage specification.

Show MeSH
Related in: MedlinePlus