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USF1 and hSET1A mediated epigenetic modifications regulate lineage differentiation and HoxB4 transcription.

Deng C, Li Y, Liang S, Cui K, Salz T, Yang H, Tang Z, Gallagher PG, Qiu Y, Roeder R, Zhao K, Bungert J, Huang S - PLoS Genet. (2013)

Bottom Line: Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation.We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation.Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA.

ABSTRACT
The interplay between polycomb and trithorax complexes has been implicated in embryonic stem cell (ESC) self-renewal and differentiation. It has been shown recently that WRD5 and Dpy-30, specific components of the SET1/MLL protein complexes, play important roles during ESC self-renewal and differentiation of neural lineages. However, not much is known about how and where specific trithorax complexes are targeted to genes involved in self-renewal or lineage-specification. Here, we report that the recruitment of the hSET1A histone H3K4 methyltransferase (HMT) complex by transcription factor USF1 is required for mesoderm specification and lineage differentiation. In undifferentiated ESCs, USF1 maintains hematopoietic stem/progenitor cell (HS/PC) associated bivalent chromatin domains and differentiation potential. Furthermore, USF1 directed recruitment of the hSET1A complex to the HoxB4 promoter governs the transcriptional activation of HoxB4 gene and regulates the formation of early hematopoietic cell populations. Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation. We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation. Finally, ectopic expression of USF1 in ESCs promotes mesoderm differentiation and enforces the endothelial-to-hematopoietic transition by inducing hematopoietic-associated transcription factors, HoxB4 and TAL1. Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

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USF1 regulates ESC fate and hematopoietic differentiation by modulating bivalent domains of HS/PC-specific genes.(A) Western blotting assay of hSET1A levels in undifferentiated (day 0) and differentiated (day 13) ESCs comparing pcDNA vector transfected control and AUSF1 overexpressing cells. (B) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium cell population in pcDNA tranfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 6. (C) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in pcDNA transfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 10. (D) Real-time RT-qPCR analysis of hematopoietic markers and transcription factors, c-Myb, CD150, CD34, Tal1, Runx1, and HoxB4 mRNA transcript levels upon hematopoietic differentiation comparing pcDNA transfected control and two AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05. (E) to (H). ChIP analyses of USF1 binding (E), hSET1A recruitment (F), H3K4me3 enrichment (G), and RNAPII loading (H) at the HoxB4 promoter in AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05.
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pgen-1003524-g006: USF1 regulates ESC fate and hematopoietic differentiation by modulating bivalent domains of HS/PC-specific genes.(A) Western blotting assay of hSET1A levels in undifferentiated (day 0) and differentiated (day 13) ESCs comparing pcDNA vector transfected control and AUSF1 overexpressing cells. (B) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium cell population in pcDNA tranfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 6. (C) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in pcDNA transfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 10. (D) Real-time RT-qPCR analysis of hematopoietic markers and transcription factors, c-Myb, CD150, CD34, Tal1, Runx1, and HoxB4 mRNA transcript levels upon hematopoietic differentiation comparing pcDNA transfected control and two AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05. (E) to (H). ChIP analyses of USF1 binding (E), hSET1A recruitment (F), H3K4me3 enrichment (G), and RNAPII loading (H) at the HoxB4 promoter in AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05.

Mentions: The mouse mesoderm derived aorta-gonad mesonephros (AGM) region is considered to be the first site of hematopoiesis [39] and hematopoietic potential is segregated within the Flk1+ compartment in the murine EB system [40], [41]. The hemogenic endothelium stage cells (Tie2hic-Kit+ population) developed from hemangioblast give rise to HS/PCs at the onset of hematopoiesis [37], [42]–[45]. AUSF1 specifically inhibited Flk1 expression in the mesoderm (Figure 5) suggesting that USF1 may be involved during early hematopoietic commitment steps. We next explored the effect of AUSF1 expression on cytokine induced hematopoietic differentiation of ES cells (Figure S4B). Ectopic expression of AUSF1 did not affect co-activator hSET1A expression in undifferentiated or differentiated ESCs (Figure 6A). However, it specifically inhibited differentiation of hematopoietic progenitors in three independent AUSF1 ESC clones (Figure 6B, C, and S7A). Compared to cells harboring the pcDNA vector control, cells expressing AUSF exhibited a reduction in c-Kit and Tie2 double positive hemogenic endothelium cell population by 64% (down to 5.4% from 15.1%; Figure 6B). These cells represent hematopoietic potential at the onset of hematopoiesis [37], [43]. Furthermore, suppression of USF1 activity significantly reduced the number of CD41+c-Kit+ HS/PCs (down from 4.7% to 0.3%) (Figure 6C) and reduced Sca1+c-Kit+ hematopoietic cells by 75% (Figure S7A and 7B). In addition to the inhibition of hematopoietic cell populations, the HS/PC markers and transcription factors associated with the onset of hematopoiesis such as HoxB4, Tal1, and Runx1 were downregulated by the inhibition of USF1 activity (Figure 6D). AUSF1 only dimerized with wt USF1 but not with TAL1 (Figure S7C), as expected [46]. Therefore, AUSF1 did not interfere with the DNA binding activity of hematopoietic specific bHLH transcription factor TAL1 (Figure S7C and D). Thus, the data show that USF1 is critical for the initial expression of hematopoietic transcription factors and markers.


USF1 and hSET1A mediated epigenetic modifications regulate lineage differentiation and HoxB4 transcription.

Deng C, Li Y, Liang S, Cui K, Salz T, Yang H, Tang Z, Gallagher PG, Qiu Y, Roeder R, Zhao K, Bungert J, Huang S - PLoS Genet. (2013)

USF1 regulates ESC fate and hematopoietic differentiation by modulating bivalent domains of HS/PC-specific genes.(A) Western blotting assay of hSET1A levels in undifferentiated (day 0) and differentiated (day 13) ESCs comparing pcDNA vector transfected control and AUSF1 overexpressing cells. (B) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium cell population in pcDNA tranfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 6. (C) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in pcDNA transfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 10. (D) Real-time RT-qPCR analysis of hematopoietic markers and transcription factors, c-Myb, CD150, CD34, Tal1, Runx1, and HoxB4 mRNA transcript levels upon hematopoietic differentiation comparing pcDNA transfected control and two AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05. (E) to (H). ChIP analyses of USF1 binding (E), hSET1A recruitment (F), H3K4me3 enrichment (G), and RNAPII loading (H) at the HoxB4 promoter in AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05.
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pgen-1003524-g006: USF1 regulates ESC fate and hematopoietic differentiation by modulating bivalent domains of HS/PC-specific genes.(A) Western blotting assay of hSET1A levels in undifferentiated (day 0) and differentiated (day 13) ESCs comparing pcDNA vector transfected control and AUSF1 overexpressing cells. (B) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium cell population in pcDNA tranfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 6. (C) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in pcDNA transfected control and AUSF1 overexpressing ESCs upon hematopoietic differentiation at day 10. (D) Real-time RT-qPCR analysis of hematopoietic markers and transcription factors, c-Myb, CD150, CD34, Tal1, Runx1, and HoxB4 mRNA transcript levels upon hematopoietic differentiation comparing pcDNA transfected control and two AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05. (E) to (H). ChIP analyses of USF1 binding (E), hSET1A recruitment (F), H3K4me3 enrichment (G), and RNAPII loading (H) at the HoxB4 promoter in AUSF1 overexpressing ES clones. Data are shown as mean ± SD. * P<0.05.
Mentions: The mouse mesoderm derived aorta-gonad mesonephros (AGM) region is considered to be the first site of hematopoiesis [39] and hematopoietic potential is segregated within the Flk1+ compartment in the murine EB system [40], [41]. The hemogenic endothelium stage cells (Tie2hic-Kit+ population) developed from hemangioblast give rise to HS/PCs at the onset of hematopoiesis [37], [42]–[45]. AUSF1 specifically inhibited Flk1 expression in the mesoderm (Figure 5) suggesting that USF1 may be involved during early hematopoietic commitment steps. We next explored the effect of AUSF1 expression on cytokine induced hematopoietic differentiation of ES cells (Figure S4B). Ectopic expression of AUSF1 did not affect co-activator hSET1A expression in undifferentiated or differentiated ESCs (Figure 6A). However, it specifically inhibited differentiation of hematopoietic progenitors in three independent AUSF1 ESC clones (Figure 6B, C, and S7A). Compared to cells harboring the pcDNA vector control, cells expressing AUSF exhibited a reduction in c-Kit and Tie2 double positive hemogenic endothelium cell population by 64% (down to 5.4% from 15.1%; Figure 6B). These cells represent hematopoietic potential at the onset of hematopoiesis [37], [43]. Furthermore, suppression of USF1 activity significantly reduced the number of CD41+c-Kit+ HS/PCs (down from 4.7% to 0.3%) (Figure 6C) and reduced Sca1+c-Kit+ hematopoietic cells by 75% (Figure S7A and 7B). In addition to the inhibition of hematopoietic cell populations, the HS/PC markers and transcription factors associated with the onset of hematopoiesis such as HoxB4, Tal1, and Runx1 were downregulated by the inhibition of USF1 activity (Figure 6D). AUSF1 only dimerized with wt USF1 but not with TAL1 (Figure S7C), as expected [46]. Therefore, AUSF1 did not interfere with the DNA binding activity of hematopoietic specific bHLH transcription factor TAL1 (Figure S7C and D). Thus, the data show that USF1 is critical for the initial expression of hematopoietic transcription factors and markers.

Bottom Line: Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation.We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation.Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, USA.

ABSTRACT
The interplay between polycomb and trithorax complexes has been implicated in embryonic stem cell (ESC) self-renewal and differentiation. It has been shown recently that WRD5 and Dpy-30, specific components of the SET1/MLL protein complexes, play important roles during ESC self-renewal and differentiation of neural lineages. However, not much is known about how and where specific trithorax complexes are targeted to genes involved in self-renewal or lineage-specification. Here, we report that the recruitment of the hSET1A histone H3K4 methyltransferase (HMT) complex by transcription factor USF1 is required for mesoderm specification and lineage differentiation. In undifferentiated ESCs, USF1 maintains hematopoietic stem/progenitor cell (HS/PC) associated bivalent chromatin domains and differentiation potential. Furthermore, USF1 directed recruitment of the hSET1A complex to the HoxB4 promoter governs the transcriptional activation of HoxB4 gene and regulates the formation of early hematopoietic cell populations. Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation. We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation. Finally, ectopic expression of USF1 in ESCs promotes mesoderm differentiation and enforces the endothelial-to-hematopoietic transition by inducing hematopoietic-associated transcription factors, HoxB4 and TAL1. Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

Show MeSH
Related in: MedlinePlus