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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 pluripotency by specifically controlling mesoderm differentiation.(A) Immunoblot assay showing the levels of endogenous USF1 and ectopically expressed AUSF1 in pcDNA transfected and AUSF1 expressing mES cells. (B) ChIP analyses of H3K4m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (C) ChIP analyses of H3K27m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (E) to (F)Time course qRT-PCR analyses of the expression levels of mesoderm (D), endoderm (E), and ectoderm (F) markers in pcDNA control and two AUSF1 overexpressing clones upon withdrawal of LIF.
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pgen-1003524-g005: USF1 regulates ESC pluripotency by specifically controlling mesoderm differentiation.(A) Immunoblot assay showing the levels of endogenous USF1 and ectopically expressed AUSF1 in pcDNA transfected and AUSF1 expressing mES cells. (B) ChIP analyses of H3K4m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (C) ChIP analyses of H3K27m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (E) to (F)Time course qRT-PCR analyses of the expression levels of mesoderm (D), endoderm (E), and ectoderm (F) markers in pcDNA control and two AUSF1 overexpressing clones upon withdrawal of LIF.

Mentions: Having shown that USF1 activity is important for HoxB4 expression in differentiation of ES cells (Figure 4C–F and S5A–C), we next examined the biological effect of USF1 on stem cell pluripotency and differentiation by enforced expression in ES cells of AUSF1, a dominant negative USF1 mutant that competes with endogenous USF for DNA-binding (Figure 5A). Disruption of USF1 DNA binding activity affected neither expression of stemness genes Oct4 and Nanog (Figure S6A) nor the self-renewal ability of ESCs as examined by AP staining (Figure S6B). In addition, we tested the relationship of enforced AUSF1 expression and changes in bivalent H3K4 and H3K27 trimethylation patterns at HS/PC-specific genes in ESCs. Suppression of USF DNA-binding activity in ESCs resulted in a specific inhibition of H3K4me3 levels within HS/PC-associated bivalent chromatin marks in the HoxB4, Tal1, and Runx1P2 gene loci (Figure 5B), but did not reduce the H3K27me3 levels at these genes (Figure 5C). Interestingly, AUSF1 embryoid bodies (EBs) appeared smaller compared to those harboring the vector control (Figure S6C), suggesting that loss of USF activity compromises lineage differentiation programs. We next examined the expression of endoderm, mesoderm, and ectoderm markers in both control and AUSF1 EBs (Figure 5D–F). To our surprise, inhibition of USF1 DNA binding activity in early embryonic development resulted in a specific reduction of primitive mesoderm markers, Brachyury (T) and FLK1, with only minor effects on endoderm and ectoderm markers, Gata4, Gata6, Fgf5, and nestin (Figure 5D–F). Further analysis indicated that USF1 does not directly regulate both T and FLK1 (Figure S6D). Several mesoderm markers, Mesp1, Snail2, Eomes, and Nkx2-5, were also significantly inhibited by ectopic expression of AUSF1 (Figure S6E). The effect of USF1 in EB differentiation was biased toward the mesoderm which can differentiate to hematopoietic lineages. This is consistent with the disruption of H3K4me3 levels at HS/PC-associated chromatin domains by enforced expression of AUSF1.


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 pluripotency by specifically controlling mesoderm differentiation.(A) Immunoblot assay showing the levels of endogenous USF1 and ectopically expressed AUSF1 in pcDNA transfected and AUSF1 expressing mES cells. (B) ChIP analyses of H3K4m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (C) ChIP analyses of H3K27m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (E) to (F)Time course qRT-PCR analyses of the expression levels of mesoderm (D), endoderm (E), and ectoderm (F) markers in pcDNA control and two AUSF1 overexpressing clones upon withdrawal of LIF.
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pgen-1003524-g005: USF1 regulates ESC pluripotency by specifically controlling mesoderm differentiation.(A) Immunoblot assay showing the levels of endogenous USF1 and ectopically expressed AUSF1 in pcDNA transfected and AUSF1 expressing mES cells. (B) ChIP analyses of H3K4m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (C) ChIP analyses of H3K27m3 levels at the HSC-associated HoxB4, TAL1, and Runx1 genes comparing the pcDNA transfected control and the dominant negative AUSF1 overexpressing ESCs. Data are shown as mean ± SD. * P<0.05 and ** P<0.01. (E) to (F)Time course qRT-PCR analyses of the expression levels of mesoderm (D), endoderm (E), and ectoderm (F) markers in pcDNA control and two AUSF1 overexpressing clones upon withdrawal of LIF.
Mentions: Having shown that USF1 activity is important for HoxB4 expression in differentiation of ES cells (Figure 4C–F and S5A–C), we next examined the biological effect of USF1 on stem cell pluripotency and differentiation by enforced expression in ES cells of AUSF1, a dominant negative USF1 mutant that competes with endogenous USF for DNA-binding (Figure 5A). Disruption of USF1 DNA binding activity affected neither expression of stemness genes Oct4 and Nanog (Figure S6A) nor the self-renewal ability of ESCs as examined by AP staining (Figure S6B). In addition, we tested the relationship of enforced AUSF1 expression and changes in bivalent H3K4 and H3K27 trimethylation patterns at HS/PC-specific genes in ESCs. Suppression of USF DNA-binding activity in ESCs resulted in a specific inhibition of H3K4me3 levels within HS/PC-associated bivalent chromatin marks in the HoxB4, Tal1, and Runx1P2 gene loci (Figure 5B), but did not reduce the H3K27me3 levels at these genes (Figure 5C). Interestingly, AUSF1 embryoid bodies (EBs) appeared smaller compared to those harboring the vector control (Figure S6C), suggesting that loss of USF activity compromises lineage differentiation programs. We next examined the expression of endoderm, mesoderm, and ectoderm markers in both control and AUSF1 EBs (Figure 5D–F). To our surprise, inhibition of USF1 DNA binding activity in early embryonic development resulted in a specific reduction of primitive mesoderm markers, Brachyury (T) and FLK1, with only minor effects on endoderm and ectoderm markers, Gata4, Gata6, Fgf5, and nestin (Figure 5D–F). Further analysis indicated that USF1 does not directly regulate both T and FLK1 (Figure S6D). Several mesoderm markers, Mesp1, Snail2, Eomes, and Nkx2-5, were also significantly inhibited by ectopic expression of AUSF1 (Figure S6E). The effect of USF1 in EB differentiation was biased toward the mesoderm which can differentiate to hematopoietic lineages. This is consistent with the disruption of H3K4me3 levels at HS/PC-associated chromatin domains by enforced expression of AUSF1.

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