Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of hSET1A depletion on ESC mesoderm differentiation and hematopoiesis.(A) Western blotting analysis of hSET1A protein levels in ESCs harboring scramble control or hSET1A specific shRNAs. (B) Hematopoietic differentiation assay. The scramble control or hSET1A KD ESCs were cultured in suspension without LIF to induce EB formation for 4 days and then cultured in the presence of SCF to induce hematopoietic differentiation for another 4 days. Shown are EBs after 8 days in culture. Scale bar, 100 µm. (C) to (E) Time course qRT-PCR analyses of the levels of mesoderm (C), endoderm (D), and ectoderm (E) markers in the scramble control and two hSET1A KD clones upon withdrawal of LIF. (F) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 6. (G) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 10.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675019&req=5

pgen-1003524-g007: Effect of hSET1A depletion on ESC mesoderm differentiation and hematopoiesis.(A) Western blotting analysis of hSET1A protein levels in ESCs harboring scramble control or hSET1A specific shRNAs. (B) Hematopoietic differentiation assay. The scramble control or hSET1A KD ESCs were cultured in suspension without LIF to induce EB formation for 4 days and then cultured in the presence of SCF to induce hematopoietic differentiation for another 4 days. Shown are EBs after 8 days in culture. Scale bar, 100 µm. (C) to (E) Time course qRT-PCR analyses of the levels of mesoderm (C), endoderm (D), and ectoderm (E) markers in the scramble control and two hSET1A KD clones upon withdrawal of LIF. (F) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 6. (G) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 10.

Mentions: The above data indicate that the activity of USF1 during mesoderm differentiation and subsequent transcription activation of HoxB4 correlates with its ability to recruit the hSET1A complex (Figure 5 and 6D–G). To ascertain whether hSET1A, but not MLL1, is required for USF1-mediated transcription activation during differentiation, hSET1A was silenced in ES cells by retroviral-mediated shRNA targeting (Figure 7A). Compared to the pSuper scramble control, three individual ES clones showed a substantial decrease in hSET1A expression (Figure 7A). ESCs transduced with retroviruses harboring the pSuper scramble control or the hSET1A-specific shRNAs were differentiated into embryoid bodies (EBs) by withdrawal of LIF (Figure S4B). Similar to the ectopic expression of AUSF1, downregulation of hSET1A did not alter ESC self-renewal properties and expression of the Oct4 and Nanog genes (Figure S8A and S8B), but impaired EB size (Figure 7B). Furthermore, we examined the expression levels of three germ layer lineage markers in the scramble and hSET1A KD EBs by qRT-PCR (Figure 7C–E). Both mesoderm and endoderm markers were largely abolished in the hSET1A KD EBs. However, ectoderm markers were not suppressed by the hSET1A KD.


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)

Effect of hSET1A depletion on ESC mesoderm differentiation and hematopoiesis.(A) Western blotting analysis of hSET1A protein levels in ESCs harboring scramble control or hSET1A specific shRNAs. (B) Hematopoietic differentiation assay. The scramble control or hSET1A KD ESCs were cultured in suspension without LIF to induce EB formation for 4 days and then cultured in the presence of SCF to induce hematopoietic differentiation for another 4 days. Shown are EBs after 8 days in culture. Scale bar, 100 µm. (C) to (E) Time course qRT-PCR analyses of the levels of mesoderm (C), endoderm (D), and ectoderm (E) markers in the scramble control and two hSET1A KD clones upon withdrawal of LIF. (F) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 6. (G) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 10.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675019&req=5

pgen-1003524-g007: Effect of hSET1A depletion on ESC mesoderm differentiation and hematopoiesis.(A) Western blotting analysis of hSET1A protein levels in ESCs harboring scramble control or hSET1A specific shRNAs. (B) Hematopoietic differentiation assay. The scramble control or hSET1A KD ESCs were cultured in suspension without LIF to induce EB formation for 4 days and then cultured in the presence of SCF to induce hematopoietic differentiation for another 4 days. Shown are EBs after 8 days in culture. Scale bar, 100 µm. (C) to (E) Time course qRT-PCR analyses of the levels of mesoderm (C), endoderm (D), and ectoderm (E) markers in the scramble control and two hSET1A KD clones upon withdrawal of LIF. (F) FACS analysis of c-Kit and Tie2 expressing hemogenic endothelium population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 6. (G) FACS analysis of CD41 and c-Kit expressing early hematopoietic stem and progenitor population in the scramble control and hSET1A KD ESCs upon hematopoietic differentiation at day 10.
Mentions: The above data indicate that the activity of USF1 during mesoderm differentiation and subsequent transcription activation of HoxB4 correlates with its ability to recruit the hSET1A complex (Figure 5 and 6D–G). To ascertain whether hSET1A, but not MLL1, is required for USF1-mediated transcription activation during differentiation, hSET1A was silenced in ES cells by retroviral-mediated shRNA targeting (Figure 7A). Compared to the pSuper scramble control, three individual ES clones showed a substantial decrease in hSET1A expression (Figure 7A). ESCs transduced with retroviruses harboring the pSuper scramble control or the hSET1A-specific shRNAs were differentiated into embryoid bodies (EBs) by withdrawal of LIF (Figure S4B). Similar to the ectopic expression of AUSF1, downregulation of hSET1A did not alter ESC self-renewal properties and expression of the Oct4 and Nanog genes (Figure S8A and S8B), but impaired EB size (Figure 7B). Furthermore, we examined the expression levels of three germ layer lineage markers in the scramble and hSET1A KD EBs by qRT-PCR (Figure 7C–E). Both mesoderm and endoderm markers were largely abolished in the hSET1A KD EBs. However, ectoderm markers were not suppressed by the hSET1A KD.

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