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Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment.

Romano O, Peano C, Tagliazucchi GM, Petiti L, Poletti V, Cocchiarella F, Rizzi E, Severgnini M, Cavazza A, Rossi C, Pagliaro P, Ambrosi A, Ferrari G, Bicciato S, De Bellis G, Mavilio F, Miccio A - Sci Rep (2016)

Bottom Line: A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment.Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers.Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT
Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

No MeSH data available.


Related in: MedlinePlus

Purification and characterization of multipotent and lineage-restricted hematopoietic progenitors.(A–C) FACS analysis and CFC assay of HSPC, EPP and MPP. Under conditions supporting erythroid differentiation, EPP differentiated into late erythrocytes (CD71+/GYPA+), while MPP remained in an undifferentiated myeloid state (CD11blow/CD14low/CD71low). In the presence of G-CSF, MPP differentiated into mature myeloid cells (CD11b+/CD14+) while EPP expressed low levels of late myeloid markers (CD11blow/CD14low) (D) HSPC, EPP and MPP gene expression profiles. Supervised analysis was performed using a fold-change threshold equal to 2 and a P-value threshold equal to 0.05, to obtain a list of differentially-expressed genes.
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f1: Purification and characterization of multipotent and lineage-restricted hematopoietic progenitors.(A–C) FACS analysis and CFC assay of HSPC, EPP and MPP. Under conditions supporting erythroid differentiation, EPP differentiated into late erythrocytes (CD71+/GYPA+), while MPP remained in an undifferentiated myeloid state (CD11blow/CD14low/CD71low). In the presence of G-CSF, MPP differentiated into mature myeloid cells (CD11b+/CD14+) while EPP expressed low levels of late myeloid markers (CD11blow/CD14low) (D) HSPC, EPP and MPP gene expression profiles. Supervised analysis was performed using a fold-change threshold equal to 2 and a P-value threshold equal to 0.05, to obtain a list of differentially-expressed genes.

Mentions: We prospectively enriched human HSPC as CD34+/CD133+ population by FACS (Fig. 1A). HSPC showed high levels of CD38, indicating that the majority of the cells were early hematopoietic progenitors42 (Fig. 1A). Committed erythroid and myeloid progenitors/precursors (EPP and MPP) were isolated as CD34low/CD36high and CD34−/CD13+ populations (Fig. 1B,C). Over 95% of EPP were CD71+ and expressed low levels of glycophorin A (GYPA), indicating that they are mainly composed by erythroid progenitors32 (Fig. 1B). MPP expressed the myeloid differentiation markers CD33 and CD11b (Fig. 1C)43 and low levels of the late differentiation marker CD14 (not shown). In a clonal progenitor assay, HSPC gave rise to mixed colonies (CFU-GEMM), and both myeloid (CFU-GM, CFU-G, CFU-M) and erythroid (BFU-E and CFU-E) colonies, thus confirming their multilineage potential (Fig. 1A). In contrast, EPP and MPP populations generated >90% erythroid and myeloid colonies, respectively (Fig. 1B,C), confirming their lineage-restricted potential. EPP and MPP populations were grown in liquid culture under conditions supporting either erythroid (+EPO) or myeloid (+G-CSF) terminal differentiation. In the presence of EPO, EPP were able to differentiate into late erythrocytes, while MPP remained in an undifferentiated myeloid state (Supplementary Fig. 1A). Conversely, in the presence of G-CSF, MPP differentiate into granulocytes and monocytes while EPP grew poorly and acquired late myeloid markers (Supplementary Fig. 1B).


Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment.

Romano O, Peano C, Tagliazucchi GM, Petiti L, Poletti V, Cocchiarella F, Rizzi E, Severgnini M, Cavazza A, Rossi C, Pagliaro P, Ambrosi A, Ferrari G, Bicciato S, De Bellis G, Mavilio F, Miccio A - Sci Rep (2016)

Purification and characterization of multipotent and lineage-restricted hematopoietic progenitors.(A–C) FACS analysis and CFC assay of HSPC, EPP and MPP. Under conditions supporting erythroid differentiation, EPP differentiated into late erythrocytes (CD71+/GYPA+), while MPP remained in an undifferentiated myeloid state (CD11blow/CD14low/CD71low). In the presence of G-CSF, MPP differentiated into mature myeloid cells (CD11b+/CD14+) while EPP expressed low levels of late myeloid markers (CD11blow/CD14low) (D) HSPC, EPP and MPP gene expression profiles. Supervised analysis was performed using a fold-change threshold equal to 2 and a P-value threshold equal to 0.05, to obtain a list of differentially-expressed genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837375&req=5

f1: Purification and characterization of multipotent and lineage-restricted hematopoietic progenitors.(A–C) FACS analysis and CFC assay of HSPC, EPP and MPP. Under conditions supporting erythroid differentiation, EPP differentiated into late erythrocytes (CD71+/GYPA+), while MPP remained in an undifferentiated myeloid state (CD11blow/CD14low/CD71low). In the presence of G-CSF, MPP differentiated into mature myeloid cells (CD11b+/CD14+) while EPP expressed low levels of late myeloid markers (CD11blow/CD14low) (D) HSPC, EPP and MPP gene expression profiles. Supervised analysis was performed using a fold-change threshold equal to 2 and a P-value threshold equal to 0.05, to obtain a list of differentially-expressed genes.
Mentions: We prospectively enriched human HSPC as CD34+/CD133+ population by FACS (Fig. 1A). HSPC showed high levels of CD38, indicating that the majority of the cells were early hematopoietic progenitors42 (Fig. 1A). Committed erythroid and myeloid progenitors/precursors (EPP and MPP) were isolated as CD34low/CD36high and CD34−/CD13+ populations (Fig. 1B,C). Over 95% of EPP were CD71+ and expressed low levels of glycophorin A (GYPA), indicating that they are mainly composed by erythroid progenitors32 (Fig. 1B). MPP expressed the myeloid differentiation markers CD33 and CD11b (Fig. 1C)43 and low levels of the late differentiation marker CD14 (not shown). In a clonal progenitor assay, HSPC gave rise to mixed colonies (CFU-GEMM), and both myeloid (CFU-GM, CFU-G, CFU-M) and erythroid (BFU-E and CFU-E) colonies, thus confirming their multilineage potential (Fig. 1A). In contrast, EPP and MPP populations generated >90% erythroid and myeloid colonies, respectively (Fig. 1B,C), confirming their lineage-restricted potential. EPP and MPP populations were grown in liquid culture under conditions supporting either erythroid (+EPO) or myeloid (+G-CSF) terminal differentiation. In the presence of EPO, EPP were able to differentiate into late erythrocytes, while MPP remained in an undifferentiated myeloid state (Supplementary Fig. 1A). Conversely, in the presence of G-CSF, MPP differentiate into granulocytes and monocytes while EPP grew poorly and acquired late myeloid markers (Supplementary Fig. 1B).

Bottom Line: A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment.Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers.Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.

ABSTRACT
Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.

No MeSH data available.


Related in: MedlinePlus