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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

Cell-specific regulatory regions targeted by MLV.(A) Validation of putative regulatory elements in hematopoietic primary cells. 8 potential EPP and MPP enhancer elements hit by MLV were cloned by PCR, inserted upstream of a basal promoter and transfected in EPP and MPP. Luciferase activity was quantitated after 18 hr. Fold induction relative to a negative control region was calculated. The log2 of the ratio between EPP and MPP fold induction for each enhancer is shown. All the putative enhancers were able to induce the transcription of the reporter gene in a cell-specific fashion. (B) MLV targets erythroid-specific regulatory regions in EPP. MLV clusters and integrations targeting the intronic enhancer of the BCL11A gene (upper panel) and the HBS1L-MYB intergenic region containing MYB enhancers (lower panel) are highlighted with red boxes. (C) Differential MLV integration preferences in HSPC, EPP and MPP inside the KIT locus. The erythroid-specific KIT enhancer (#4) is highlighted with a red box. (D) CAGE expression levels of the KIT promoter in HSPC, EPP and MPP. (E) Fold luciferase induction of the erythroid-specific KIT enhancer (#4) compared to a negative control region (37- and 3.5-fold luciferase induction in EPP and MPP, respectively). (F) The erythroid master regulator GATA1 binds the erythroid-specific KIT enhancer. ChIP assay was performed in EPP to analyze GATA1 binding to the KIT enhancer (#4). Two genomic regions were used as negative and positive controls for GATA1 binding, respectively (Neg Ctrl and Pos Ctrl).
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f7: Cell-specific regulatory regions targeted by MLV.(A) Validation of putative regulatory elements in hematopoietic primary cells. 8 potential EPP and MPP enhancer elements hit by MLV were cloned by PCR, inserted upstream of a basal promoter and transfected in EPP and MPP. Luciferase activity was quantitated after 18 hr. Fold induction relative to a negative control region was calculated. The log2 of the ratio between EPP and MPP fold induction for each enhancer is shown. All the putative enhancers were able to induce the transcription of the reporter gene in a cell-specific fashion. (B) MLV targets erythroid-specific regulatory regions in EPP. MLV clusters and integrations targeting the intronic enhancer of the BCL11A gene (upper panel) and the HBS1L-MYB intergenic region containing MYB enhancers (lower panel) are highlighted with red boxes. (C) Differential MLV integration preferences in HSPC, EPP and MPP inside the KIT locus. The erythroid-specific KIT enhancer (#4) is highlighted with a red box. (D) CAGE expression levels of the KIT promoter in HSPC, EPP and MPP. (E) Fold luciferase induction of the erythroid-specific KIT enhancer (#4) compared to a negative control region (37- and 3.5-fold luciferase induction in EPP and MPP, respectively). (F) The erythroid master regulator GATA1 binds the erythroid-specific KIT enhancer. ChIP assay was performed in EPP to analyze GATA1 binding to the KIT enhancer (#4). Two genomic regions were used as negative and positive controls for GATA1 binding, respectively (Neg Ctrl and Pos Ctrl).

Mentions: To determine whether the putative enhancers identified by combining ChIP-seq and retroviral scanning have transcriptional activity in a functional assay, we tested 8 MLV-targeted erythroid and myeloid enhancers in a reporter assay in EPP and MPP respectively. As expected, erythroid-specific MLV-targeted regions had higher activity in EPP than in MPP and vice versa (Fig. 7A), confirming that MLV identifies cell-specific enhancers, possibly controlling the expression of nearby genes. As examples, MLV was able to target known cell-specific regulatory regions, such as the intronic enhancer of the BCL11A gene46 and the HBS1L-MYB intergenic region containing erythroid-specific MYB enhancers47 (Fig. 7B and Supplementary Fig. 13). MLV scanning identified also novel enhancers in a cell-specific fashion (Supplementary Fig. 13), such as the integration clusters mapping to different regions of the KIT locus in HSPC, EPP and MPP (Fig. 7C). These clusters most likely identify enhancers used to exert a lineage-specific control of the locus during hematopoietic differentiation (Fig. 7D and Supplementary Fig. 14), such as the erythroid-specific enhancer #4, which is primarily active in EPP (Fig. 7E) and is targeted by the erythroid master regulator GATA1 (Fig. 7F).


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)

Cell-specific regulatory regions targeted by MLV.(A) Validation of putative regulatory elements in hematopoietic primary cells. 8 potential EPP and MPP enhancer elements hit by MLV were cloned by PCR, inserted upstream of a basal promoter and transfected in EPP and MPP. Luciferase activity was quantitated after 18 hr. Fold induction relative to a negative control region was calculated. The log2 of the ratio between EPP and MPP fold induction for each enhancer is shown. All the putative enhancers were able to induce the transcription of the reporter gene in a cell-specific fashion. (B) MLV targets erythroid-specific regulatory regions in EPP. MLV clusters and integrations targeting the intronic enhancer of the BCL11A gene (upper panel) and the HBS1L-MYB intergenic region containing MYB enhancers (lower panel) are highlighted with red boxes. (C) Differential MLV integration preferences in HSPC, EPP and MPP inside the KIT locus. The erythroid-specific KIT enhancer (#4) is highlighted with a red box. (D) CAGE expression levels of the KIT promoter in HSPC, EPP and MPP. (E) Fold luciferase induction of the erythroid-specific KIT enhancer (#4) compared to a negative control region (37- and 3.5-fold luciferase induction in EPP and MPP, respectively). (F) The erythroid master regulator GATA1 binds the erythroid-specific KIT enhancer. ChIP assay was performed in EPP to analyze GATA1 binding to the KIT enhancer (#4). Two genomic regions were used as negative and positive controls for GATA1 binding, respectively (Neg Ctrl and Pos Ctrl).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Cell-specific regulatory regions targeted by MLV.(A) Validation of putative regulatory elements in hematopoietic primary cells. 8 potential EPP and MPP enhancer elements hit by MLV were cloned by PCR, inserted upstream of a basal promoter and transfected in EPP and MPP. Luciferase activity was quantitated after 18 hr. Fold induction relative to a negative control region was calculated. The log2 of the ratio between EPP and MPP fold induction for each enhancer is shown. All the putative enhancers were able to induce the transcription of the reporter gene in a cell-specific fashion. (B) MLV targets erythroid-specific regulatory regions in EPP. MLV clusters and integrations targeting the intronic enhancer of the BCL11A gene (upper panel) and the HBS1L-MYB intergenic region containing MYB enhancers (lower panel) are highlighted with red boxes. (C) Differential MLV integration preferences in HSPC, EPP and MPP inside the KIT locus. The erythroid-specific KIT enhancer (#4) is highlighted with a red box. (D) CAGE expression levels of the KIT promoter in HSPC, EPP and MPP. (E) Fold luciferase induction of the erythroid-specific KIT enhancer (#4) compared to a negative control region (37- and 3.5-fold luciferase induction in EPP and MPP, respectively). (F) The erythroid master regulator GATA1 binds the erythroid-specific KIT enhancer. ChIP assay was performed in EPP to analyze GATA1 binding to the KIT enhancer (#4). Two genomic regions were used as negative and positive controls for GATA1 binding, respectively (Neg Ctrl and Pos Ctrl).
Mentions: To determine whether the putative enhancers identified by combining ChIP-seq and retroviral scanning have transcriptional activity in a functional assay, we tested 8 MLV-targeted erythroid and myeloid enhancers in a reporter assay in EPP and MPP respectively. As expected, erythroid-specific MLV-targeted regions had higher activity in EPP than in MPP and vice versa (Fig. 7A), confirming that MLV identifies cell-specific enhancers, possibly controlling the expression of nearby genes. As examples, MLV was able to target known cell-specific regulatory regions, such as the intronic enhancer of the BCL11A gene46 and the HBS1L-MYB intergenic region containing erythroid-specific MYB enhancers47 (Fig. 7B and Supplementary Fig. 13). MLV scanning identified also novel enhancers in a cell-specific fashion (Supplementary Fig. 13), such as the integration clusters mapping to different regions of the KIT locus in HSPC, EPP and MPP (Fig. 7C). These clusters most likely identify enhancers used to exert a lineage-specific control of the locus during hematopoietic differentiation (Fig. 7D and Supplementary Fig. 14), such as the erythroid-specific enhancer #4, which is primarily active in EPP (Fig. 7E) and is targeted by the erythroid master regulator GATA1 (Fig. 7F).

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