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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

Characterization of MLV-targeted regulatory regions.(A,B) Fraction of MLV clusters overlapping with epigenetically defined H3K27ac+, H3K27ac−, transcribed and untranscribed regulatory regions. (C) Dynamics of MLV-targeted promoters and enhancers upon HSPC commitment. Venn diagrams show the overlap of MLV-targeted strong (H3K27ac+) and weak/inactive (H3K27ac−) promoters (H3K4me3 > H3K4me1) and enhancers (H3K4me1 > H3K4me3) identified in HSPC, EPP and MPP. The fraction of non-overlapping HSPC, EPP and MPP regulatory regions hit by MLV is indicated. We defined a total of 1,241 HSPC-specific, 1,998 EPP-specific and 1,833 MPP-specific MLV clusters. (D) Distribution of expression levels of CAGE promoters in a ±5 kb interval centered on cell-specific enhancers hit by MLV. As control, expression levels of total HSPC, EPP and MPP CAGE promoters were analyzed. A t-test was performed as described in Fig. 4C legend (*P < 0.05; **P < 0.01; ***P < 0.001). A similar correlation was observed from promoters in a 100-Kb window around the enhancers (data not shown). (E) GREAT was used to assign a biological meaning to cell-specific MLV clusters. The analysis showed that MLV is able to target genomic regions involved in cell-specific functions. (F) Top enriched TF motifs in cell-specific MLV clusters. HOMER was used to predict TFBS in cell-specific genomic regions targeted by MLV. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
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f6: Characterization of MLV-targeted regulatory regions.(A,B) Fraction of MLV clusters overlapping with epigenetically defined H3K27ac+, H3K27ac−, transcribed and untranscribed regulatory regions. (C) Dynamics of MLV-targeted promoters and enhancers upon HSPC commitment. Venn diagrams show the overlap of MLV-targeted strong (H3K27ac+) and weak/inactive (H3K27ac−) promoters (H3K4me3 > H3K4me1) and enhancers (H3K4me1 > H3K4me3) identified in HSPC, EPP and MPP. The fraction of non-overlapping HSPC, EPP and MPP regulatory regions hit by MLV is indicated. We defined a total of 1,241 HSPC-specific, 1,998 EPP-specific and 1,833 MPP-specific MLV clusters. (D) Distribution of expression levels of CAGE promoters in a ±5 kb interval centered on cell-specific enhancers hit by MLV. As control, expression levels of total HSPC, EPP and MPP CAGE promoters were analyzed. A t-test was performed as described in Fig. 4C legend (*P < 0.05; **P < 0.01; ***P < 0.001). A similar correlation was observed from promoters in a 100-Kb window around the enhancers (data not shown). (E) GREAT was used to assign a biological meaning to cell-specific MLV clusters. The analysis showed that MLV is able to target genomic regions involved in cell-specific functions. (F) Top enriched TF motifs in cell-specific MLV clusters. HOMER was used to predict TFBS in cell-specific genomic regions targeted by MLV. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.

Mentions: We mapped and analyzed the distribution of >27,000 MLV integration sites in each cell population. About 22% of MLV integrations occurred in TSS-proximal regions and the remaining ones were equally distributed in intergenic and intragenic regions (Supplementary Fig. 10A). Statistical comparison with a random dataset identified a total of 3,498, 2,989 and 4,103 integration clusters in HSPC, EPP and MPP, respectively, with a comparable median span of 5.9, 6.0 and 4.8 kb (seeSupplementary Methods). Virtually all clusters overlapped with epigenetically defined regulatory regions, two-thirds in enhancers and one-third in promoters (Fig. 6A). However, integration clusters targeted only a small fraction of ChIP-defined regulatory regions, i.e., 6% of promoters and 4% of enhancers. Virtually all promoters (97%) and three quarters of the enhancers targeted by MLV integrations were acetylated (compared to ~60 and 15% H3K27ac+ non-targeted promoters and enhancers, respectively; Fig. 6A and Supplementary Fig. 10B), and most of the targeted promoters were associated with CAGE transcripts (compared to ~55% transcribed non-targeted promoters; Fig. 6B and Supplementary Fig. 10C). Strikingly, 10 to 13% of the MLV integrations targeted transcribed enhancers, which represented <1% of the total enhancer population (Fig. 6B and Supplementary Table 5). In addition, MLV clusters targeted SEs at a significantly higher frequency compared to the fraction of total active enhancers (53 vs. 12%, 57 vs. 11% and 73 vs. 17% in HSPC, EPP and MPP, respectively, p < 0.0001).


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)

Characterization of MLV-targeted regulatory regions.(A,B) Fraction of MLV clusters overlapping with epigenetically defined H3K27ac+, H3K27ac−, transcribed and untranscribed regulatory regions. (C) Dynamics of MLV-targeted promoters and enhancers upon HSPC commitment. Venn diagrams show the overlap of MLV-targeted strong (H3K27ac+) and weak/inactive (H3K27ac−) promoters (H3K4me3 > H3K4me1) and enhancers (H3K4me1 > H3K4me3) identified in HSPC, EPP and MPP. The fraction of non-overlapping HSPC, EPP and MPP regulatory regions hit by MLV is indicated. We defined a total of 1,241 HSPC-specific, 1,998 EPP-specific and 1,833 MPP-specific MLV clusters. (D) Distribution of expression levels of CAGE promoters in a ±5 kb interval centered on cell-specific enhancers hit by MLV. As control, expression levels of total HSPC, EPP and MPP CAGE promoters were analyzed. A t-test was performed as described in Fig. 4C legend (*P < 0.05; **P < 0.01; ***P < 0.001). A similar correlation was observed from promoters in a 100-Kb window around the enhancers (data not shown). (E) GREAT was used to assign a biological meaning to cell-specific MLV clusters. The analysis showed that MLV is able to target genomic regions involved in cell-specific functions. (F) Top enriched TF motifs in cell-specific MLV clusters. HOMER was used to predict TFBS in cell-specific genomic regions targeted by MLV. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
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f6: Characterization of MLV-targeted regulatory regions.(A,B) Fraction of MLV clusters overlapping with epigenetically defined H3K27ac+, H3K27ac−, transcribed and untranscribed regulatory regions. (C) Dynamics of MLV-targeted promoters and enhancers upon HSPC commitment. Venn diagrams show the overlap of MLV-targeted strong (H3K27ac+) and weak/inactive (H3K27ac−) promoters (H3K4me3 > H3K4me1) and enhancers (H3K4me1 > H3K4me3) identified in HSPC, EPP and MPP. The fraction of non-overlapping HSPC, EPP and MPP regulatory regions hit by MLV is indicated. We defined a total of 1,241 HSPC-specific, 1,998 EPP-specific and 1,833 MPP-specific MLV clusters. (D) Distribution of expression levels of CAGE promoters in a ±5 kb interval centered on cell-specific enhancers hit by MLV. As control, expression levels of total HSPC, EPP and MPP CAGE promoters were analyzed. A t-test was performed as described in Fig. 4C legend (*P < 0.05; **P < 0.01; ***P < 0.001). A similar correlation was observed from promoters in a 100-Kb window around the enhancers (data not shown). (E) GREAT was used to assign a biological meaning to cell-specific MLV clusters. The analysis showed that MLV is able to target genomic regions involved in cell-specific functions. (F) Top enriched TF motifs in cell-specific MLV clusters. HOMER was used to predict TFBS in cell-specific genomic regions targeted by MLV. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
Mentions: We mapped and analyzed the distribution of >27,000 MLV integration sites in each cell population. About 22% of MLV integrations occurred in TSS-proximal regions and the remaining ones were equally distributed in intergenic and intragenic regions (Supplementary Fig. 10A). Statistical comparison with a random dataset identified a total of 3,498, 2,989 and 4,103 integration clusters in HSPC, EPP and MPP, respectively, with a comparable median span of 5.9, 6.0 and 4.8 kb (seeSupplementary Methods). Virtually all clusters overlapped with epigenetically defined regulatory regions, two-thirds in enhancers and one-third in promoters (Fig. 6A). However, integration clusters targeted only a small fraction of ChIP-defined regulatory regions, i.e., 6% of promoters and 4% of enhancers. Virtually all promoters (97%) and three quarters of the enhancers targeted by MLV integrations were acetylated (compared to ~60 and 15% H3K27ac+ non-targeted promoters and enhancers, respectively; Fig. 6A and Supplementary Fig. 10B), and most of the targeted promoters were associated with CAGE transcripts (compared to ~55% transcribed non-targeted promoters; Fig. 6B and Supplementary Fig. 10C). Strikingly, 10 to 13% of the MLV integrations targeted transcribed enhancers, which represented <1% of the total enhancer population (Fig. 6B and Supplementary Table 5). In addition, MLV clusters targeted SEs at a significantly higher frequency compared to the fraction of total active enhancers (53 vs. 12%, 57 vs. 11% and 73 vs. 17% in HSPC, EPP and MPP, respectively, p < 0.0001).

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