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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

Analysis of CAGE promoters.(A) Genomic distribution of CAGE TSSs in HSPC, EPP and MPP. TSSs were mapped to regions annotated as promoters (500 bp-long regions upstream annotated TSSs), 5′ UTR, exon, intron and 3′ UTR of coding and noncoding genes (in sense or antisense orientation) or as intergenic regions. For each category, frequency is indicated. (B) Distribution of total and differentially used CAGE promoters overlapping with epigenetically defined promoters and enhancers. (C) Annotation of total and differentially used CAGE promoters. The graphs show the proportions of total and differentially used CAGE promoters associated to coding RNA and ncRNA (miRNA, rRNA, snoRNA and snRNAand lincRNA). (D) Distribution of total and differentially used CAGE promoters amongst the different classes of repetitive elements, defined by RepeatMasker. Total CAGE promoters: HSPC, EPP and MPP. Differentially used CAGE promoters: HSPC/EPP and HSPC/MPP. (E) Top enriched TF motifs within CAGE promoters (−300 to +100 bp from TSSs). Transcription factor motif finding in cell-specific promoters was performed using HOMER software. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
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f2: Analysis of CAGE promoters.(A) Genomic distribution of CAGE TSSs in HSPC, EPP and MPP. TSSs were mapped to regions annotated as promoters (500 bp-long regions upstream annotated TSSs), 5′ UTR, exon, intron and 3′ UTR of coding and noncoding genes (in sense or antisense orientation) or as intergenic regions. For each category, frequency is indicated. (B) Distribution of total and differentially used CAGE promoters overlapping with epigenetically defined promoters and enhancers. (C) Annotation of total and differentially used CAGE promoters. The graphs show the proportions of total and differentially used CAGE promoters associated to coding RNA and ncRNA (miRNA, rRNA, snoRNA and snRNAand lincRNA). (D) Distribution of total and differentially used CAGE promoters amongst the different classes of repetitive elements, defined by RepeatMasker. Total CAGE promoters: HSPC, EPP and MPP. Differentially used CAGE promoters: HSPC/EPP and HSPC/MPP. (E) Top enriched TF motifs within CAGE promoters (−300 to +100 bp from TSSs). Transcription factor motif finding in cell-specific promoters was performed using HOMER software. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.

Mentions: To define the promoter usage in HSPC and their committed progeny, we used Cap Analysis of Gene Expression (CAGE), a technique that identifies active transcription start sites (TSSs) at single base-pair resolution and measures the expression level of each transcript44. We clustered CAGE tags into 2 levels: Level-1 promoters (“TSSs”) were created by summing the weighted number of CAGE tags that have an identical 5′ start site, and were then clustered in Level-2 promoters (“CAGE promoters”) if they were within 20 bp of each other and had similar expression levels. We mapped by CAGE ~0.6 × 106 TSSs in each cell population, typically scattered over short genomic regions due to the inherent variability of transcription initiation45. As an example, 3 TSSs were mainly used to drive transcription of the human beta globin gene (HBB) in EPP, which started at low frequency at 13 nucleotide positions in a 58-bp region encompassing the 5′ UTR of HBB (Supplementary Fig. 2). We mapped most of the TSSs (>70%) to regions annotated as promoters and 5′ UTR of known transcripts (Fig. 2A). Interestingly, 23% of TSSs were mapped to intergenic regions, exons, introns, and 3′ UTR, suggesting the presence of alternative or novel, yet unannotated promoters (Fig. 2A). Notably, TSSs mapping to exons, introns and 3′ UTRs of coding genes had lower expression levels compared to those mapping to promoters and 5′ UTRs (Supplementary Fig. 3A). About 3.5% of TSSs mapped to the antisense strand of known genes, mostly in promoters and introns (Fig. 2A).


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)

Analysis of CAGE promoters.(A) Genomic distribution of CAGE TSSs in HSPC, EPP and MPP. TSSs were mapped to regions annotated as promoters (500 bp-long regions upstream annotated TSSs), 5′ UTR, exon, intron and 3′ UTR of coding and noncoding genes (in sense or antisense orientation) or as intergenic regions. For each category, frequency is indicated. (B) Distribution of total and differentially used CAGE promoters overlapping with epigenetically defined promoters and enhancers. (C) Annotation of total and differentially used CAGE promoters. The graphs show the proportions of total and differentially used CAGE promoters associated to coding RNA and ncRNA (miRNA, rRNA, snoRNA and snRNAand lincRNA). (D) Distribution of total and differentially used CAGE promoters amongst the different classes of repetitive elements, defined by RepeatMasker. Total CAGE promoters: HSPC, EPP and MPP. Differentially used CAGE promoters: HSPC/EPP and HSPC/MPP. (E) Top enriched TF motifs within CAGE promoters (−300 to +100 bp from TSSs). Transcription factor motif finding in cell-specific promoters was performed using HOMER software. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Analysis of CAGE promoters.(A) Genomic distribution of CAGE TSSs in HSPC, EPP and MPP. TSSs were mapped to regions annotated as promoters (500 bp-long regions upstream annotated TSSs), 5′ UTR, exon, intron and 3′ UTR of coding and noncoding genes (in sense or antisense orientation) or as intergenic regions. For each category, frequency is indicated. (B) Distribution of total and differentially used CAGE promoters overlapping with epigenetically defined promoters and enhancers. (C) Annotation of total and differentially used CAGE promoters. The graphs show the proportions of total and differentially used CAGE promoters associated to coding RNA and ncRNA (miRNA, rRNA, snoRNA and snRNAand lincRNA). (D) Distribution of total and differentially used CAGE promoters amongst the different classes of repetitive elements, defined by RepeatMasker. Total CAGE promoters: HSPC, EPP and MPP. Differentially used CAGE promoters: HSPC/EPP and HSPC/MPP. (E) Top enriched TF motifs within CAGE promoters (−300 to +100 bp from TSSs). Transcription factor motif finding in cell-specific promoters was performed using HOMER software. The frequency of target (background) sequences enriched in TF motifs and p-values are indicated.
Mentions: To define the promoter usage in HSPC and their committed progeny, we used Cap Analysis of Gene Expression (CAGE), a technique that identifies active transcription start sites (TSSs) at single base-pair resolution and measures the expression level of each transcript44. We clustered CAGE tags into 2 levels: Level-1 promoters (“TSSs”) were created by summing the weighted number of CAGE tags that have an identical 5′ start site, and were then clustered in Level-2 promoters (“CAGE promoters”) if they were within 20 bp of each other and had similar expression levels. We mapped by CAGE ~0.6 × 106 TSSs in each cell population, typically scattered over short genomic regions due to the inherent variability of transcription initiation45. As an example, 3 TSSs were mainly used to drive transcription of the human beta globin gene (HBB) in EPP, which started at low frequency at 13 nucleotide positions in a 58-bp region encompassing the 5′ UTR of HBB (Supplementary Fig. 2). We mapped most of the TSSs (>70%) to regions annotated as promoters and 5′ UTR of known transcripts (Fig. 2A). Interestingly, 23% of TSSs were mapped to intergenic regions, exons, introns, and 3′ UTR, suggesting the presence of alternative or novel, yet unannotated promoters (Fig. 2A). Notably, TSSs mapping to exons, introns and 3′ UTRs of coding genes had lower expression levels compared to those mapping to promoters and 5′ UTRs (Supplementary Fig. 3A). About 3.5% of TSSs mapped to the antisense strand of known genes, mostly in promoters and introns (Fig. 2A).

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