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BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE - Nature (2015)

Bottom Line: Despite conserved function of the composite enhancers, their architecture diverges.The crucial human sequences appear to be primate-specific.The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

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Functional sequences at the Bcl11a erythroid enhancera-c, HMM segmentation of active functional states at m+55, m+58, and m+62 orthologs. HbF enrichment scores shown as gray lines and circles with blue line representing smoothed enrichment score. DNase I sequencing from mouse fetal liver erythroid precursors28. PhyloP (scale from -3.3 to 2.1) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 30 vertebrates. d, Top, BCL11A expression determined by RT-qPCR displayed as a heatmap in 108 hemizygous m+62 ortholog deletion clones ordered by genomic position of deletion midpoint. Each bar demonstrates the genomic position of the deletion breakpoints and the associated color demonstrates the level of BCL11A expression. Bottom, BCL11A expression determined by RT-qPCR in 108 hemizygous m+62 ortholog deletion clones. Per nucleotide mean effect size was calculated as the mean fold change in BCL11A expression from all clones in which that nucleotide was deleted. Gray shading represents one s.d. The BCL11A expression data are shown with same x-axis as in Extended Data Fig. 8c immediately above.
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Figure 8: Functional sequences at the Bcl11a erythroid enhancera-c, HMM segmentation of active functional states at m+55, m+58, and m+62 orthologs. HbF enrichment scores shown as gray lines and circles with blue line representing smoothed enrichment score. DNase I sequencing from mouse fetal liver erythroid precursors28. PhyloP (scale from -3.3 to 2.1) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 30 vertebrates. d, Top, BCL11A expression determined by RT-qPCR displayed as a heatmap in 108 hemizygous m+62 ortholog deletion clones ordered by genomic position of deletion midpoint. Each bar demonstrates the genomic position of the deletion breakpoints and the associated color demonstrates the level of BCL11A expression. Bottom, BCL11A expression determined by RT-qPCR in 108 hemizygous m+62 ortholog deletion clones. Per nucleotide mean effect size was calculated as the mean fold change in BCL11A expression from all clones in which that nucleotide was deleted. Gray shading represents one s.d. The BCL11A expression data are shown with same x-axis as in Extended Data Fig. 8c immediately above.

Mentions: We used pairs of sgRNAs in the presence of Cas9 to produce MEL clones with deletions of various substituent elements at the Bcl11a enhancer (Fig. 5b). Deletion of the DNase-insensitive m+58 ortholog had no apparent effect on BCL11A expression consistent with the pooled screen result. Deletion of the m+55 ortholog led to an approximately two-fold reduction in BCL11A expression (mean residual level 49%, p<0.0001), whereas deletion of the m+62 ortholog approached deletion of the entire composite enhancer in terms of reduction in BCL11A expression (mean residual levels of 8% (p<0.0001) and 6% (p<0.0001) respectively, Fig. 5b; also see Supplementary Information and Extended Data Fig. 8, 9). In addition, clones in which the m+62 ortholog was inverted showed no change in BCL11A expression, suggesting that the mouse, like the human, enhancer functions independent of orientation in situ (Fig. 2c-e; 5b).


BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE - Nature (2015)

Functional sequences at the Bcl11a erythroid enhancera-c, HMM segmentation of active functional states at m+55, m+58, and m+62 orthologs. HbF enrichment scores shown as gray lines and circles with blue line representing smoothed enrichment score. DNase I sequencing from mouse fetal liver erythroid precursors28. PhyloP (scale from -3.3 to 2.1) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 30 vertebrates. d, Top, BCL11A expression determined by RT-qPCR displayed as a heatmap in 108 hemizygous m+62 ortholog deletion clones ordered by genomic position of deletion midpoint. Each bar demonstrates the genomic position of the deletion breakpoints and the associated color demonstrates the level of BCL11A expression. Bottom, BCL11A expression determined by RT-qPCR in 108 hemizygous m+62 ortholog deletion clones. Per nucleotide mean effect size was calculated as the mean fold change in BCL11A expression from all clones in which that nucleotide was deleted. Gray shading represents one s.d. The BCL11A expression data are shown with same x-axis as in Extended Data Fig. 8c immediately above.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4644101&req=5

Figure 8: Functional sequences at the Bcl11a erythroid enhancera-c, HMM segmentation of active functional states at m+55, m+58, and m+62 orthologs. HbF enrichment scores shown as gray lines and circles with blue line representing smoothed enrichment score. DNase I sequencing from mouse fetal liver erythroid precursors28. PhyloP (scale from -3.3 to 2.1) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 30 vertebrates. d, Top, BCL11A expression determined by RT-qPCR displayed as a heatmap in 108 hemizygous m+62 ortholog deletion clones ordered by genomic position of deletion midpoint. Each bar demonstrates the genomic position of the deletion breakpoints and the associated color demonstrates the level of BCL11A expression. Bottom, BCL11A expression determined by RT-qPCR in 108 hemizygous m+62 ortholog deletion clones. Per nucleotide mean effect size was calculated as the mean fold change in BCL11A expression from all clones in which that nucleotide was deleted. Gray shading represents one s.d. The BCL11A expression data are shown with same x-axis as in Extended Data Fig. 8c immediately above.
Mentions: We used pairs of sgRNAs in the presence of Cas9 to produce MEL clones with deletions of various substituent elements at the Bcl11a enhancer (Fig. 5b). Deletion of the DNase-insensitive m+58 ortholog had no apparent effect on BCL11A expression consistent with the pooled screen result. Deletion of the m+55 ortholog led to an approximately two-fold reduction in BCL11A expression (mean residual level 49%, p<0.0001), whereas deletion of the m+62 ortholog approached deletion of the entire composite enhancer in terms of reduction in BCL11A expression (mean residual levels of 8% (p<0.0001) and 6% (p<0.0001) respectively, Fig. 5b; also see Supplementary Information and Extended Data Fig. 8, 9). In addition, clones in which the m+62 ortholog was inverted showed no change in BCL11A expression, suggesting that the mouse, like the human, enhancer functions independent of orientation in situ (Fig. 2c-e; 5b).

Bottom Line: Despite conserved function of the composite enhancers, their architecture diverges.The crucial human sequences appear to be primate-specific.The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.

Show MeSH