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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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Primate-specific BCL11A enhancer functional coreDHS h+58 functional core defined by maximal HbF enrichment score and Active HMM state. HbF enrichment scores shown by gray lines and circles. HbF indel enrichment per nucleotide based on amplicon genomic sequencing of sorted cells exposed to either sgRNA-1617 or -1621. No common SNPs (MAF>1%) present at this region. JASPAR motifs (P < 10-4) depicted in black with selected motifs annotated by TF based on known erythroid-specific function or genomic position. Gata1 motif LOGO at sgRNA-1617 cleavage position as described in text. Orthologous sequences listed from representative primates and nonprimates of distributed phylogeny. PhyloP (scale from -4.5 to 4.88) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 100 vertebrates.
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Figure 14: Primate-specific BCL11A enhancer functional coreDHS h+58 functional core defined by maximal HbF enrichment score and Active HMM state. HbF enrichment scores shown by gray lines and circles. HbF indel enrichment per nucleotide based on amplicon genomic sequencing of sorted cells exposed to either sgRNA-1617 or -1621. No common SNPs (MAF>1%) present at this region. JASPAR motifs (P < 10-4) depicted in black with selected motifs annotated by TF based on known erythroid-specific function or genomic position. Gata1 motif LOGO at sgRNA-1617 cleavage position as described in text. Orthologous sequences listed from representative primates and nonprimates of distributed phylogeny. PhyloP (scale from -4.5 to 4.88) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 100 vertebrates.

Mentions: The overall sequence conservation at the h+58 Active region appears both less intense and less distinct from flanking sequences as compared to those of h+62 and h+55 (Fig. 3a-c). The top-scoring sgRNAs in the screen colocalize to 42 bp within h+58 (Fig. 4, Extended Data Fig. 5b). The third-highest scoring enhancer-targeted sgRNA (sgRNA-1617) mapped directly onto an apparent GATA1 motif, though below a genome-scale significance threshold (P = 3.74 × 10-4). The mouse orthologous sequence has a GATA1 motif P-value only modestly higher than the human (p = 4.33 × 10-4). This GATA1 motif appears to have relatively high vertebrate conservation, with exact human sequence identity in rabbits, pigs, dogs, and elephants. The top-scoring sgRNA (sgRNA-1621) mapped to a position 15 bp from this GATA1 motif (Fig. 4). An additional four sgRNAs mapping between sgRNA-1621 and 1617 each had substantially elevated HbF enrichment scores. Underlying these sgRNAs were additional predicted motifs (i.e. RXRA, EHF, ELF1, and STAT1). Although these sequences showed a high level of conservation among primates, they showed high degeneracy among nonprimate vertebrates (Fig. 4).


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)

Primate-specific BCL11A enhancer functional coreDHS h+58 functional core defined by maximal HbF enrichment score and Active HMM state. HbF enrichment scores shown by gray lines and circles. HbF indel enrichment per nucleotide based on amplicon genomic sequencing of sorted cells exposed to either sgRNA-1617 or -1621. No common SNPs (MAF>1%) present at this region. JASPAR motifs (P < 10-4) depicted in black with selected motifs annotated by TF based on known erythroid-specific function or genomic position. Gata1 motif LOGO at sgRNA-1617 cleavage position as described in text. Orthologous sequences listed from representative primates and nonprimates of distributed phylogeny. PhyloP (scale from -4.5 to 4.88) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 100 vertebrates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644101&req=5

Figure 14: Primate-specific BCL11A enhancer functional coreDHS h+58 functional core defined by maximal HbF enrichment score and Active HMM state. HbF enrichment scores shown by gray lines and circles. HbF indel enrichment per nucleotide based on amplicon genomic sequencing of sorted cells exposed to either sgRNA-1617 or -1621. No common SNPs (MAF>1%) present at this region. JASPAR motifs (P < 10-4) depicted in black with selected motifs annotated by TF based on known erythroid-specific function or genomic position. Gata1 motif LOGO at sgRNA-1617 cleavage position as described in text. Orthologous sequences listed from representative primates and nonprimates of distributed phylogeny. PhyloP (scale from -4.5 to 4.88) and PhastCons (from 0 to 1) estimates of evolutionary conservation among 100 vertebrates.
Mentions: The overall sequence conservation at the h+58 Active region appears both less intense and less distinct from flanking sequences as compared to those of h+62 and h+55 (Fig. 3a-c). The top-scoring sgRNAs in the screen colocalize to 42 bp within h+58 (Fig. 4, Extended Data Fig. 5b). The third-highest scoring enhancer-targeted sgRNA (sgRNA-1617) mapped directly onto an apparent GATA1 motif, though below a genome-scale significance threshold (P = 3.74 × 10-4). The mouse orthologous sequence has a GATA1 motif P-value only modestly higher than the human (p = 4.33 × 10-4). This GATA1 motif appears to have relatively high vertebrate conservation, with exact human sequence identity in rabbits, pigs, dogs, and elephants. The top-scoring sgRNA (sgRNA-1621) mapped to a position 15 bp from this GATA1 motif (Fig. 4). An additional four sgRNAs mapping between sgRNA-1621 and 1617 each had substantially elevated HbF enrichment scores. Underlying these sgRNAs were additional predicted motifs (i.e. RXRA, EHF, ELF1, and STAT1). Although these sequences showed a high level of conservation among primates, they showed high degeneracy among nonprimate vertebrates (Fig. 4).

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