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  1. Evaluation Summary:

    The authors investigate the contribution of a CTCF binding site located 3' of the beta-globin locus to the relative expression of the genes within the cluster. Their results serve to further clarify a longstanding model for how relatively high levels of fetal beta-globin are observed in some individuals harboring a deletion of this region, and contribute to existing models of the function of CTCF binding sites in genome organization.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    The authors investigate the contribution of a CTCF binding site located 3' of the beta-globin gene cluster to the relative expression of different genes within the cluster, in erythroid cell lines. They determine that deletion of 3'HS1, which harbors the first CTCF binding site downstream of the cluster, results in upregulation of the fetal HBG genes in both the K562 and HUDEP-2 cell lines. Hi-C analysis indicates that, as might be expected, the patterns of nuclear colocalization/interactions between the CTCF binding sites in the region change upon deletion of 3'HS1. Moreover, the authors also examine an inversion of the 3'HS1 binding site at its normal location, and this manipulation results in decreases in HBE/G expression associated with an increased frequency of interaction between 3'HS1 and another CTCF site further downstream of the locus, as determined by Hi-C. Additional disruption of the HBG transcriptional repressor BCL11A results in a further increase in HBG expression, suggesting that the upregulation that occurs upon 3'HS1 deletion results from a distinct mechanism. Deletion of a putative enhancer located downstream of the 3'HS1 site, marked by GATA-1 binding, in the 3'HS1-deletion background results in partial loss of increased HBG expression. Deletion of 48 kb of DNA between this enhancer and 3'HS1 results in a modest (2-fold) increase in HBG and HBE expression. Finally, the authors perform the 3'HS1 deletion in primary human CD34+ erythroid cell cultures, and demonstrate a similar effect on gene expression, as measured by HbF+ cells, albeit not as dramatic as that observed in the HUDEP-2 cell line.

    In this study, the authors revisit a decades-old line of research related to deletional HPFH. A number of prior studies had indicated that 3' deletions in the human beta-globin locus resulted in significant upregulation of fetal beta-globin expression. The bulk of this effect has always been ascribed to the loss of the adult beta-globin gene, which then presumably allows the beta-globin LCR to activate the remaining genes within the locus, but there were prior suggestions of a downstream enhancer as well. The current study pinpoints the identity of this downstream enhancer and suggests a contribution for this element, as well as for its relative proximity to the genes after deletion of intervening sequences, in fetal globin activation. The study also clarifies the absence of any effect when 3'HS1 was deleted in the mouse, in that the enhancer is human-specific. In this light, the absence of an accompanying deletion of the HBB and/or HBD genes in combination with the deletion of 3'HS1 is notable; this would have provided a more complete investigation of the relationship with deletional HPFH. The authors potentially provide a wider scope of interest, outside the realm of beta-globin gene expression, by demonstrating specific effects, associated with physiologically relevant phenomena, upon manipulation (deletion and inversion) of a single CTCF binding site. My only complaint about the data is that beta-globin gene expression is not examined comprehensively for each cell line, and also is not presented consistently.

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  3. Reviewer #2 (Public Review):

    The authors analyzed the functional role of a CTCF binding site in the β-globin gene locus in Hudep-2 and differentiating CD34+ cells. Previous studies have shown that CTCF sites flanking the globin gene locus interact and form chromosomal loops. The authors found that deleting 3'HS1 specifically increased expression of the γ-globin gene and reduced expression of the β-globin gene. This seems independent from the levels of the known γ-globin repressor BCL11a. Through analysis of ATAC seq. and GATA1-ChIP-seq. data, they identified an enhancer that upon deletion reduced activation of globin in the 3'HS1 deficient cells. Deletion of a GATA1 site within this enhancer also reduced globin expression.

    This is an interesting manuscript that provides functional insight into regulatory DNA elements located downstream of the β-globin gene cluster. This study is also significant with respect to potentially improving therapeutic fetal globin production. Overall, the experiments include appropriate controls and statistics.

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