Thread by @bloodgenes

Wonderful to have our work on using genome editing in single cells to better understand hemoglobin switching published in @NatureComms today. Terrific work by Yong Shen, @JeffreyVerboon, and colleagues from the @JianXuLab and Orkin labs: go.nature.com/3k3OanG
👇Short thread

13 years ago, we had identified BCL11A as a key regulator of fetal hemoglobin (HbF) expression: science.sciencemag.org/content/322/59…

But how was it acting to silence HbF? (1/n)

10 years ago we characterized a number of HPFH and delta-beta-thalassemia deletions and showed that there may be roles for long-range elements in the silencing of HbF: nejm.org/doi/full/10.10…

But definitive perturbations were not done then (2/n)

More recently, the Orkin lab and @MerlinCrossley's lab have shown that BCL11A occupies chromatin in the proximal promoters of the HbF encoding HBG1/2 (gamma-globin) genes... But how much does this functionally contribute to HbF silencing? (3/n)

Answering this question has been challenging for two reasons: (1) Rare mutations that have a major impact on HbF levels are almost never found together in an individual in vivo and (2) transformed cell lines like HUDEP-2 cells do not always faithfully mimic in vivo data (4/n)

By performing genome editing in human hematopoietic stem/progenitor cells and allowing single cells to mature into erythroid colonies, we could perform single and combinatorial editing and get a read out on globin expression as a result due to these edits as a result. (5/n)

This approach allowed us to make some interesting observations... (6/n)

We wanted to recapitulate all major categories of rare human genetic variation impacting HbF levels using genome editing in this system. (7/n)

If we dissect the role of HPFH deletions that cause high HbF, we find non-additive interactions between a region upstream of HBD and deletion of the adult globin promoters. This validates the promoter competition model as being critical for high HbF expression. (8/n)

If we combine edits of these distal regulatory elements and BCL11A, we see epistatic interactions, demonstrating that BCL11A does require long-range interactions for its silencing activity. (9/n)

Similarly, if we combine editing of the HBG1/2 proximal promoters and BCL11A, we also observe interactions, supporting both distal and proximal roles for BCL11A in HbF silencing. (10/n)

We also explore the role of another key trans-acting factor, ZBTB7A. We show that ZBTB7A primarily silences HBG1/2 and that perturbation of it results in supra-physiological HBG1/2 expression. Moreover, ZBTB7A and BCL11A silencing HbF in an independent manner. (11/n)

We couple these findings with long-range chromatin interaction data using the elegant CAPTURE approach developed by @JianXuLab. (12/n)

Collectively, this provides a refined view of how distinct regulatory factors nominated by human genetic variation can act in a collective manner to alter HbF silencing. (13/n)

This will hopefully be an approach that can be widely used. Moreover, this approach may not only inform our understanding of the mechanisms of HbF silencing, but can also help improve therapeutic approaches for #SickleCell disease and #thalassemia. (fin)