LRF Promotes Indirectly Advantageous Chromatin Conformation via BGLT3-lncRNA Expression and Switch from Fetal to Adult Hemoglobin

The hemoglobin switch from fetal (HbF) to adult (HbA) has been studied intensively as an essential model for gene expression regulation, but also as a beneficial therapeutic approach for beta-hemoglobinopathies, towards the objective of reactivating HbF. The transcription factor LRF (Leukemia/lymphoma-related), encoded from the ZBTB7A gene has been implicated in fetal hemoglobin silencing, though has a wide range of functions that have not been fully clarified. We thus established the LRF/ZBTB7A-overexpressing and ZBTB7A-knockdown K562 (human erythroleukemia cell line) clones to assess fetal vs. adult hemoglobin production pre- and post-induction. Transgenic K562 clones were further developed and studied under the influence of epigenetic chromatin regulators, such as DNA methyl transferase 3 (DNMT3) and Histone Deacetylase 1 (HDAC1), to evaluate LRF's potential disturbance upon the aberrant epigenetic background and provide valuable information of the preferable epigenetic frame, in which LRF unfolds its action on the beta-type globin's expression. The ChIP-seq analysis demonstrated that LRF binds to gamma-globin genes (HBG2/1) and apparently associates BCL11A for their silencing, but also during erythropoiesis induction, LRF binds the BGLT3 gene, promoting BGLT3-lncRNA production through the gamma-delta intergenic region of beta-type globin's locus, triggering the transcriptional events from gamma- to beta-globin switch. Our findings are supported by an up-to-date looping model, which highlights chromatin alterations during erythropoiesis at late stages of gestation, to establish an "open" chromatin conformation across the gamma-delta intergenic region and accomplish beta-globin expression and hemoglobin switch.