Papayannopoulou, T. | bioRxiv Science

bioRxiv2024

The glucocorticoid receptor elicited proliferative response in human erythropoiesis is BCL11A-dependent

Prior evidence indicates that the erythroid cellular response to glucocorticoids (GC) has developmental specificity, namely, that developmentally more advanced cells that are undergoing or have undergone fetal to adult globin switching are more responsive to GC-induced expansion. To investigate the molecular underpinnings of this, we focused on the major developmental globin regulator BCL11A. We compared: a) levels of expression and nuclear content of BCL11A in adult erythroid cells upon GC stimulation; b) response to GC of CD34+ cells from patients with BCL11A microdeletions and reduced BCL11A expression, and; c) response to GC of two cellular models (HUDEP-2 and adult CD34+ cells) before and after reduction of BCL11A expression by shRNA. We observed that: a) GC-expanded erythroid cells from a large cohort of blood donors displayed amplified expression and nuclear accumulation of BCL11A; b) CD34+ cells from BCL11A microdeletion patients generated fewer erythroid cells when cultured with GC compared to their parents, while the erythroid expansion of the patients was similar to that of their parents in cultures without GC, and; c) adult CD34+ cells and HUDEP-2 cells with shRNA-depleted expression of BCL11A exhibit reduced expansion in response to GC. In addition, RNA-seq profiling of shRNA-BCL11A CD34+ cells cultured with and without GC was similar (very few differentially expressed genes), while GC-specific responses (differential expression of GILZ and of numerous additional genes) were observed only in controls cells with unperturbed BCL11A expression. These data indicate that BCL11A is an important participant of certain aspects of the stress pathway sustained by GC.

cell biology↗

bioRxiv2023

Large-scale discovery of potent, compact and lineage specific enhancers for gene therapy vectors

Regulation of gene expression during cell development and differentiation is chiefly orchestrated by distal noncoding regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors rely on the cellular and context specificity of regulatory DNA elements to express therapeutic transgenes in the correct location and time. Here, we develop a straight-forward, one-shot approach to screen putative regulatory sequences identified in large-scale epigenomics profiling experiments for precise and programmable control of transgenes encoded within gene therapy viral vectors. We designed a library of 15,000 short sequences ([~]200bp) derived from a set of developmentally active DHS elements during human ex vivo erythropoiesis and cloned them into a GFP reporter lentiviral vector. In an erythroid progenitor cell line, these elements display a gradient of transcriptional enhancer activity, with some demonstrating equivalent activity to the canonical {beta}-globin LCR despite a 9-fold smaller size. We show that these elements are both highly cell type restricted and developmental stage specific both in vitro and in vivo. Finally, we replace the LCR element with one of the novel short enhancers in a {beta}-thalassemia lentiviral therapeutic vector and efficiently correct the thalassemic phenotype in patient-derived HSPCs. More broadly, our approach provides further insights into enhancer biology with wider implications into the development of highly cell type specific and efficacious viral vectors for human gene therapy.

genomics↗