Recapitulation of erythropoiesis in congenital dyserythropoietic anemia type I (CDA-I) identifies defects in differentiation and nucleolar abnormalities

Abstract

The investigation of inherited disorders of erythropoiesis has elucidated many of the principles underlying the production of normal red blood cells and how this is perturbed in human disease. Congenital Dyserythropoietic Anaemia type 1 (CDA-I) is a rare form of anaemia caused by mutations in two genes of unknown function: CDAN1 and CDIN1 (previously called C15orf41), whilst in some cases, the underlying genetic abnormality is completely unknown. Consequently, the pathways affected in CDA-I remain to be discovered. To enable detailed analysis of this rare disorder we have validated a culture system which recapitulates all of the cardinal haematological features of CDA-I, including the formation of the pathognomonic ‘spongy’ heterochromatin seen by electron microscopy. Using a variety of cell and molecular biological approaches we discovered that erythroid cells in this condition show a delay during terminal erythroid differentiation, associated with increased proliferation and widespread changes in chromatin accessibility. We also show that the proteins encoded by CDAN1 and CDIN1 are enriched in nucleoli which are structurally and functionally abnormal in CDA-I. Together these findings provide important pointers to the pathways affected in CDA-I which for the first time can now be pursued in the tractable culture system utilised here.

Identification of LZTFL1 as a candidate effector gene at a COVID-19 risk locus

“…HUVECs were fixed in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FCS. For erythroid cells, CD34 + hematopoietic stem and progenitor cells were isolated from the peripheral blood of 2 healthy males and 1 healthy female and differentiated ex vivo for 13 d as described previously 82 . CD4 + T cells were enriched from whole blood (93-99% pure, RosetteSep Human CD4 + T Cell Enrichment Cocktail; STEMCELL Technologies) and were plated at 250,000 cells per well in U-96 well plates (Greiner) and cultured in medium alone or stimulated with anti-CD3/ CD28 T-activator beads (Dynabeads; Thermo Fisher Scientific) at a ratio of 0.3 beads per cell for 4 h at 37 °C in X-VIVO 15 (Lonza), 1% AB serum (Lonza) and penicillin-streptomycin (Thermo Fisher Scientific).…”

High-resolution targeted 3C interrogation of cis-regulatory element organization at genome-wide scale

“…Invitrogen], 1000 U/mL Leukemia Inhibition Factor) and resuspended with 0.05% trypsin for 5 min 37°C before washing with PBS. Human erythroid cells were generated from CD34+ cells as described 51,60 with ethics approval (MREC 03/08/097) and stored according to HTA guidelines (License 12433). Mouse erythroid and ESC were resuspended in RPMI (11875093; Invitrogen) with 15% FBS for fixation.…”

The congenital dyserythropoieitic anemias: genetics and pathophysiology

“…Initial efforts aimed at studying the function of Codanin-1 were performed using a cervical cancer cell line (HeLa) and an osteosarcoma cell line (U-2-OS); these studies demonstrated that Codanin-1 localizes to the heterochromatin during interphase and cytokinesis, but that Codanin-1 was phosphorylated during mitosis resulting in its exclusion from condensed chromosomes [28]. In contrast, three independent reports [27,29,30 & ], two of which used validated antibodies [27,29], showed that Codanin-1 is localized mostly to the cytosol [27,29,30 & ], a finding confirmed at the endogenous expression level in primary human erythroblasts, CDA I erythroblasts, and murine erythroblasts [27]. Although one of the latter reports showed that the Golgi apparatus of CDA I erythroblasts aberrantly accumulates HP1" (a member of the heterochromatin protein family) [27], another report showed that Codanin-1 interacts with and sequesters Asf1 in the cytoplasm, resulting in…”