SATB1 Chromatin Loops Regulate Megakaryocyte/Erythroid Progenitor Expansion by Facilitating HSP70 and GATA1 Induction

Abstract

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with severe anemia, congenital malformations, and an increased risk of developing cancer. The chromatin-binding special AT-rich sequence-binding protein-1 (SATB1) is downregulated in megakaryocyte/erythroid progenitors (MEPs) in patients and cell models of DBA, leading to a reduction in MEP expansion. Here we demonstrate that SATB1 expression is required for the upregulation of the critical erythroid factors heat shock protein 70 (HSP70) and GATA1 which accompanies MEP differentiation. SATB1 binding to specific sites surrounding the HSP70 genes promotes chromatin loops that are required for the induction of HSP70, which, in turn, promotes GATA1 induction. This demonstrates that SATB1, although gradually downregulated during myelopoiesis, maintains a biological function in early myeloid progenitors.

Keywords: CLOuD9; Diamond Blackfan anemia; GATA1; HSP70; SATB1; chromatin; erythropoiesis; hematopoiesis; megakaryocytes.

Graphical Abstract

Figure 1.

Special AT-rich sequence-binding protein-1 (SATB1) is required for heat shock protein 70 (HSP70) induction and binds chromatin surrounding HSP70 genes. (A) cord blood (CB) CD34⁺ HSPCs were transduced with siRNA against SATB1 and grown in the presence or absence of doxycycline for 5 days. HSPA1A and HSPA1B transcripts were quantified by qRT-PCR at days 0 and 5. (B) After 5 days, protein expression of HSP70, SATB1, RPS19, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Western blot analysis. (C) CB CD34⁺ HSPCs were transduced with shRNA against luciferase (shLuc) or RPS19 (shRPS19) with or without SATB1 cDNA. HSPA1A and HSPA1B mRNA expression was quantified by qRT-PCR. (D) HSP70, SATB1, RPS19, and GAPDH protein expression was analyzed from cultures at both day 0 and day 5. Experiments were performed in triplicate and repeated a minimum of 5 times. Data are represented as mean with individual data points presented. (E) CB CD34⁺ were differentiated for 5 days prior to SATB1 chromatin immunoprecipitation. The extent of SATB1 binding to each 1 kb fragment for a 40 kb region surrounding the HSP70 gene loci was quantified by qRT-PCR. Data are represented as mean ± SD.

Figure 2.

Special AT-rich sequence-binding protein-1 (SATB1) induces looping surrounding heat shock protein 70 (HSP70) genes in early hematopoiesis. (A) CB CD34⁺ HSPCs transduced with shRNA against luciferase, SATB1, RPS19, and/or cDNA for SATB1 were cultured for 5 days. A 30 kb region including the 3 SATB1-binding sites, HSPA1A and HSPA1B, was analyzed by semi-quantitative chromatin conformation capture (3-C) assay, followed by qRT-PCR examining the frequency of fragments separated by 1 kb. Ligation product reads were normalized for copy frequency based on proximity to the reference point. A point 1 kb downstream of the SATB1-binding site proximal to HSPA1B promoter was defined as the reference point. (B) Schematic depicting genomic region surrounding HSPA1A and A1B, SATB1-binding sites, the 3-C reference point, and our proposed model of SATB1-induced chromatin looping. (C) Un-transduced CD34+ HSPCs, or HSPCs that had been cultured in liquid media for 5 or 8 days, were subjected to 3-C qRT-PCR. (D) Expression of SATB1 mRNA was examined by qRT-PCR and was analyzed in parallel.

Figure 3.

Special AT-rich sequence-binding protein-1 (SATB1)-mediated chromatin looping required to facilitate heat shock protein 70 (HSP70) induction during hematopoiesis. Control and RPS19-insufficient CB CD34⁺ HSPCs at day 5 were co-transduced with CLOuD9 gRNA dimer pairs and subjected to 3-C qRT-PCR analysis. To the left are schematic representations of the predicted looping patterns. (A) RPS19-insufficiency data (in blue) is superimposed over control (in gray). HSPA1A and HSP70A1B induction was assessed at days 0 and 5 by qRT-PCR. (B–) Transduced CLOuD9 components facilitate loops among the 3 SATB1-binding sites and are designated loop 1, loop 2, and loop 3. Corresponding 3-C and qRT-PCR values are superimposed over controls in purple (B), green (C). and orange (D) to correspond with cells inducing loops 1, 2, and 3, respectively. (E) Concurrently, cells were lysed and HSP70 and GAPDH protein expression was determined by Western blot analysis. Data are represented as mean and variance is indicated as SD or individual data points.

Figure 4.

Special AT-rich sequence-binding protein-1 (SATB1)-driven heat shock protein 70 (HSP70) induction is required for early induction of GATA1 but does not significantly impact later GATA1 induction. (A) CB CD34⁺ HSPCs were transduced with doxycycline-inducible siRNA against SATB1 and CLOuD9 loop 1, loop 2, or loop 3 components and differentiated for 12 days. Cells were FACs sorted, lysed, and GATA1 and GAPDH protein expression was scrutinized by Western blot analysis. Quantification of 3 independent experiments is presented above while representative images are presented below. (B) CB CD34⁺ HSPCs were transduced with doxycycline-inducible siRNA against SATB1 and CLOuD9 E-P1, E-P2, or P1-P2 looping components and differentiated in the presence or absence of doxycycline for 5 days. Western blot analysis was performed to determine GATA1 and GAPDH expression. Quantification is presented above with representative images provided below. (C) Lin⁻CD34⁺CD38⁺CD45RA⁻FLT3^-MPL⁺CD36⁻CD41⁻ common myeloid progenitors (CMPs) were enriched from CB CD34⁺ HSPCs and transduced as above. After 4 days of differentiation, Lin^-CD34⁺CD38^midCD45RA⁻FLT3^-MPL⁺CD36⁻CD41⁻ megakaryocyte/erythroid progenitor (MEP) populations were purified and GATA1 was assessed by qRT-PCR. (D) The population of MEPs was calculated by multiplying the total number of viable cells by the percentage of Lin^-CD34⁺CD38^midCD45RA^-FLT3^-MPL⁺CD36⁻CD41⁻ cells as evaluated by flow cytometry. Data are represented as mean ± SD and significance is defined as P < .05. (E) Diagrammatic representation of protein expression dynamics in normal and siRNA- and RPS19-insufficiency mediated SATB1 downregulation. In controls, SATB1 is gradually downregulated while GATA1 is sequentially induced at the HSP-CMP, CMP-MEP, and CFU-E-proerythroblast transitions. HSP70 induction precedes/correlates with the second GATA1 induction event. When SATB1 expression is downregulated, HSP70 induction does not occur, and the second GATA1 induction is absent.