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, 34 (18), 5232-7

BP1 Is a Negative Modulator of Definitive Erythropoiesis

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BP1 Is a Negative Modulator of Definitive Erythropoiesis

Marthe-Sandrine Eiymo Mwa Mpollo et al. Nucleic Acids Res.

Abstract

Beta protein 1 (BP1), a human homeotic transcription factor, is expressed during hematopoeisis in the erythroid lineage. To determine the in vivo role of BP1 in erythropoiesis, we have undertaken two complementary approaches using enforced BP1 expression in both transgenic mice and embryonic stem (ES) cells. Despite repeated attempts, only one adult transgenic BP1 founder mouse among 121 mice was obtained. This mouse presumably survived due to transgene mosaicism because the transgene could not be transmitted. This mouse expressed BP1 and displayed splenomegaly, extramedullary erythropoiesis and severe amyloidosis A in the kidney, a phenotype compatible with thalassemia. Consistently, the presence of BP1 transgene in fetuses was associated with paleness and lethality. In ES cells, BP1 expression in primary differentiation appeared to antagonize adult beta-globin expression. In secondary differentiation, BP1 expression reduced significantly beta-globin gene expression in both primitive and definitive erythroid cells, whereas it impaired only the definitive erythroid cell differentiation. These studies showed that BP1 can negatively modulate adult beta-globin gene expression and definitive erythroid cell differentiation, and suggest that BP1 could play a role in thalassemia.

Figures

Figure 1
Figure 1
LCRBP1 transgene structure and expression in transgenic mouse. (a) The LCRBP1 construct was produced in a bluescript vector by joining the human β-globin promoter (pr) lacking the two silencer sequences (−264/SnaB1 to +48/NcoI) to the 1.1 kb BP1 cDNA (SmaI–XbaI). The human β-globin 8 kb midi LCR was linked to this DNA upstream, and downstream 2.8 kb of the β-globin gene including 3′ sequences of exon 2, all of exon 3, and 3′ flanking sequences [BamHI (nt 62613) to XbaI (nt 65421), GenBank accession no. U01317), containing the two globin 3′ enhancers (18). The probe BP1-β-globin (SnaBI–XbaI, 3.2 kb) used for transgenic mouse screening by Southern blot analysis is indicated above the LCRBP1 construct. (b) Expression analysis in peripheral blood using RT–PCR. Three RNA blood samples from control H2O (C), transgenic mouse (tg BP1) and control identical mouse strain (WT) were monitored for BP1 and S16 expression. Human BP1 expression was readily detectable in the blood of the transgenic founder mouse and undetectable in the blood of the control identical mouse strain. Expression of the ribosomal protein S16 served as internal control.
Figure 2
Figure 2
Histopathological analysis of the LCRBP1 transgenic founder mouse. (a) Normal histology of the kidney displaying small regular glomeruli and surrounding tubulo-interstitial compartment (H&E, ×100). Inset shows glomerulus of normal size and cellularity with patent capillaries and basement membranes of normal thickness (H&E, ×400). (b) Renal histology of the transgenic LCRBP1 mouse showed diffuse and uniform enlargement of the glomeruli due to expansion of the glomerular tuft by amorphous pale eosinophilic material. The outlying tubules, interstitium and blood vessels are uninvolved (H&E, ×100). Inset shows a representative glomerulus of the transgenic LCRBP1 mouse with expansion of the mesangium and narrowing of the glomerular capillary lumina by voluminous deposits of amorphous material typical of amyloid. The glomerular cellularity appeared reduced (H&E, ×400). (c) Kidney of the transgenic LCRBP1 mouse viewed under polarized light displayed apple green birefringence diagnostic of amyloid deposits in a diffuse and global glomerular distribution (Congo red, polarized light, ×100). (d) Kidney of the transgenic LCRBP1 mouse showed strong diffuse and global glomerular positivity for serum amyloid A protein (SAA immunostain, ×250). (e) Ultrastructural analysis of a normal renal glomerular capillary showed the normal glomerular architecture, without fibrillar deposits (EM, ×6000). (f) Ultrastructure of a renal glomerulus from the transgenic LCRBP1 mouse showed abundant randomly oriented 8–10 nm fibrils expanding the mesangial regions and narrowing the adjacent glomerular capillary lumen (EM, ×6000). Inset shows fibrils at higher magnitude (EM, ×20 000).
Figure 3
Figure 3
Integrity of the BP1 transgene in ES cells. Southern blot analysis of electroporated ES cells with LCRΔBP1 and LCRBP1 constructs. Restriction enzyme digestion was carried out for LCRΔBP1 and LCRBP1 clones using SalI/BamHI. Probe used is depicted in Figure 1a and spanned the adult β-globin and BP1 sequences. Expected fragments are 6.1 and 2.8 kb for the LCRΔBP1clones, and 7.2 and 2.8 kb for the LCRBP1 clones. The intensity of the 6.1 kb band of LCRΔBP1 clones is weaker than the 2.8 kb or the 7.2 kb of the LCRBP1 clones since only ∼0.3 kb of the probe is homologous to globin. Extra bands correspond to junction of the constructs with genomic sequences. M, molecular weight marker λ digested with HindIII and C, copy of LCRBP1 unit length.
Figure 4
Figure 4
Expression analysis of BP1 and β-globin genes in EBs at various stages of primary differentiation. (a) Demonstration of linearity of RT–PCR amplification. Varying quantities of total cDNA aliquots were amplified with primers specific for BP1, β-globin and S16 as internal control to ensure that conditions were within linear range of amplification. The reactions were evaluated semiquantitatively on polyacrylamide gels scanned by phosphoimager and values plotted on linear graphs. (b) Parental ES cells (WT), ES cells containing the empty vector (LCRΔBP1) or ES cells containing BP1 (LCRBP1) were differentiated into embryoid bodies (EBs). Expression analysis of BP1 and of the endogeneous β-globin gene was carried out by RT–PCR on the indicated days of differentiation, using S16 expression as an internal control. Days indicated were selected for analysis of the kinetics of erythroid differentiation since primitive erythroid expression is maximum on day 7 and definitive erythroid lineage expression is high from day 5 to day 12 (11). Semi-quantitative evaluation was carried out for each sample run on the same gel and normalized to the ribosomal RNA control.

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