Page 66 - Haematologica March 2020

C. Zhang et al.
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Figure 6. SNP rs31528793 associates with Lxn promoter activity and hematopoietic stem cell (HSC) number. (A) SNP rs31528793 affects Lxn promoter activity. G allele is associated with the higher promoter activity whereas C allele lowers its activity. HMGB2 has transcription suppressor activity in both alleles. The luciferase reporter assay was performed on G or C containing vectors (G or C) and on the vectors co-transfected with HMGB2 plasmids (G+ HMGB2; or C+ HMGB2). The average values of three independent experiments are shown with standard deviation (SD) (n=12). (B) HMGB2 binds to G/C containing Lxn promoter. Electrophoretic mobility shift assay (EMSA) was performed with HMGB2 polyclonal antibody and biotin labeled oligonucleotides containing G or C allele. One representative experiment shown out of three independent experiments. (C) G/C allele in SNP rs31528793 indicates LXN protein level. Western blot measured LXN protein in bone marrow cells of different mouse strains carrying either G or C allele. One representative experiment shown out of three independent experiments. (D) G/C allele in SNP rs31528793 indicates HSC number. Long-term HSC and frequency (%) were determined by the markers lineage-Sca-1+c-kit+flk2-CD34-. Results are average (± 1 Standard Deviation) of nine mice from three independent experiments. *P<0.05; **P<0.01. (E) Model of transcriptional regulation of Lxn by HMGB2 and the effect of SNP rs31528793 on the natural variation of Lxn expression and HSC number. HMGB2 binds to the Lxn promoter and acts as a transcriptional suppressor. SNP rs31528793 in the Lxn promoter sequence causes the differential promoter activity in which G allele is associated with higher Lxn expression and lower HSC number whereas C allele is associated with higher Lxn expression and lower HSC number. HMGB2 is involved in Lxn transcriptional regulation either by itself or by modifying chromatin structure in the Lxn promoter region, thereby facilitating access of other transcription factors to the region.
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haematologica | 2020; 105(3)