Exploring The Mechanisms To Reveal The Contribution Of LMO2 To The Transcriptional Regulation In Human Erythroblasts

Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA1 and SCL/TAL1), chromatin looping factor LDB1 (referred as GATA-SCL/TAL1 complex) (Wadman et al. EMBO J. 1997). Recently, we have demonstrated that LMO2 contributes to the expression of GATA-1 target genes such as HBB and SLC4A1, through modulating the assembly of GATA-1 as well as the components of SCL/TAL1 complex at the endogeneous loci (ASH 2012). To gain new mechanistic insights, we have extended our study to reveal the contribution of LMO2 to the GATA-1 activity in human erythroblasts.

For LMO2, TAL1 and LDB1 knockdown, anti-LMO2, anti-TAL1, anti-LDB1 siRNA (Thermo Scientific Dharmacon) were used. Western blotting and quantitative ChIP analyses were performed using antibodies for GATA-1 (CST and abcam), LMO2, 6×His tag (abcam), TAL1 and LDB1 (Santa Cruz). Human induced pluripotent stem cell (iPS)-derived erythroid progenitor cells (HiDEP), which have a capacity to differentiate into enucleated red blood cells (Kurita et al. PLOS ONE. 2013), were included for the analysis. For the exogeneous expression of 6×His tagged wild-type GATA-1 and mutant GATA-1 in K562 cells, pBABEpuro retroviral vector and PLAT-GP packaging cell line were used (Fujiwara et al. Exp Hematol. 2013).

We previously demonstrated that transient LMO2 knockdown in K562 cells, which did not affect the expression of GATA-1, SCL/TAL1 and LDB1, resulted in the significantly decreased chromatin occupancy of GATA-1 and the components of SCL/TAL1 complex at beta-globin locus control region (LCR) and SLC4A1 loci (ASH 2012). Based on iPS-derived erythroblasts (HiDEP), we further confirmed the significant downregulation of GATA-1-target genes (HBB, HBA and SLC4A1), and concomitant decrease in GATA-1 chromatin occupancy at the target gene loci, by siRNA-mediated LMO2 knockdown. To reveal the molecular mechanism linking LMO2 and GATA-1, we first expressed 6×His tagged wild-type GATA-1 or mutated GATA-1, including R202Q and R217D, which impaired direct binding with LMO2 (Wilkinson-White et al. PNAS. 2011), in K562 cells. Quantitative ChIP analysis anti-6×His tag antibody revealed significantly diminished occupancy of the mutated GATA-1 (R202Q and R217D) at the beta-globin LCR, HBA and SLC4A1 loci. Next, in addition to the direct interaction between GATA-1 and LMO2, we examined whether the knockdown of each individual component of the SCL/TAL1 complex, such as SCL/TAL1 and LDB1, could affect GATA-1 chromatin occupancy. The expression of GATA-1 target genes, such as HBB, HBA, and SLC4A1, were downregulated by either SCL/TAL1 or LDB1 transient knockdown, whereas the expression of GATA-1 was unaffected. Under the condition, GATA-1 chromatin occupancy was significantly reduced, suggesting that impaired assembly of the individual component of SCL/TAL1 complex may also affect GATA-1 chromatin occupancy.

LMO2 contributes to the assembly of components of the GATA-SCL/TAL1 complex at endogenous loci in erythroblasts, which may lead to dysregulation of a subset of GATA-1 target genes. Our results may lead to the identification of novel disease mechanisms involving anemia as well as leukemia.

No relevant conflicts of interest to declare.