The PcG Protein L3MBTL1 Transcriptionally Represses Human Embryonic and Fetal Globin Genes: a Novel Prospect for HbF Activation

Abstract 1014

L3MBTL1 is the human homolog of the Drosophila Polycomb Group tumor suppressor gene, lethal(3)malignant brain tumor. We demonstrated that human L3MBTL1 functions as a transcriptional repressor and after crystallizing the MBT repeat domain determined that L3MBTL1 compacts chromatin by binding mono- and di-methylated lysine residues in histones H1 (H1K26) and H4 (H4K20).

Despite the known role of L3MBTL1 in affecting chromatin structure, the function of L3MBTL1 in human hematopoiesis has remained largely unknown. We recently demonstrated that L3MBTL1 enforces cell fate decision toward the erythroid lineage and that knockdown of L3MBTL1 accelerates the erythroid differentiation of human hematopoietic stem/progenitor cells, suggesting that its deletion contributes to the pathogenesis of 20q- erythroid malignancies. Consistently with its role in erythropoiesis, here we reveal that L3MBTL1 is a novel transcriptional repressor of fetal globin genes and it may work in concert with BCL11A and EKLF to control globin gene expression.

By utilizing RNA interference to reduce L3MBTL1 expression, we have found that knockdown of L3MBTL1 in human cord blood hematopoietic stem/progenitor cells consistently upregulates the expression of the epsilon, gamma, and zeta globin genes, but not the beta globin gene. Similar effects were seen following knockdown of L3MBTL1 in the human erythroleukemia cell line K562, and knockdown of L3MBTL1 in human embryonic stem cells (ESCs) led to the inappropriate expression of fetal and embryonic globin genes (which increases more than 50-fold after the L3MBTL1-KD). These data suggest a role for L3MBTL1 in regulating the globin switch.

To investigate the mechanism by which L3MBTL1 silences embryonic and fetal globin gene expression, we used chromatin immunoprecipitation (ChIP) assays to show that L3MBTL1 directly associates with the human β-globin locus. L3MBTL1 occupies several discrete regions within the human β-globin cluster and colocalizes with H4K20me within the Locus Control Region (LCR), a primary attachment site for chromatin modifiers. As confirmation, we found that treatment of K562 cells with hemin, which broadly increases H3K9 acetylation over the β-globin locus and activates the transcription of globin genes, leads to decreases in expression of the repressive H4K20me2 methylmark and L3MBTL1 to the beta-globin cluster.

Given the recent identification of the repressor of gamma globin gene expression, BCL11A, we investigated a potential relationship between L3MBTL1 and BCL11A. We found that knockdown of L3MBTL1 led to downregulation of BCL11A mRNA. Accordingly, we have also found that overexpression of L3MBTL1 is associated with an upregulation of BCL11A mRNA, suggesting that L3MBTL1 and BCL11A may function cooperatively to silence globin gene expression. Knockdown of L3MBTL1 also upregulated EKLF mRNA levels which could relate to the decreased BCL11A expression.

In summary our data demonstrate that knock-down of L3MBTL1 upregulates embryonic and fetal globin genes in cell contexts where they are usually silenced, indicating the functional importance of this Polycomb protein for repressing the globin gene locus. The clearance of L3MBTL1 and its associated histone mark (H4K20me2) during treatments that induce potent transcriptional activation of globin genes suggest that repression induced by L3MBTL1 is dynamic and may be involved in the fetal-to-adult globin switch. L3MBTL1 therefore emerges as a novel transcriptional repressor of fetal globin genes whose expression may be coordinated with that of BCL11A and EKLF. Understanding the role of L3MBTL1 and the H4K20 methylmark in globin gene switching offers the prospect of the targeted activation of HbF in erythroid cells of patients with hemoglobin disorders.