Protein arginine methyltransferase 6 controls erythroid gene expression and differentiation of human CD34+ progenitor cells.

Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.

PRMT6 activates cyclin D1 expression in conjunction with the transcription factor LEF1.

The establishment of cell type specific gene expression by transcription factors and their epigenetic cofactors is central for cell fate decisions. Protein arginine methyltransferase 6 (PRMT6) is an epigenetic regulator of gene expression mainly through methylating arginines at histone H3. This way it influences cellular differentiation and proliferation. PRMT6 lacks DNA-binding capability but is recruited by transcription factors to regulate gene expression. However, currently only a limited number of transcription factors have been identified, which facilitate recruitment of PRMT6 to key cell cycle related target genes. Here, we show that LEF1 contributes to the recruitment of PRMT6 to the central cell cycle regulator CCND1 (Cyclin D1). We identified LEF1 as an interaction partner of PRMT6. Knockdown of LEF1 or PRMT6 reduces CCND1 expression. This is in line with our observation that knockdown of PRMT6 increases the number of cells in G1 phase of the cell cycle and decreases proliferation. These results improve the understanding of PRMT6 activity in cell cycle regulation. We expect that these insights will foster the rational development and usage of specific PRMT6 inhibitors for cancer therapy.

The transcription factor TAL1 and miR-17-92 create a regulatory loop in hematopoiesis.

A network of gene regulatory factors such as transcription factors and microRNAs establish and maintain gene expression patterns during hematopoiesis. In this network, transcription factors regulate each other and are involved in regulatory loops with microRNAs. The microRNA cluster miR-17-92 is located within the MIR17HG gene and encodes six mature microRNAs. It is important for hematopoietic differentiation and plays a central role in malignant disease. However, the transcription factors downstream of miR-17-92 are largely elusive and the transcriptional regulation of miR-17-92 is not fully understood. Here we show that miR-17-92 forms a regulatory loop with the transcription factor TAL1. The miR-17-92 cluster inhibits expression of TAL1 and indirectly leads to decreased stability of the TAL1 transcriptional complex. We found that TAL1 and its heterodimerization partner E47 regulate miR-17-92 transcriptionally. Furthermore, miR-17-92 negatively influences erythroid differentiation, a process that depends on gene activation by the TAL1 complex. Our data give example of how transcription factor activity is fine-tuned during normal hematopoiesis. We postulate that disturbance of the regulatory loop between TAL1 and the miR-17-92 cluster could be an important step in cancer development and progression.

FUSE binding protein 1 (FUBP1) expression is upregulated by T-cell acute lymphocytic leukemia protein 1 (TAL1) and required for efficient erythroid differentiation.

During erythropoiesis, haematopoietic stem cells (HSCs) differentiate in successive steps of commitment and specification to mature erythrocytes. This differentiation process is controlled by transcription factors that establish stage- and cell type-specific gene expression. In this study, we demonstrate that FUSE binding protein 1 (FUBP1), a transcriptional regulator important for HSC self-renewal and survival, is regulated by T-cell acute lymphocytic leukaemia 1 (TAL1) in erythroid progenitor cells. TAL1 directly activates the FUBP1 promoter, leading to increased FUBP1 expression during erythroid differentiation. The binding of TAL1 to the FUBP1 promoter is highly dependent on an intact GATA sequence in a combined E-box/GATA motif. We found that FUBP1 expression is required for efficient erythropoiesis, as FUBP1-deficient progenitor cells were limited in their potential of erythroid differentiation. Thus, the finding of an interconnection between GATA1/TAL1 and FUBP1 reveals a molecular mechanism that is part of the switch from progenitor- to erythrocyte-specific gene expression. In summary, we identified a TAL1/FUBP1 transcriptional relationship, whose physiological function in haematopoiesis is connected to proper erythropoiesis.

Hematopoietic transcription factors and differential cofactor binding regulate PRKACB isoform expression.

Hematopoietic differentiation is controlled by key transcription factors, which regulate stem cell functions and differentiation. TAL1 is a central transcription factor for hematopoietic stem cell development in the embryo and for gene regulation during erythroid/megakaryocytic differentiation. Knowledge of the target genes controlled by a given transcription factor is important to understand its contribution to normal development and disease. To uncover direct target genes of TAL1 we used high affinity streptavidin/biotin-based chromatin precipitation (Strep-CP) followed by Strep-CP on ChIP analysis using ChIP promoter arrays. We identified 451 TAL1 target genes in K562 cells. Furthermore, we analysed the regulation of one of these genes, the catalytic subunit beta of protein kinase A (PRKACB), during megakaryopoiesis of K562 and primary human CD34+ stem cell/progenitor cells. We found that TAL1 together with hematopoietic transcription factors RUNX1 and GATA1 binds to the promoter of the isoform 3 of PRKACB (Cß3). During megakaryocytic differentiation a coactivator complex on the Cß3 promoter, which includes WDR5 and p300, is replaced with a corepressor complex. In this manner, activating chromatin modifications are removed and expression of the PRKACB-Cß3 isoform during megakaryocytic differentiation is reduced. Our data uncover a role of the TAL1 complex in controlling differential isoform expression of PRKACB. These results reveal a novel function of TAL1, RUNX1 and GATA1 in the transcriptional control of protein kinase A activity, with implications for cellular signalling control during differentiation and disease.