Histone modifications play an important role in the process of transcription. However, in contrast to lysine methylation, the role of arginine methylation in chromatin structure and transcription has been underexplored. The globin genes are regulated by a highly organized chromatin structure that juxtaposes the locus control region (LCR) with downstream globin genes. We report here that the targeted recruitment of asymmetric dimethyl H4R3 catalyzed by PRMT1 (protein arginine methyltransferase 1) facilitates histone H3 acetylation on Lys9/Lys14. Dimethyl H4R3 provides a binding surface for P300/ CBP-associated factor (PCAF) and directly enhances histone H3 acetylation in vitro. We show that these active modifications are essential for efficient interactions between the LCR and the ␤ maj -promoter as well as transcription of the ␤-globin gene. Furthermore, knockdown (KD) of PRMT1 by RNA interference in erythroid progenitor cells prevents histone acetylation, enhancer and promoter interaction, and recruitment of transcription complexes to the active ␤-globin promoter. Reintroducing rat PRMT1 into the PRMT1 KD MEL cells rescues PRMT1 binding, ␤-globin transcription, and erythroid differentiation. Taken together, our data suggest that PRMT1-mediated dimethyl H4R3 facilitates histone acetylation and enhancer/ promoter communications, which lead to the efficient recruitment of transcription preinitiation complexes to active promoters. (Blood. 2010;115:2028-2037 IntroductionCovalent modifications of N-terminal histone tails are critically involved in transcriptional activation and repression. 1 The interplay between individual modifications may exert distinct regulatory effects on different gene loci during development and cellular differentiation. For example, H3K9 and H3K27 methylations are generally linked to gene repression, whereas methylation of H3K4 correlates with transcriptionally active euchromatin. 2 However, in the ␤-globin locus H3K9 methylation was also detected in the active globin genes. 3 Arginine methylation of histones is associated with both transcriptional repression and activation. 4 PRMT6-mediated H3R2 dimethylation negatively regulates deposition of H3K4 trimethylation at active promoters, 5 whereas dimethyl H4R3 correlates with transcriptional activation. 6,7 Asymmetric dimethylation of H4R3 residues by protein arginine methyltransferase PRMT1 is essential in vitro and in vivo for the establishment or maintenance of active histone acetylation patterns. 7,8 The interdependence of these modifications appears to be important for the transcription of a p53-dependent reporter gene in a cell-free system with reconstituted chromatin templates. 9 Furthermore, PRMT1 was associated with the activation of HNF4 and HoxA9 genes during tissue development and oncogenesis, respectively. 10, 11 We showed recently that PRMT1 directly interacts with transcription factor USF1 (upstream regulatory factor 1), which has been implicated in chromatin barrier function and ␤-globin gene regulation. [12][13][14][15] The ...

The interplay between polycomb and trithorax complexes has been implicated in embryonic stem cell (ESC) self-renewal and differentiation. It has been shown recently that WRD5 and Dpy-30, specific components of the SET1/MLL protein complexes, play important roles during ESC self-renewal and differentiation of neural lineages. However, not much is known about how and where specific trithorax complexes are targeted to genes involved in self-renewal or lineage-specification. Here, we report that the recruitment of the hSET1A histone H3K4 methyltransferase (HMT) complex by transcription factor USF1 is required for mesoderm specification and lineage differentiation. In undifferentiated ESCs, USF1 maintains hematopoietic stem/progenitor cell (HS/PC) associated bivalent chromatin domains and differentiation potential. Furthermore, USF1 directed recruitment of the hSET1A complex to the HoxB4 promoter governs the transcriptional activation of HoxB4 gene and regulates the formation of early hematopoietic cell populations. Disruption of USF or hSET1A function by overexpression of a dominant-negative AUSF1 mutant or by RNA-interference-mediated knockdown, respectively, led to reduced expression of mesoderm markers and inhibition of lineage differentiation. We show that USF1 and hSET1A together regulate H3K4me3 modifications and transcription preinitiation complex assembly at the hematopoietic-associated HoxB4 gene during differentiation. Finally, ectopic expression of USF1 in ESCs promotes mesoderm differentiation and enforces the endothelial-to-hematopoietic transition by inducing hematopoietic-associated transcription factors, HoxB4 and TAL1. Taken together, our findings reveal that the guided-recruitment of the hSET1A histone methyltransferase complex and its H3K4 methyltransferase activity by transcription regulator USF1 safeguards hematopoietic transcription programs and enhances mesoderm/hematopoietic differentiation.

Rhabdoid sarcomas are highly malignant tumors that usually occur in young children. A key to the genesis of this tumor is the mutational loss of the BAF47 gene as well as the widespread epigenetic suppression of other key anticancer genes. The BRM gene is one such epigenetically silenced gene in Rhabdoid tumors. This gene codes for an ATPase catalytic subunit that shifts histones and opens the chromatin. We show that BRM is an epigenetically silenced gene in 10/11 Rhabdoid cell lines and in 70% of Rhabdoid tumors. Moreover, BRM can be induced by BAF47 re-expression and by Flavopiridol. By selective shRNAi knockdown of BRM, we show that BRM re-expression is necessary for growth inhibition by BAF47 re-expression or Flavopiridol application. Similar to lung cancer cell lines, we found that HDAC3, HDAC9, MEF2D and GATA3 controlled BRM silencing and that HDAC9 was overexpressed in Rhabdoid cancer cell lines. In primary BRM-deficient Rhabdoid tumors, HDAC9 was also found to be highly overexpressed. Two insertional BRM promoter polymorphisms contribute to BRM silencing, but only the -1321 polymorphism correlated with BRM silencing in Rhabdoid cell lines. To determine how these polymorphisms were tied to BRM silencing, we conducted ChIP assays and found that both HDAC9 and MEF2D bound to the BRM promoter at or near these polymorphic sites. Using BRM promoter swap experiments, we indirectly showed that both HDAC9 and MEF2D bound to these polymorphic sites. Together, these data show that the mechanism of BRM silencing contributes to the pathogenesis of Rhabdoid tumors and appears to be conserved among tumor types.

2Helix-loop-helix (HLH) proteins play a profound role in the process of development and cellular differentiation. Among the HLH proteins expressed in differentiating erythroid cells are the ubiquitous proteins Myc, USF1, USF2, and TFII-I, as well as the hematopoiesis-specific transcription factor Tal1/SCL. All of these HLH proteins exhibit distinct functions during the differentiation of erythroid cells. For example, Myc stimulates the proliferation of erythroid progenitor cells, while the USF proteins and Tal1 regulate genes that specify the differentiated phenotype. This minireview summarizes the known activities of Myc, USF, TFII-I, and Tal11/ SCL and discusses how they may function sequentially, cooperatively, or antagonistically in regulating expression programs during the differentiation of erythroid cells.Adult erythroid cells differentiate from hematopoietic stem cells (HSCs) through a cascade of steps (18,132). The most primitive HSC is called a long-term HSC (LT-HSC) for its ability to reconstitute HSCs in the bone marrow of irradiated mice over a long period of time. These slowly dividing cells are attached to a niche in the bone marrow and give rise to shortterm HSCs, which then differentiate into common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs). The CMPs go on to differentiate into granulocyte/monocyte precursors (GMPs) or into megakaryocyte/erythroid cell precursors (MEPs). MEPs further differentiate into erythropoietin-responsive BFU-E (blast-forming unit-erythroid) and then CFU-E (CFU-erythroid). The CFU-E cells differentiate to form orthochromatic normoblasts, then reticulocytes, and finally enucleated mature erythrocytes (125). The process of erythropoiesis has been extensively studied in vitro and in vivo and led to the identification of key erythroid cell transcription factors that regulate gene expression programs at the various steps of differentiation. The availability of erythroid cells representing different stages of maturation has rendered this system ideal for studying gene regulatory mechanisms.Transcription factors are classified based on the presence of specific protein-protein and protein-DNA interaction motifs which allow them to regulate gene expression by binding to DNA in a sequence-specific manner and to recruit coregulator complexes (98). The class of helix-loop-helix (HLH) transcription factors encompasses many proteins that play important roles during development and differentiation (89). The HLH motif is a characteristic dimerization domain which is accompanied by a basic (b) DNA-binding domain. Some HLH proteins contain an additional leucine zipper (ZIP) protein interaction module; these proteins are referred to as bHLHZIP proteins (89). Erythroid cells express many different HLH proteins. Here, we will review the well-characterized proteins USF1, USF2, Myc, TFII-I, and Tal1/SCL but will also discuss how inhibitor of DNA binding (ID) proteins, which only contain the HLH domain, may interfere with the function of HLH transcription factors in erythro...