PRDI-BF1, the human ortholog of mouse Blimp-1, is a DNA-binding protein involved in postinduction repression of interferon-beta gene transcription in response to viral infection. PRDI-BF1 also has an essential function in driving terminal differentiation of B lymphocytes and therein silences multiple genes. Here we show PRDI-BF1 assembles silent chromatin over the interferon-beta promoter in the osteosarcoma cell line U2OS through recruitment of the histone H3 lysine methyltransferase G9a. G9a is recruited only when in a complex with PRDI-BF1. G9a catalytic activity is required for the accumulation of methylated histone H3 and transcriptional silencing mediated by PRDI-BF1 in vivo. This establishes a mechanism for the recruitment of G9a, the main mammalian euchromatic methyltransferase, and defines nonembryonic targets of G9a.

The transcription factor early B cell factor-1 (Ebf1) is a key determinant of B lineage specification and differentiation. To gain insight into the molecular basis of Ebf1 function in early-stage B cells, we combined a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Among 565 genes that are occupied and transcriptionally regulated by Ebf1, we identified large sets involved in (pre)-B cell receptor and Akt signaling, cell adhesion, and migration. Interestingly, a third of previously described Pax5 targets was found to be occupied by Ebf1. In addition to Ebf1-activated and -repressed genes, we identified targets at which Ebf1 induces chromatin changes that poise the genes for expression at subsequent stages of differentiation. Poised chromatin states on specific targets could also be established by Ebf1 expression in T cells but not in NIH 3T3 cells, suggesting that Ebf1 acts as a "pioneer" factor in a hematopoietic chromatin context.

Early differentiation of B lymphocytes requires the function of multiple transcription factors that regulate the specification and commitment of the lineage. Loss-and gain-of-function experiments have provided important insight into the transcriptional control of B lymphopoiesis, whereby E2A was suggested to act upstream of EBF1 and Pax5 downstream of EBF1. However, this simple hierarchy cannot account for all observations, and our understanding of a presumed regulatory network, in which transcription factors and signaling pathways operate, is limited. Here, we show that the expression of the Ebf1 gene involves two promoters that are differentially regulated and generate distinct protein isoforms. We find that interleukin-7 signaling, E2A, and EBF1 activate the distal Ebf1 promoter, whereas Pax5, together with Ets1 and Pu.1, regulates the stronger proximal promoter. In the absence of Pax5, the function of the proximal Ebf1 promoter and accumulation of EBF1 protein are impaired and the replication timing and subcellular localization of the Ebf1 locus are altered. Taken together, these data suggest that the regulation of Ebf1 via distinct promoters allows for the generation of several feedback loops and the coordination of multiple determinants of B lymphopoiesis in a regulatory network.

The transcription factors EBF1 and Pax5 have been linked to activation of the B cell lineage program and irreversible loss of alternative lineage potential (commitment), respectively. Here we conditionally deleted Ebf1 in committed pro-B cells after transfer into alymphoid mice. We found that those cells converted into innate lymphoid cells (ILCs) and T cells with variable-diversity-joining (VDJ) rearrangements of loci encoding both B cell and T cell antigen receptors. As intermediates in lineage conversion, Ebf1-deficient CD19(+) cells expressing Pax5 and transcriptional regulators of the ILC and T cell fates were detectable. In particular, genes encoding the transcription factors Id2 and TCF-1 were bound and repressed by EBF1. Thus, both EBF1 and Pax5 are required for B lineage commitment by repressing distinct and common determinants of alternative cell fates.

The transcription factor Ebf1 is an important determinant of early B lymphopoiesis. To gain insight into the functions of Ebf1 at distinct stages of differentiation, we conditionally inactivated Ebf1. We found that Ebf1 is

Methylation of specific residues within the N-terminal histone tails plays a critical role in regulating eukaryotic gene expression. Although great advances have been made toward identifying histone methyltransferases (HMTs) and elucidating the consequences of histone methylation, little is known about the recruitment of HMTs to regulatory regions of chromatin. Here we report that the sequence-specific DNA-binding transcription factor Yin Yang 1 (YY1) binds to and recruits the histone H4 (Arg 3)-specific methyltransferase, PRMT1, to a YY1-activated promoter. Our data confirm that histone methylation does not occur randomly but rather is a targeted event and provides one mechanism by which HMTs can be recruited to chromatin to activate gene expression.

Significance Macrophages—cells crucially involved in defense against infections—exhibit, depending on their anatomical location, distinct biological properties. Studies of the underlying mechanisms are of scientific and clinical interest, but are hampered by the difficulty of obtaining primary tissue macrophages in sufficient numbers and purity. Here, we report the generation of nontransformed murine macrophages, which are similar to alveolar macrophages and can be grown continuously without change of phenotype and in unlimited amounts. Such macrophages helped us to recognize several innate immune properties of alveolar macrophages that are involved in the pathogenesis of infectious lung inflammation.

B cell differentiation into a plasma cell requires expression of the positive regulatory domain zinc finger protein 1 gene (PRDM1) that encodes the positive regulatory domain I binding factor 1 (PRDI-BF1 or Blimp-1) protein. It represses the transcription of specific target genes, including c-myc, the MHC class II trans-activator, Pax-5, and CD23b. In this study we demonstrate the presence of an alternative protein product of the PRDM1 gene. The new protein, PRDI-BF1β, has a disrupted PR domain and lacks the amino-terminal 101 aa of the originally described protein. PRDI-BF1β has a dramatic loss of repressive function on multiple target genes, but maintains normal DNA-binding activity, nuclear localization, and association with histone deacetylases and deacetylase activity. Myeloma cell lines express the highest levels of PRDM1β mRNA relative to the full-length form, while primary cells and several other cell lines have very low, but detectable, levels of PRDM1β. RNA analysis and analysis of the PRDM1 promoters demonstrate that PRDI-BF1β is generated from the same gene by alternative transcription initiation using an internal promoter. These newly described features of the PRDM1 gene are highly analogous to the PRDM2 (RIZ) and PRDM3 (MDS1-EVI1) genes, in which each express a truncated protein missing the PR domain. The expression of each of the truncated proteins is elevated in cancerous cells and may play an important role in the disease.