BAP1 regulation of the key adaptor protein NCoR1 is critical for γ-globin gene repression.

Human globin gene production transcriptionally "switches" from fetal to adult synthesis shortly after birth and is controlled by macromolecular complexes that enhance or suppress transcription by cis elements scattered throughout the locus. The DRED (direct repeat erythroid-definitive) repressor is recruited to the ε-globin and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA-binding and epigenetic enzyme components (e.g., DNA methyltransferase 1 [DNMT1] and lysine-specific demethylase 1 [LSD1]) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: The deubiquitinase BRCA1-associated protein-1 (BAP1) is a component of the repressor complex whose activity maintains NCoR1 at sites in the ß-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.

The orphan nuclear receptor TR4 regulates erythroid cell proliferation and maturation.

The orphan nuclear receptors TR4 (NR2C2) and TR2 (NR2C1) are the DNA-binding subunits of the macromolecular complex, direct repeat erythroid-definitive, which has been shown to repress ε- and γ-globin transcription during adult definitive erythropoiesis. Previous studies implied that TR2 and TR4 act largely in a redundant manner during erythroid differentiation; however, during the course of routine genetic studies, we observed multiple variably penetrant phenotypes in the Tr4 mutants, suggesting that indirect effects of the mutation might be masked by multiple modifying genes. To test this hypothesis, Tr4+/- mutant mice were bred into a congenic C57BL/6 background and their phenotypes were reexamined. Surprisingly, we found that homozygous Tr4 null mutant mice expired early during embryogenesis, around embryonic day 7.0, and well before erythropoiesis commences. We further found that Tr4+/- erythroid cells failed to fully differentiate and exhibited diminished proliferative capacity. Analysis of Tr4+/- mutant erythroid cells revealed that reduced TR4 abundance resulted in decreased expression of genes required for heme biosynthesis and erythroid differentiation (Alad and Alas2), but led to significantly increased expression of the proliferation inhibitory factor, cyclin dependent kinase inhibitor (Cdkn1c) These studies support a vital role for TR4 in promoting erythroid maturation and proliferation, and demonstrate that TR4 and TR2 execute distinct, individual functions during embryogenesis and erythroid differentiation.

Genome-wide Twist1 occupancy in endocardial cushion cells, embryonic limb buds, and peripheral nerve sheath tumor cells.

BACKGROUND: The basic helix-loop-helix transcription factor Twist1 has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions (ECC) and limb buds, and also in cancer. Whether Twist1 regulates the same transcriptional targets in different tissue types is largely unknown. RESULTS: The tissue-specificity of Twist1 genomic occupancy was examined in mouse ECCs, limb buds, and peripheral nerve sheath tumor (PNST) cells using chromatin immunoprecipitation followed by sequencing (Chip-seq) analysis. Consistent with known Twist1 functions during development and in cancer cells, Twist1-DNA binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. However, the vast majority of Twist1 binding regions were specific to individual tissue types. Thus, while Twist1 has similar functions in ECCs, limb buds, and PNST cells, the specific genomic sequences occupied by Twist1 were different depending on cellular context. Subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ECC, limb buds and PNST cells. Twist1 genomic occupancy was detected for six binding regions in all tissue types, and Twist1-binding sequences associated with Chst11, Litaf, Ror2, and Spata5 also bound the potential Twist1 cofactor RREB1. Pathway analysis of the genes associated with Twist1 binding suggests that Twist1 may regulate genes associated with the Wnt signaling pathway in ECCs and limb buds. CONCLUSIONS: Together, these data indicate that Twist1 interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. In addition, there is a small subset of genes occupied by Twist1 in all three tissues that may represent a core group of Twist1 target genes in multiple cell types.

Twist1 directly regulates genes that promote cell proliferation and migration in developing heart valves.

Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC) valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive extracellular matrix (ECM) molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs) containing E-box consensus sequences that are potential Twist1 binding sites. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation (ChIP) assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo, which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1-responsive regulatory sequences.