Cyp1b1-mediated suppression of lymphoid progenitors in bone marrow by polycyclic aromatic hydrocarbons coordinately impacts spleen and thymus: a selective role for the Ah Receptor.

Bone marrow (BM) hematopoietic stem cells differentiate to common lymphoid progenitors (CLP) that emigrate to the thymus to form T cells or differentiate into immature B cells that then migrate to the spleen for maturation. Rapid in vivo suppression of BM progenitor cells by a single oral or intraperitoneal dose of 7,12-dimethylbenz(a)anthracene (DMBA) subsequently decreased mature lymphoid populations in BM, spleen, and thymus. These suppressions depended on BM CYP1B1, but not on aryl hydrocarbon receptor (AhR) activity. Suppression of pre-B colony formation at 6 h, correlated with subsequent decreases in mature BM, spleen, and thymus populations (48-168 h). Thymus T-cell ratios were unaffected, suggesting low local toxicity. DMBA treatment suppressed progenitor cells 24-h post treatment in wild type (WT), AhRb mice, but not in Cyp1b1-ko mice. The stem cell populations were sustained. Benzo(a)pyrene (BP) mediated a similar progenitor suppression up to 6 h, but reversal rapidly ensued. This recovery was absent in mice with a polycyclic aromatic hydrocarbon (PAH)-resistant, AhRd genotype. This AhR-dependent progenitor recovery with BP induction accounts for the absence of suppression of B220+ BM and spleen populations at 48-168 h. However, DMBA and BP produced similar profiles for thymus cell suppression, independent of AhR genotype. Thus, lymphoid progenitors may exit the BM to the thymus prior to the BP reversal. This progenitor recovery is associated with elevated chemokines and cytokines that depend on AhR-mediated induction of CYP1A1. This response increased constitutively in Cyp1b1-ko BM, demonstrating that CYP1B1 metabolizes local stimulants that impact a basal progenitor protection process.

Histone acetylation beyond promoters: long-range acetylation patterns in the chromatin world.

Histone acetylation is an important regulatory mechanism that controls transcription and diverse nuclear processes. While great progress has been made in understanding how localized acetylation and deacetylation control promoter activity, virtually nothing is known about the consequences of acetylation throughout entire chromosomal regions. An increasing number of genes have been found to reside in large chromatin domains that are controlled by regulatory elements many kilobases away. Recent studies have shown that broad histone acetylation patterns are hallmarks of chromatin domains. The purpose of this review is to discuss how such patterns are established and their implications for regulating gene expression.

Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain.

We have defined the histone acetylation pattern of the endogenous murine beta-globin domain, which contains the erythroidspecific beta-globin genes. The beta-globin locus control region (LCR) and transcriptionally active promoters were enriched in acetylated histones in fetal liver relative to fetal brain, whereas the inactive promoters were hypoacetylated. In contrast, the LCR and both active and inactive promoters were hyperacetylated in yolk sac. Hypersensitive site two of the LCR was also hyperacetylated in murine embryonic stem cells, whereas beta-globin promoters were hypoacetylated. Thus, the acetylation pattern varied at different developmental stages. Histone deacetylase inhibition selectively increased acetylation at a hypoacetylated promoter in fetal liver, suggesting that active deacetylation contributes to silencing of promoters. We propose that dynamic histone acetylation and deacetylation play an important role in the developmental control of beta-globin gene expression.

Direct interaction of NF-E2 with hypersensitive site 2 of the beta-globin locus control region in living cells.

The human beta-globin locus control region (LCR) confers high-level, tissue-specific expression to the beta-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for beta-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in alpha- and beta-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of beta-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate beta-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of beta-globin gene expression through activation of the LCR.

Requirement of an E1A-sensitive coactivator for long-range transactivation by the beta-globin locus control region.

Four erythroid-specific DNase I-hypersensitive sites at the 5'-end of the beta-globin locus confer high-level transcription to the beta-globin genes. To identify coactivators that mediate long-range transactivation by this locus control region (LCR), we assessed the influence of E1A, an inhibitor of the CBP/p300 histone acetylase, on LCR function. E1A strongly inhibited transactivation of Agamma- and beta-globin promoters by the HS2, HS2-HS3, and HS1-HS4 subregions of the LCR in human K562 and mouse erythroleukemia cells. Short- and long-range transactivation mediated by the LCR were equally sensitive to E1A. The E1A sensitivity was apparent in transient and stable transfection assays, and E1A inhibited expression of the endogenous gamma-globin genes. Only sites for NF-E2 within HS2 were required for E1A sensitivity in K562 cells, and E1A abolished transactivation mediated by the activation domain of NF-E2. E1A mutants defective in CBP/p300 binding only weakly inhibited HS2-mediated transactivation, whereas a mutant defective in retinoblastoma protein binding strongly inhibited transactivation. Expression of CBP/p300 potentiated HS2-mediated transactivation. Moreover, expression of GAL4-CBP strongly increased transactivation of a reporter containing HS2 with a GAL4 site substituted for the NF-E2 sites. Thus, we propose that a CBP/p300-containing coactivator complex is the E1A-sensitive factor important for LCR function.

Enhancement of beta-globin locus control region-mediated transactivation by mitogen-activated protein kinases through stochastic and graded mechanisms.

Activation of the mitogen-activated protein kinase (MAPK) pathway enhances long-range transactivation by the beta-globin locus control region (LCR) (W. K. Versaw, V. Blank, N. M. Andrews, and E. H. Bresnick, Proc. Natl. Acad. Sci. USA 95:8756-8760, 1998). The enhancement requires tandem recognition sites for the hematopoietic transcription factor NF-E2 within the hypersensitive site 2 (HS2) subregion of the LCR. To distinguish between mechanisms of induction involving the activation of silent promoters or the increased efficacy of active promoters, we analyzed basal and MAPK-stimulated HS2 enhancer activity in single, living cells. K562 erythroleukemia cells stably transfected with constructs containing the human Agamma-globin promoter linked to an enhanced green fluorescent protein (EGFP) reporter, with or without HS2, were analyzed for EGFP expression by flow cytometry. When most cells in a population expressed EGFP, MAPK augmented the activity of active promoters. However, under conditions of silencing, in which cells reverted to a state with no measurable EGFP expression, MAPK activated silent promoters. Furthermore, studies of populations of EGFP-expressing and non-EGFP-expressing cells isolated by flow cytometry showed that MAPK activation converted nonexpressing cells into expressing cells and increased expression in expressing cells. These results support a model in which MAPK elicits both graded and stochastic responses to increase HS2-mediated transactivation from single chromatin templates.

Physical and functional interactions between the transactivation domain of the hematopoietic transcription factor NF-E2 and WW domains.

Tandem binding sites for the hematopoietic transcription factor NF-E2 in the beta-globin locus control region activate high-level beta-globin gene expression in transgenic mice. NF-E2 is a heterodimer consisting of a hematopoietic subunit p45 and a ubiquitous subunit p18. Gavva et al. [Gavva, N. R., Gavva, R., Ermekova, K., Sudol, M., and Shen, J. C. (1997) J. Biol. Chem. 272, 24105-24108] reported that human p45 contains a PPXY motif that binds WW domains. We show that murine NF-E2, which contains two PPXY motifs (PPXY-1 and -2) within its transactivation domain, differentially interacted with nine GST-WW domain fusion proteins. Quantitative analysis revealed high-affinity binding (KD = 5.7 nM) of p45 to a WW domain from a novel human ubiquitin ligase homologue (WWP1) expressed in hematopoietic tissues. The amino-terminal WW domain of WWP1 formed a multimeric complex with DNA-bound NF-E2. A WWP1 ligand peptide, isolated by phage display, and a peptide spanning PPXY-1 inhibited p45 binding, whereas an SH3 domain-interacting peptide and a peptide spanning PPXY-2 did not. Mutation of PPXY-1, but not PPXY-2, inhibited the transactivation function of NF-E2, providing support for the hypothesis that WW domain interactions are important for NF-E2-mediated transactivation.

Human histone acetyltransferase GCN5 exists in a stable macromolecular complex lacking the adapter ADA2.

Acetylation of core histones is an important regulatory step in transcriptional activation from chromatin templates. The yeast transcriptional coactivator protein GCN5 was recently shown to be a nuclear histone acetyltransferase (HAT). Genetic and biochemical studies in yeast suggest that GCN5 functions with the adapter proteins ADA1, ADA2, ADA3, and ADA5 in a heteromeric complex. We have established conditions for chromatographic fractionation of HATs and ADA2 from human K562 erythroleukemia cells. Gel-filtration chromatography revealed two populations of GCN5 with Stokes' radii of 67 and 33 A, consistent with a large macromolecular complex and a monomer, respectively. The GCN5-related HAT, PCAF, was resolved as a stable complex with a Stokes' radius of 74 A. The HAT complexes were resistant to 0.3 M NaCl and DNase I. ADA2 was characterized by a Stokes' radius of 35 A, consistent with a monomer. Thus, in contrast to the stable GCN5-adapter complex in yeast, human GCN5 and ADA2 are not stably associated with each other. The implications of this result are discussed vis-a-vis the mechanism of recruitment of GCN5 to regulatory regions of genes.