Changing the Guard at a Prostate Cancer SNP.

The molecular underpinnings of a prostate-cancer-associated SNP are investigated in a pair of papers in this issue of Cell. Together, Gao et al. and Hua et al. paint a picture of competition between transcription factors that, in turn, toggle the function of a regulatory region from a promoter to an enhancer and induce a switch in noncoding RNA isoforms.

Functional and mechanistic diversity of distal transcription enhancers.

Biological differences among metazoans and between cell types in a given organism arise in large part due to differences in gene expression patterns. Gene-distal enhancers are key contributors to these expression patterns, exhibiting both sequence diversity and cell type specificity. Studies of long-range interactions indicate that enhancers are often important determinants of nuclear organization, contributing to a general model for enhancer function that involves direct enhancer-promoter contact. However, mechanisms for enhancer function are emerging that do not fit solely within such a model, suggesting that enhancers as a class of DNA regulatory element may be functionally and mechanistically diverse.

Environmentally-defined enhancer populations regulate diversity of tissue-resident macrophages.

Macrophages represent a class of cells specialized for phagocytosis that occurs in multiple, phenotypically distinct populations throughout the body. Two studies published in Cell demonstrate that these phenotypic differences reflect drastic differences in the populations of enhancers that regulate transcription, and that this epigenomic diversity is, in fact, highly plastic and sensitive to environmental cues.

A transient definitive erythroid lineage with unique regulation of the ß-globin locus in the mammalian embryo.

A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult ß-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" ßH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with ßH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human ß-globin locus, like human fetal erythroblasts, express predominately human γ-, low ß-, and no ε-globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only ß-globin transcripts. We conclude that yolk sac-derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

An embryonic stage-specific enhancer within the murine ß-globin locus mediates domain-wide histone hyperacetylation.

In mammalian nuclei, a select number of tissue-specific gene loci exhibit broadly distributed patterns of histone modifications, such as histone hyperacetylation, that are normally associated with active gene promoters. Previously, we characterized such hyperacetylated domains within mammalian ß-globin gene loci, and determined that within the murine locus, neither the ß-globin locus control region nor the gene promoters were required for domain formation. Here, we identify a developmentally specific erythroid enhancer, hypersensitive site-embryonic 1 (HS-E1), located within the embryonic ß-globin domain in mouse, which is homologous to a region located downstream of the human embryonic ε-globin gene. This sequence exhibits nuclease hypersensitivity in primitive erythroid cells and acts as an enhancer in gain-of-function assays. Deletion of HS-E1 from the endogenous murine ß-globin locus results in significant decrease in the expression of the embryonic ß-globin genes and loss of the domain-wide pattern of histone hyperacetylation. The data suggest that HS-E1 is an enhancer that is uniquely required for ß-like globin expression in primitive erythroid cells, and that it defines a novel class of enhancer that works in part by domain-wide modulation of chromatin structure.

Enhancers: the abundance and function of regulatory sequences beyond promoters.

Transcriptional control in mammals and Drosophila is often mediated by regulatory sequences located far from gene promoters. Different classes of such elements - particularly enhancers, but also locus control regions and insulators - have been defined by specific functional assays, although it is not always clear how these assays relate to the function of these elements within their native loci. Recent advances in genomics suggest, however, that such elements are highly abundant within the genome and may represent the primary mechanism by which cell- and developmental-specific gene expression is accomplished. In this review, we discuss the functional parameters of enhancers as defined by specific assays, along with the frequency with which they occur in the genome. In addition, we examine the available evidence for the mechanism by which such elements communicate or interact with the promoters they regulate.

Histone hyperacetylation within the beta-globin locus is context-dependent and precedes high-level gene expression.

Active gene promoters are associated with covalent histone modifications, such as hyperacetylation, which can modulate chromatin structure and stabilize binding of transcription factors that recognize these modifications. At the beta-globin locus and several other loci, however, histone hyperacetylation extends beyond the promoter, over tens of kilobases; we term such patterns of histone modifications "hyperacetylated domains." Little is known of either the mechanism by which these domains form or their function. Here, we show that domain formation within the murine beta-globin locus occurs before either high-level gene expression or erythroid commitment. Analysis of beta-globin alleles harboring deletions of promoters or the locus control region demonstrates that these sequences are not required for domain formation, suggesting the existence of additional regulatory sequences within the locus. Deletion of embryonic globin gene promoters, however, resulted in the formation of a hyperacetylated domain over these genes in definitive erythroid cells, where they are otherwise inactive. Finally, sequences within beta-globin domains exhibit hyperacetylation in a context-dependent manner, and domains are maintained when transcriptional elongation is inhibited. These data narrow the range of possible mechanisms by which hyperacetylated domains form.

Histone H2A.X phosphorylation and Caspase-Initiated Chromatin Condensation in late-stage erythropoiesis.

BACKGROUND: Condensation of chromatin prior to enucleation is an essential component of terminal erythroid maturation, and defects in this process are associated with inefficient erythropoiesis and anemia. However, the mechanisms involved in this phenomenon are not well understood. Here, we describe a potential role for the histone variant H2A.X in erythropoiesis. RESULTS: We find in multiple model systems that this histone is essential for normal maturation, and that the loss of H2A.X in erythroid cells results in dysregulation in expression of erythroid-specific genes as well as a nuclear condensation defect. In addition, we demonstrate that erythroid maturation is characterized by phosphorylation at both S139 and Y142 on the C-terminal tail of H2A.X during late-stage erythropoiesis. Knockout of the kinase BAZ1B/WSTF results in loss of Y142 phosphorylation and a defect in nuclear condensation, but does not replicate extensive transcriptional changes to erythroid-specific genes observed in the absence of H2A.X. CONCLUSIONS: We relate these findings to Caspase-Initiated Chromatin Condensation (CICC) in terminal erythroid maturation, where aspects of the apoptotic pathway are invoked while apoptosis is specifically suppressed.

Hyperacetylated chromatin domains mark cell type-specific genes and suggest distinct modes of enhancer function.

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.

RelB is differentially regulated by IkappaB Kinase-alpha in B cells and mouse lung by cigarette smoke.

The activation of transcription factor NF-kappaB is controlled by two main pathways: the classical canonical (RelA/p65-p50)- and the alternative noncanonical (RelB/p52)-NF-kappaB pathways. RelB has been shown to play a protective role in RelA/p65-mediated proinflammatory cytokine release in immune-inflammatory lymphoid cells. Increased infiltration of macrophages and lymphoid cells occurs in lungs of patients with chronic obstructive pulmonary disease, leading to abnormal inflammation. We hypothesized that RelB, and its signaling pathway, is differentially regulated in macrophages and B cells and in lung cells, leading to differential regulation of proinflammatory cytokines in response to cigarette smoke (CS). CS exposure increased the levels of RelB and NF-kappaB-inducing kinase associated with recruitment of RelB on promoters of the IL-6 and macrophage inflammatory protein-2 genes in mouse lung. Treatment of macrophage cell line, MonoMac6, with CS extract showed activation of RelB. In contrast, RelB was degraded by a proteasome-dependent mechanism in B lymphocytes (human Ramos, mouse WEHI-231, and primary mouse spleen B cells), suggesting that RelB is differentially regulated in lung inflammatory and lymphoid cells in response to CS exposure. Transient transfection of dominant negative IkappaB-kinase-alpha and double mutants of NF-kappaB-inducing kinase partially attenuated the CS extract-mediated loss of RelB in B cells and normalized the increased RelB level in macrophages. Taken together, these data suggest that RelB is differentially regulated in response to CS exposure in macrophages, B cells, and in lung cells by IkappaB-kinase-alpha-dependent mechanism. Rapid degradation of RelB signals for RelA/p65 activation and loss of its protective ability to suppress the proinflammatory cytokine release in lymphoid B cells.

A spectrum of gene regulatory phenomena at mammalian beta-globin gene loci.

The beta-globin gene cluster in mammals, consisting of a set of erythroid-specific, developmentally activated, and (or) silenced genes, has long presented a model system for the investigation of gene regulation. As the number and complexity of models of gene activation and repression have expanded, so too has the complexity of phenomena associated with the regulation of the beta-globin genes. Models for expression from within the locus must account for local (promoter-proximal), distal (enhancer-mediated), and domain-wide components of the regulatory pathways that proceed through mammalian development and erythroid differentiation. In this review, we provide an overview of transcriptional activation, silencing, chromatin structure, and the function of distal regulatory elements involved in the normal developmental regulation of beta-globin gene expression.

Author Correction: Transcriptional Regulation on Aneuploid Chromosomes in Diverse Candida albicans Mutants.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

IKK alpha causes chromatin modification on pro-inflammatory genes by cigarette smoke in mouse lung.

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.

ChIPs of the beta-globin locus: unraveling gene regulation within an active domain.

Recent studies of beta-globin gene expression have concentrated on the analysis of factor binding and chromatin structure within the endogenous locus. These studies have more precisely defined the extent and nature of the active chromosomal domain and the elements that organize it. Surprisingly, the beta-globin locus control region (LCR), although critical for high-level gene expression, plays little role in the overall architecture of the active locus. Analysis of the effects of targeted deletion of the beta-globin LCR, along with emerging knowledge of the behavior of the erythroid transcription factor NF-E2, leads to a new perspective on factor binding and LCR function.

Transcriptional Regulation on Aneuploid Chromosomes in Divers Candida albicans Mutants.

Candida albicans is a diploid fungus and a predominant opportunistic human pathogen. Notably, C. albicans employs reversible chromosomal aneuploidies as a means of survival in adverse environments. We previously characterized transcription on the monosomic chromosome 5 (Ch5) that arises with adaptation to growth on the toxic sugar sorbose in the mutant Sor125(55). We now extend this analysis to the trisomic hybrid Ch4/7 within Sor125(55) and a diverse group of three mutants harboring a single Ch5. We find a similar pattern of transcriptional changes on either type of aneuploid chromosome within these mutants wherein expression of many genes follows chromosome ploidy, consistent with a direct mechanism to regulate genes important for adaptation to growth. In contrast, a significant number of genes are expressed at the disomic level, implying distinct mechanisms compensating for gene dose on monosomic or trisomic chromosomes consistent with maintaining cell homeostasis. Finally, we find evidence for an additional mechanism that elevates expression of genes on normal disomic Ch4 and Ch7 in mutants to levels commensurate with that found on the trisomic Ch4/7b in Sor125(55). Several of these genes are similarly differentially regulated among mutants, suggesting they play key functions in either maintaining aneuploidy or adaptation to growth conditions.

A complex chromatin landscape revealed by patterns of nuclease sensitivity and histone modification within the mouse beta-globin locus.

In order to create an extended map of chromatin features within a mammalian multigene locus, we have determined the extent of nuclease sensitivity and the pattern of histone modifications associated with the mouse beta-globin genes in adult erythroid tissue. We show that the nuclease-sensitive domain encompasses the beta-globin genes along with several flanking olfactory receptor genes that are inactive in erythroid cells. We describe enhancer-blocking or boundary elements on either side of the locus that are bound in vivo by the transcription factor CTCF, but we found that they do not coincide with transitions in nuclease sensitivity flanking the locus or with patterns of histone modifications within it. In addition, histone hyperacetylation and dimethylation of histone H3 K4 are not uniform features of the nuclease-sensitive mouse beta-globin domain but rather define distinct subdomains within it. Our results reveal a complex chromatin landscape for the active beta-globin locus and illustrate the complexity of broad structural changes that accompany gene activation.

NuA4 histone acetyltransferase activity is required for H4 acetylation on a dosage-compensated monosomic chromosome that confers resistance to fungal toxins.

BACKGROUND: The major human fungal pathogen Candida albicans possesses a diploid genome, but responds to growth in challenging environments by employing chromosome aneuploidy as an adaptation mechanism. For example, we have shown that C. albicans adapts to growth on the toxic sugar L-sorbose by transitioning to a state in which one chromosome (chromosome 5, Ch5) becomes monosomic. Moreover, analysis showed that while expression of many genes on the monosomic Ch5 is altered in accordance with the chromosome ploidy, expression of a large fraction of genes is increased to the normal diploid level, presumably compensating for gene dose. RESULTS: In order to understand the mechanism of the apparent dosage compensation, we now report genome-wide ChIP-microarray assays for a sorbose-resistant strain harboring a monosomic Ch5. These data show a significant chromosome-wide elevation in histone H4 acetylation on the mCh5, but not on any other chromosome. Importantly, strains lacking subunits of the NuA4 H4 histone acetyltransferase complex, orthologous to a complex previously shown in Drosophila to be associated with a similar gene dosage compensation mechanism, did not show an increase in H4 acetylation. Moreover, loss of NuA4 subunits severely compromised the adaptation to growth on sorbose. CONCLUSIONS: Our results are consistent with a model wherein chromosome-wide elevation of H4 acetylation mediated by the NuA4 complex plays a role in increasing gene expression in compensation for gene dose and adaption to growth in a toxic environment.

Correction to: NuA4 histone acetyltransferase activity is required for H4 acetylation on a dosage-compensated monosomic chromosome that confers resistance to fungal toxins.

After the publication of this work [1], it was noticed that an initial was missing from the author name: Jeffrey Hayes. His name should be written as: Jeffrey J. Hayes.

Expression of adducin genes during erythropoiesis: a novel erythroid promoter for ADD2.

OBJECTIVE: The first objective of this study was to examine the differences in levels of adducin (ADD1, ADD2, ADD3) mRNA expression during human erythropoiesis. The second objective was to determine whether the rapid induction of ADD2 expression could be attributed to a novel erythroid-specific promoter. METHODS: Expression of mRNA was quantified using real-time RT-PCR. Primary erythroid precursors were isolated from normal human bone marrow using fluorescence-activated cell sorting. Two model systems were compared: CD34(+) hematopoietic stem cells induced to differentiate with erythropoietin and HEL cells induced to differentiate with hemin. 5'RACE analysis was performed using primary human erythroblasts as starting material. RESULTS: All three adducin genes showed different patterns of expression during erythropoietic differentiation of cultured CD34(+) stem cells. Levels of ADD3 mRNA were higher than levels of ADD2 mRNA at early stages of erythropoiesis. Expression of ADD2 was induced to very high levels (100 times baseline) in erythropoietin-stimulated cultures. 5'RACE analysis identified a novel starting exon and putative erythroid promoter for ADD2. CONCLUSION: These results suggest that expression of each adducin gene is regulated in a gene-specific manner during erythropoiesis. The early expression of ADD3 suggests that it may have a role in erythroblasts but is replaced by ADD2 in later stages of erythropoiesis. The very high levels of expression of ADD2 suggest that its promoter may be useful for directing erythroid-specific gene expression.