UpSET recruits HDAC complexes and restricts chromatin accessibility and acetylation at promoter regions.

Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.

Something silent this way forms: the functional organization of the repressive nuclear compartment.

The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization.

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.

The nucleus inside out--through a rod darkly.

In the nuclei of eukaryotic cells, euchromatin is located at the center, whereas heterochromatin is found at the periphery and is interspersed in the nucleoplasm. Solovei et al. (2009) now reveal that this normal pattern is reversed in the retinal rod cells of mice. This inversion might serve to maximize light transmission to photoreceptors in nocturnal mammals.

Conservation of trans-acting circuitry during mammalian regulatory evolution.

The basic body plan and major physiological axes have been highly conserved during mammalian evolution, yet only a small fraction of the human genome sequence appears to be subject to evolutionary constraint. To quantify cis- versus trans-acting contributions to mammalian regulatory evolution, we performed genomic DNase I footprinting of the mouse genome across 25 cell and tissue types, collectively defining ∼8.6 million transcription factor (TF) occupancy sites at nucleotide resolution. Here we show that mouse TF footprints conjointly encode a regulatory lexicon that is ∼95% similar with that derived from human TF footprints. However, only ∼20% of mouse TF footprints have human orthologues. Despite substantial turnover of the cis-regulatory landscape, nearly half of all pairwise regulatory interactions connecting mouse TF genes have been maintained in orthologous human cell types through evolutionary innovation of TF recognition sequences. Furthermore, the higher-level organization of mouse TF-to-TF connections into cellular network architectures is nearly identical with human. Our results indicate that evolutionary selection on mammalian gene regulation is targeted chiefly at the level of trans-regulatory circuitry, enabling and potentiating cis-regulatory plasticity.

A comparative encyclopedia of DNA elements in the mouse genome.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

An expansive human regulatory lexicon encoded in transcription factor footprints.

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis-regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein-DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

Olfactory receptor genes expressed in distinct lineages are sequestered in different nuclear compartments.

The olfactory system translates a vast array of volatile chemicals into diverse odor perceptions and innate behaviors. Odor detection in the mouse nose is mediated by 1,000 different odorant receptors (ORs) and 14 trace amine-associated receptors (TAARs). ORs are used in a combinatorial manner to encode the unique identities of myriad odorants. However, some TAARs appear to be linked to innate responses, raising questions about regulatory mechanisms that might segregate OR and TAAR expression in appropriate subsets of olfactory sensory neurons (OSNs). Here, we report that OSNs that express TAARs comprise at least two subsets that are biased to express TAARs rather than ORs. The two subsets are further biased in Taar gene choice and their distribution within the sensory epithelium, with each subset preferentially expressing a subgroup of Taar genes within a particular spatial domain in the epithelium. Our studies reveal one mechanism that may regulate the segregation of Olfr (OR) and Taar expression in different OSNs: the sequestration of Olfr and Taar genes in different nuclear compartments. Although most Olfr genes colocalize near large central heterochromatin aggregates in the OSN nucleus, Taar genes are located primarily at the nuclear periphery, coincident with a thin rim of heterochromatin. Taar-expressing OSNs show a shift of one Taar allele away from the nuclear periphery. Furthermore, examination of hemizygous mice with a single Taar allele suggests that the activation of a Taar gene is accompanied by an escape from the peripheral repressive heterochromatin environment to a more permissive interior chromatin environment.

Molecular basis for chromatin binding and regulation of MLL5.

The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His-Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.

Developmental and species-divergent globin switching are driven by BCL11A.

The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian beta-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human beta-globin locus, consisting of the linked embryonic (epsilon), fetal (gamma) and adult (beta) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human gamma-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human gamma-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human gamma-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of beta-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.

A functional element necessary for fetal hemoglobin silencing.

BACKGROUND: An improved understanding of the regulation of the fetal hemoglobin genes holds promise for the development of targeted therapeutic approaches for fetal hemoglobin induction in the ß-hemoglobinopathies. Although recent studies have uncovered trans-acting factors necessary for this regulation, limited insight has been gained into the cis-regulatory elements involved. METHODS: We identified three families with unusual patterns of hemoglobin expression, suggestive of deletions in the locus of the ß-globin gene (ß-globin locus). We performed array comparative genomic hybridization to map these deletions and confirmed breakpoints by means of polymerase-chain-reaction assays and DNA sequencing. We compared these deletions, along with previously mapped deletions, and studied the trans-acting factors binding to these sites in the ß-globin locus by using chromatin immunoprecipitation. RESULTS: We found a new (δß)(0)-thalassemia deletion and a rare hereditary persistence of fetal hemoglobin deletion with identical downstream breakpoints. Comparison of the two deletions resulted in the identification of a small intergenic region required for γ-globin (fetal hemoglobin) gene silencing. We mapped a Kurdish ß(0)-thalassemia deletion, which retains the required intergenic region, deletes other surrounding sequences, and maintains fetal hemoglobin silencing. By comparing these deletions and other previously mapped deletions, we elucidated a 3.5-kb intergenic region near the 5' end of the δ-globin gene that is necessary for γ-globin silencing. We found that a critical fetal hemoglobin silencing factor, BCL11A, and its partners bind within this region in the chromatin of adult erythroid cells. CONCLUSIONS: By studying three families with unusual deletions in the ß-globin locus, we identified an intergenic region near the δ-globin gene that is necessary for fetal hemoglobin silencing. (Funded by the National Institutes of Health and others.).

The hypersensitive sites of the murine ß-globin locus control region act independently to affect nuclear localization and transcriptional elongation.

The ß-globin locus control region (LCR) is necessary for high-level ß-globin gene transcription and differentiation-dependent relocation of the ß-globin locus from the nuclear periphery to the central nucleoplasm and to foci of hyperphosphorylated Pol II "transcription factories" (TFys). To determine the contribution of individual LCR DNaseI hypersensitive sites (HSs) to transcription and nuclear location, in the present study, we compared ß-globin gene activity and location in erythroid cells derived from mice with deletions of individual HSs, deletions of 2 HSs, and deletion of the whole LCR and found all of the HSs had a similar spectrum of activities, albeit to different degrees. Each HS acts as an independent module to activate expression in an additive manner, and this is correlated with relocation away from the nuclear periphery. In contrast, HSs have redundant activities with respect to association with TFys and the probability that an allele is actively transcribed, as measured by primary RNA transcript FISH. The limiting effect on RNA levels occurs after ß-globin genes associate with TFys, at which time HSs contribute to the amount of RNA arising from each burst of transcription by stimulating transcriptional elongation.

Dynamics and control of state-dependent networks for probing genomic organization.

A state-dependent dynamic network is a collection of elements that interact through a network, whose geometry evolves as the state of the elements changes over time. The genome is an intriguing example of a state-dependent network, where chromosomal geometry directly relates to genomic activity, which in turn strongly correlates with geometry. Here we examine various aspects of a genomic state-dependent dynamic network. In particular, we elaborate on one of the important ramifications of viewing genomic networks as being state-dependent, namely, their controllability during processes of genomic reorganization such as in cell differentiation.

Genome-wide DNA replication profile for Drosophila melanogaster: a link between transcription and replication timing.

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes.

Cohesin mediates chromatin interactions that regulate mammalian ß-globin expression.

The ß-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.

Multiple functions of Ldb1 required for beta-globin activation during erythroid differentiation.

Ldb1 and erythroid partners SCL, GATA-1, and LMO2 form a complex that is required to establish spatial proximity between the ß-globin locus control region and gene and for transcription activation during erythroid differentiation. Here we show that Ldb1 controls gene expression at multiple levels. Ldb1 stabilizes its erythroid complex partners on ß-globin chromatin, even though it is not one of the DNA-binding components. In addition, Ldb1 is necessary for enrichment of key transcriptional components in the locus, including P-TEFb, which phosphorylates Ser2 of the RNA polymerase C-terminal domain for efficient elongation. Furthermore, reduction of Ldb1 results in the inability of the locus to migrate away from the nuclear periphery, which is necessary to achieve robust transcription of ß-globin in nuclear transcription factories. Ldb1 contributes these critical functions at both embryonic and adult stages of globin gene expression. These results implicate Ldb1 as a factor that facilitates nuclear relocation for transcription activation.

The emergence of lineage-specific chromosomal topologies from coordinate gene regulation.

Although the importance of chromosome organization during mitosis is clear, it remains to be determined whether the nucleus assumes other functionally relevant chromosomal topologies. We have previously shown that homologous chromosomes have a tendency to associate during hematopoiesis according to their distribution of coregulated genes, suggesting cell-specific nuclear organization. Here, using the mathematical approaches of distance matrices and coupled oscillators, we model the dynamic relationship between gene expression and chromosomal associations during the differentiation of a multipotential hematopoietic progenitor. Our analysis reveals dramatic changes in total genomic order: Commitment of the progenitor results in an initial increase in entropy at both the level of gene coregulation and chromosomal organization, which we suggest represents a phase transition, followed by a progressive decline in entropy during differentiation. The stabilization of a highly ordered state in the differentiated cell types results in lineage-specific chromosomal topologies and is related to the emergence of coherence-or self-organization-between chromosomal associations and coordinate gene regulation. We discuss how these observations may be generally relevant to cell fate decisions encountered by progenitor/stem cells.

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.

Networking the nucleus.

The nuclei of differentiating cells exhibit several fundamental principles of self-organization. They are composed of many dynamical units connected physically and functionally to each other--a complex network--and the different parts of the system are mutually adapted and produce a characteristic end state. A unique cell-specific signature emerges over time from complex interactions among constituent elements that delineate coordinate gene expression and chromosome topology. Each element itself consists of many interacting components, all dynamical in nature. Self-organizing systems can be simplified while retaining complex information using approaches that examine the relationship between elements, such as spatial relationships and transcriptional information. These relationships can be represented using well-defined networks. We hypothesize that during the process of differentiation, networks within the cell nucleus rewire according to simple rules, from which a higher level of order emerges. Studying the interaction within and among networks provides a useful framework for investigating the complex organization and dynamic function of the nucleus.

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.