We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

/pdf/comprehensive-mapping-of-long-range-interactions-reveals-15wp5ww9ic.pdf

Comprehensive mapping of long-range interactions reveals folding principles of the human genome.

There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFκB, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.

https://www.med.upenn.edu/feldserlab/resources/Publications/2009-Guttman-et-al.pdf

Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals

http://web.mit.edu/biophysics/papers/CELL2008.pdf

Nature, Nurture, or Chance: Stochastic Gene Expression and Its Consequences

BACKGROUND Living cells contain distinct subcompartments to facilitate spatiotemporal regulation of biological reactions. In addition to canonical membrane-bound organelles such as secretory vesicles and endoplasmic reticulum, there are many organelles that do not have an enclosing membrane yet remain coherent structures that can compartmentalize and concentrate specific sets of molecules. Examples include assemblies in the nucleus such as the nucleolus, Cajal bodies, and nuclear speckles and also cytoplasmic structures such as stress granules, P-bodies, and germ granules. These structures play diverse roles in various biological processes and are also increasingly implicated in protein aggregation diseases. ADVANCES A number of studies have shown that membrane-less assemblies exhibit remarkable liquid-like features. As with conventional liquids, they typically adopt round morphologies and coalesce into a single droplet upon contact with one another and also wet intracellular surfaces such as the nuclear envelope. Moreover, component molecules exhibit dynamic exchange with the surrounding nucleoplasm and cytoplasm. These findings together suggest that these structures represent liquid-phase condensates, which form via a biologically regulated (liquid-liquid) phase separation process. Liquid phase condensation increasingly appears to be a fundamental mechanism for organizing intracellular space. Consistent with this concept, several membrane-less organelles have been shown to exhibit a concentration threshold for assembly, a hallmark of phase separation. At the molecular level, weak, transient interactions between molecules with multivalent domains or intrinsically disordered regions (IDRs) are a driving force for phase separation. In cells, condensation of liquid-phase assemblies can be regulated by active processes, including transcription and various posttranslational modifications. The simplest physical picture of a homogeneous liquid phase is often not enough to capture the full complexity of intracellular condensates, which frequently exhibit heterogeneous multilayered structures with partially solid-like characters. However, recent studies have shown that multiple distinct liquid phases can coexist and give rise to richly structured droplet architectures determined by the relative liquid surface tensions. Moreover, solid-like phases can emerge from metastable liquid condensates via multiple routes of potentially both kinetic and thermodynamic origins, which has important implications for the role of intracellular liquids in protein aggregation pathologies. OUTLOOK The list of intracellular assemblies driven by liquid phase condensation is growing rapidly, but our understanding of their sequence-encoded biological function and dysfunction lags behind. Moreover, unlike equilibrium phases of nonliving matter, living cells are far from equilibrium, with intracellular condensates subject to various posttranslational regulation and other adenosine triphosphate–dependent biological activity. Efforts using in vitro reconstitution, combined with traditional cell biology approaches and quantitative biophysical tools, are required to elucidate how such nonequilibrium features of living cells control intracellular phase behavior. The functional consequences of forming liquid condensates are likely multifaceted and may include facilitated reaction, sequestration of specific factors, and organization of associated intracellular structures. Liquid phase condensation is particularly interesting in the nucleus, given the growing interest in the impact of nuclear phase behavior on the flow of genetic information; nuclear condensates range from micrometer-sized bodies such as the nucleolus to submicrometer structures such as transcriptional assemblies, all of which directly interact with and regulate the genome. Deepening our understanding of these intracellular states of matter not only will shed light on the basic biology of cellular organization but also may enable therapeutic intervention in protein aggregation disease by targeting intracellular phase behavior.

Liquid phase condensation in cell physiology and disease.

Leu-M

The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes migrate to preassembled transcription sites.

/pdf/active-genes-dynamically-colocalize-to-shared-sites-of-534wdjswft.pdf

Active genes dynamically colocalize to shared sites of ongoing transcription.

As the relationship between nuclear structure and function begins to unfold, a picture is emerging of a dynamic landscape that is centred on the two main processes that execute the regulated use and propagation of the genome. Rather than being subservient enzymatic activities, the replication and transcriptional machineries provide potent forces that organize the genome in three-dimensional nuclear space. Their activities provide opportunities for epigenetic changes that are required for differentiation and development. In addition, they impose physical constraints on the genome that might help to shape its evolution.

Replication and transcription: Shaping the landscape of the genome

In female mammals, one of the two X chromosomes in each cell becomes transcriptionally inactive early in embryonic development. This process is known as X inactivation and is closely associated with cellular differentiation (see reference 22 for a review). The initiation and spread of X inactivation are dependent on a unique region of the X chromosome, the X-inactivation center (Xic). The Xic is also thought to be involved in determining how many (counting) and which (choice) X chromosomes to inactivate (see reference 2 for a review). The Xist gene, which lies within the Xic region, encodes a large nuclear, untranslated RNA that coats the inactive X chromosome at interphase (5–8). The developmental expression pattern of Xist suggests a causal role in X inactivation: prior to inactivation, Xist is expressed at low levels from every X chromosome in a cell (27), and at the onset of inactivation, steady-state levels of Xist RNA increase and the transcript coats the X chromosome that will be inactivated. This transition is associated with stabilization of Xist RNA on the X chromosome that will become inactive (36, 42). The regulated changes in stability of the Xist transcript and the coating of the X chromosome are thought to be concomitant with a switch in promoter use (26). The elements that regulate all of these changes remain to be defined functionally, however.

Targeted deletions of the Xist gene have demonstrated that it is essential for inactivation in cis (33, 37), but since counting is not abolished in these mutants, it has been suggested that elements outside Xist itself may also be involved in overall Xic function. Indeed, when a 65-kb region 3′ to Xist exon 6 is deleted, cis inactivation is not affected but, instead, the deleted X is systematically inactivated upon differentiation, even in the absence of a second X chromosome (12). Elements lying outside Xist that have been proposed to be implicated in the X-inactivation process include the Xce locus (10), which is known to affect choice and has been shown to be genetically separable from Xist (37a, 43). Another is the DXPas34 locus, which lies 15 kb 3′ to Xist (13).

The DXPas34 locus is a CpG-rich region consisting of a SalI site and a neighboring cluster of HpaII sites lying within a 34-mer minisatellite repeat, which was originally identified as a result of its unusual methylation profile. Methylation of DXPas34 is specifically associated with the active X chromosome and the transcriptionally silent Xist gene in somatic tissues and differentiated embryonic stem (ES) cells (3, 13). Analysis of this methylation profile in strains carrying different Xce alleles suggested a correlation between the Xce allele present in cis and the methylation status of the region, although the Xce locus actually maps distal to DXPas34 (37a, 43). Given its proximity to the Xist gene and its unusual methylation profile, it has been proposed that the DXPas34 region might play a role in regulating Xist expression and the initiation of X inactivation. The DXPas34 locus also displays a number of features that are strongly reminiscent of the differentially methylated regions (DMRs) frequently found in the vicinity of imprinted genes (38). Like DXPas34, DMRs show differential methylation between alleles, occur in or near CpG-rich regions, and are often associated with blocks of short tandem repeats. Elements involved in the establishment of imprinted gene expression lie within such DMRs in several cases. There are also an increasing number of examples where DMRs overlap with the transcribed region of unusual noncoding RNAs. In this context, Lee et al. (28) recently described the presence of an antisense transcript initiating close to the DXPas34 region and stretching as far as the 5′ end of Xist. As part of the study we present here, we show that the antisense transcription in this region is actually more widespread and complex than was originally thought.

To investigate the potential role of the DXPas34 locus in Xist regulation and the initiation of X inactivation, we have deleted it in the context of yeast artificial chromosome (YAC) transgenes in ES cells. We have chosen a YAC transgene-based approach for the ease and rapidity with which transgene mutations can be generated in yeast. Transgenes containing Xist are known to function as ectopic Xics when introduced into ES cells (29–31), provided that they are present as multicopy arrays (20). Both inactivation of autosomal sequences in cis around the transgene and inactivation of the single X chromosome (counting) can be observed in such transgenic male ES cell lines. This parallels the process seen in XX ES cells, where in vitro differentiation is accompanied by inactivation of one of the two X chromosomes. Although single-copy Xist YAC transgenes do not seem to be able to induce inactivation, they correctly express Xist prior to differentiation in ES cells (20). Thus, even single-copy Xist transgenes can be used to study the effects of a DXPas34 deletion on Xist regulation prior to inactivation.

Based on the analysis of several different ES cell lines carrying YAC transgenes with DXPas34 deleted, we demonstrate that this locus is essential for Xist expression in undifferentiated ES cells and may affect the normally random nature of X inactivation in ES cells. We also show that the presence of this locus is associated with widespread antisense transcription. Finally, we show that transcription at the DXPas34 locus during early development is imprinted, suggesting that this locus may play a regulatory role in the early steps of Xist expression and X inactivation.

Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

X inactivation in female mammals is controlled by a key locus on the X chromosome, the X-inactivation center (Xic). The Xic controls the initiation and propagation of inactivation in cis. It also ensures that the correct number of X chromosomes undergo inactivation (counting) and determines which X chromosome becomes inactivated (choice). The Xist gene maps to the Xic region and is essential for the initiation of X inactivation in cis. Regulatory elements of X inactivation have been proposed to lie 3* to Xist. One such element, lying 15 kb downstream of Xist ,i s theDXPas34 locus, which was first identified as a result of its hypermethylation on the active X chromosome and the correlation of its methylation level with allelism at the X-controlling element (Xce), a locus known to affect choice. In this study, we have tested the potential function of the DXPas34 locus in Xist regulation and X-inactivation initiation by deleting it in the context of large Xistcontaining yeast artificial chromosome transgenes. Deletion of DXPas34 eliminates both Xist expression and antisense transcription present in this region in undifferentiated ES cells. It also leads to nonrandom inactivation of the deleted transgene upon differentiation. DXPas34 thus appears to be a critical regulator of Xist activity and X inactivation. The expression pattern of DXPas34 during early embryonic development, which we report here, further suggests that it could be implicated in the regulation of imprinted Xist expression. In female mammals, one of the two X chromosomes in each cell becomes transcriptionally inactive early in embryonic development. This process is known as X inactivation and is closely associated with cellular differentiation (see reference 22 for a review). The initiation and spread of X inactivation are dependent on a unique region of the X chromosome, the X-inactivation center (Xic). The Xic is also thought to be involved in determining how many (counting) and which (choice) X chromosomes to inactivate (see reference 2 for a review). The Xist gene, which lies within the Xic region, encodes a large nuclear, untranslated RNA that coats the inactive X chromosome at interphase (5‐8). The developmental expression pattern of Xist suggests a causal role in X inactivation: prior to inactivation, Xist is expressed at low levels from every X chromosome in a cell (27), and at the onset of inactivation, steady-state levels of Xist RNA increase and the transcript coats the X chromosome that will be inactivated. This transition is associated with stabilization of Xist RNA on the X chromosome that will become inactive (36, 42). The regulated changes in stability of the Xist transcript and the coating of the X chromosome are thought to be concomitant with a switch in promoter use (26). The elements that regulate all of these changes remain to be defined functionally, however.

Functional Analysis of the DXPas34 Locus, a 39 Regulator of Xist Expression

A counting process senses the X chromosome/autosome ratio and ensures that X chromosome inactivation (XCI) initiates in the female (XX) but not in the male (XY) mouse embryo. Counting is regulated by the X-inactivation centre, which contains the Xist gene. Deleting 65 kb 3′ to Xist in XO embryonic stem (ES) cells affects counting and results in inappropriate XCI upon differentiation. We show here that normal counting can be rescued in these deleted ES cells using cre/loxP re-insertion, and refine the location of elements controlling counting within a 20 kb bipartite domain. Furthermore, we show that the 65 kb deletion also leads to inappropriate XCI in XY differentiated ES cells, which excludes the involvement of sex-specific mechanisms in the initiation of XCI. At the chromatin level, we have found that the Xist gene corresponds to a peak of H3 Lys-4 dimethylation, which is dramatically and specifically affected by the deletion 3′ to Xist. Our results raise the possibility that H3 Lys-4 dimethylation within Xist may be functionally implicated in the counting process.

/pdf/the-region-3-to-xist-mediates-x-chromosome-counting-and-h3-w4pdkn9ku5.pdf

Emmanuel Debrand

Papers

Active genes dynamically colocalize to shared sites of ongoing transcription.

Replication and transcription: Shaping the landscape of the genome

Functional analysis of the DXPas34 locus, a 3' regulator of Xist expression.

Functional Analysis of the DXPas34 Locus, a 39 Regulator of Xist Expression

The region 3' to Xist mediates X chromosome counting and H3 Lys-4 dimethylation within the Xist gene.