Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability1, and silencing of transposon and gene expression2. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA3. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and extracellular compartments4. We show that Drosophila HP1a protein undergoes liquid–liquid demixing in vitro, and nucleates into foci that display liquid properties during the first stages of heterochromatin domain formation in early Drosophila embryos. Furthermore, in both Drosophila and mammalian cells, heterochromatin domains exhibit dynamics that are characteristic of liquid phase-separation, including sensitivity to the disruption of weak hydrophobic interactions, and reduced diffusion, increased coordinated movement and inert probe exclusion at the domain boundary. We conclude that heterochromatic domains form via phase separation, and mature into a structure that includes liquid and stable compartments. We propose that emergent biophysical properties associated with phase-separated systems are critical to understanding the unusual behaviours of heterochromatin, and how chromatin domains in general regulate essential nuclear functions.

The central serotonin (5-HT) neurotransmitter system is an important modulator of diverse physiological processes and behaviors; however, the transcriptional mechanisms controlling its development are largely unknown. The Pet-1 ETS factor is a precise marker of developing and adult 5-HT neurons and is expressed shortly before 5-HT appears in the hindbrain. Here we show that in mice lacking Pet-1, the majority of 5-HT neurons fail to differentiate. Remaining ones show deficient expression of genes required for 5-HT synthesis, uptake, and storage. Significantly, defective development of the 5-HT system is followed by heightened anxiety-like and aggressive behavior in adults. These findings indicate that Pet-1 is a critical determinant of 5-HT neuron identity and implicate a Pet-1-dependent program in serotonergic modulation of behavior.

Together with core histones, which make up the nucleosome, the linker histone (H1) is one of the five main histone protein families present in chromatin in eukaryotic cells. H1 binds to the nucleosome to form the next structural unit of metazoan chromatin, the chromatosome, which may help chromatin to fold into higher-order structures. Despite their important roles in regulating the structure and function of chromatin, linker histones have not been studied as extensively as core histones. Nevertheless, substantial progress has been made recently. The first near-atomic resolution crystal structure of a chromatosome core particle and an 11 Å resolution cryo-electron microscopy-derived structure of the 30 nm nucleosome array have been determined, revealing unprecedented details about how linker histones interact with the nucleosome and organize higher-order chromatin structures. Moreover, several new functions of linker histones have been discovered, including their roles in epigenetic regulation and the regulation of DNA replication, DNA repair and genome stability. Studies of the molecular mechanisms of H1 action in these processes suggest a new paradigm for linker histone function beyond its architectural roles in chromatin.

The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is ∼3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.[Key Words: Chromatin assembly; ACF; Acf1; bromodomain; PHD finger; ISWI]Received April 26, 1999; revised version accepted May 6, 1999. Nucleosome assembly is a fundamental biological process that is required for the replication and maintenance of chromatin in the eukaryotic nucleus (for reviews, see

Serotonin (5-HT) plays a crucial neuromodulatory role in numerous physiological and behavioral functions, and dysfunction of the serotonergic system has been implicated in several psychiatric disorders. Despite the widespread importance of the central serotonergic neurotransmitter system, little is known about the molecular mechanisms controlling the development of 5-HT neurons. We previously identified an ETS domain transcription factor, Pet-1, that is expressed in a small number of tissues, including the brain. Here, we show that expression of Pet-1 RNA in the brain is restricted to, and marks, the entire rostrocaudal extent of rat serotonergic hindbrain raphe nuclei. Remarkably, Pet-1 RNA colocalizes with tryptophan hydroxylasepositive neurons in raphe nuclei but not with their nonserotonergic neuron or non-neuronal neighbors. Pet-1 RNA is limited to two domains in the developing hindbrain, which precedes the appearance of 5-HT in each domain by approximately a half day. Conserved Pet-1 binding sites are present in or near the promoter regions of the human and mouse 5-HT1a receptor, serotonin transporter, tryptophan hydroxylase, and aromatic L-amino acid decarboxylase genes whose expression is characteristic of the serotonergic neuron phenotype. These sites are capable of supporting transcriptional activation through interactions with the Pet-1 ETS domain and can function as enhancers. Together, our findings establish Pet-1 as an early and precise marker of 5-HT neurons and suggest that it functions specifically in the differentiation and maintenance of these neurons. Key words: serotonin; ETS factor; raphe nuclei; transcription; binding site; neurotransmitter phenotypeThe central serotonin (5-HT) neurotransmitter system consists of a relatively small population of morphologically diverse neurons whose cell bodies are present primarily within the limits of the midbrain -hindbrain raphe nuclei and particular regions of the reticular formation (Steinbusch, 1981). Although there are only ϳ20,000 serotonergic neurons in the rat brain, the extensive axonal projection system arising from these cells bears a tremendous number of collateral branches so that the 5-HT system densely innervates nearly all regions of the C NS (Jacobs and Azmitia, 1992;Halliday et al., 1995). Given its widespread distribution, it is not surprising that 5-HT has been implicated in the control of numerous neural systems, including those that mediate cognition, affect, aggression, and perception (Heninger, 1997). Abnormal f unction of the central 5-HT system has been implicated in several psychiatric maladies, such as depression, anxiety, and eating disorders. Moreover, this system is the target of several highly effective pharmacological agents that are used widely to treat these conditions. Despite the clear importance of the central 5-HT system in a wide range of CNS processes and clinical disorders, little is known about the genetic mechanisms that control the specification and differentiation of serotonergic neurons.ETS domain transcrip...

multiple cell types, with gene-rich euchromatin more centrally disposed of than gene-poor heterochromatin. Such territories are not randomly positioned and both gene-density-based and size-based radial positioning schemes have been proposed to describe the data. We propose that any realistic model of these and other large-scale features of nuclear architecture must account for ATP-fueled non-equilibrium activity, associated with transcriptional processes that are inhomogeneous within and across chromosomes. We describe a biophysical model for human cell nuclei which incorporates such activity. The model predicts the statistics of the shapes, positioning, and contact maps of individual chromosomes, with the differential positioning of the inactive and active X chromosomes in female (XX), cells emerging as a natural consequence, and our results compare favorably to a broad spectrum of experimental data. We argue that the consequences, in mechanical terms, of the distribution of transcriptional activity across chromosomes should be the primary determinant of a chromosome positioning code. Constitutive heterochromatin is made of repetitive sequences and is epigenetically identified by methylation of H3K9 and binding of Heterochromatin Protein 1a (HP1a). In cells, repetitive sequences from multiple chromosomes are organized into spherical domains important for maintaining transcriptional silencing and preventing aberrant recombination. Canonically, these functions of heterochromatin are attributed to tight compaction of nucleosomes and consequent exclusion of large protein complexes like polymerases and recombinases. However, proteins within the domain are mobile, and size is not the only factor that defines whether a protein can enter heterochromatin. We investigated whether the heterochromatin domain is similar to other membraneless cellular compartments like nucleoli in that it is formed via phase separation. We purified recombinant Drosophila HP1a and found that in vitro, this protein is able to demix from aqueous solution to form droplets that fuse and flow like a liquid. In vivo, we observe similar droplet formation and fusion during the initial establishment of heterochromatin in the early Drosophila embryo. We used Fluorescence Correlation Spectroscopyderived imaging methods to observe bulk movement of HP1a at the heteroeuchromatin interface, and found that proteins exhibit specific dynamic properties associated with phase interfaces, indicating they are held in the heterochromatin domain by surface tension. Additionally, an inert probe (three tandem YFPs) is excluded from the domain in a similar manner, indicating that physicochemical properties of the heterochromatin domain define access to these sequences. The phase separation model we propose here is consistent with historical data about heterochromatin domains, including the tendency for distal regions of heterochromatin to loop back and contact the main domain. We believe this work represents a shift in perspective of how we should view the nucleus; as a...

The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of chromatin remodeling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses, and leads to the development of haploid embryos. Furthermore, CHD1, but not ISWI, interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of adenosine triphosphate (ATP)-utilizing motor proteins for histone deposition in vivo.H istone-DNA interactions constantly change during various processes of DNA metabolism. Recent studies have highlighted the importance of histone variants, such as H3.3, CENP-A (centromere protein A), or H2A.Z, in chromatin dynamics (1, 2). Incorporation of replacement histones into chromatin occurs throughout the cell cycle, whereas nucleosomes containing canonical histones are assembled exclusively during DNA replication. A thorough understanding of the replication-independent mechanisms of chromatin assembly, however, is lacking.In vitro, chromatin assembly requires the action of histone chaperones and adenosine triphosphate (ATP)-utilizing factors (3). Histone chaperones may specialize for certain histone variants. For example, H3.3 associates with a complex containing HIRA, whereas canonical H3 is in a complex with CAF-1 (chromatin assembly factor 1) (4). The molecular motors known to assemble nucleosomes are ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (nucleosome-remodeling and spacing factor), which contain the Snf2 family member ISWI as the catalytic subunit (5-7), and CHD1, which belongs to the CHD subfamily of Snf2-like adenosine triphosphatases (ATPases) (8). These factors have not been shown to mediate deposition of histones in vivo. We previously demonstrated that CHD1, together with the chaperone NAP-1, assembles nucleosome arrays from DNA and histones in vitro (9). Here, we investigated the role of CHD1 in chromatin assembly in vivo in Drosophila.We generated Chd1 alleles by P elementmediated mutagenesis (Fig. 1A) (10). Two exci- 2] with Df(2L)Exel7014 affect both copies of the Chd1 gene only (Fig. 1B). We also identified a single point mutation that results in premature translation termination of Chd1 (Q1394*) in a previously described lethal allele, l(2)23Cd[A7-4] (11). Hence, l(2)23Cd[A7-4] was renamed Chd1 [3].sions, Df(2L)Chd1[1] and Df(2L)Chd1[2], deleted fragments of the Chd1 gene and fragments of unrelated adjacent genes. Heterozygous combinations, however, of Chd1[1] or Chd1[Analysis of Western blots of embryos from heterozyg...

Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. Here we investigate the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus. The assembly of chromatin is a fundamental biological process that occurs in proliferating cells during DNA replication and in quiescent cells during maintenance and repair of chromosomes (for recent reviews, see Verreault 2000;Mello and Almouzni 2001;Tyler 2002;Haushalter and Kadonaga 2003). During DNA replication, chromatin structure is transiently disrupted at the replication fork (Gruss et al. 1993), and the preexisting nucleosomes are segregated randomly between the daughter DNA strands (Sogo et al. 1986). Then, additional nucleosomes are formed with newly synthesized histones. In this process, it appears that histones H3 and H4 are deposited prior to the incorporation of histones H2A and H2B (Worcel et al. 1978;Smith et al. 1984).