Dmitry V. Fyodorov

The ETS domain factor Pet-1 is an early and precise marker of central serotonin neurons and interacts with a conserved element in serotonergic genes

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 hydroxylase-positive 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.

CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo.

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.

The Many Faces of Chromatin Remodeling: SWItching beyond Transcription

Here, we have presented a handful of examples of chromatin remodeling in processes other than transcription. These studies are likely to be the proverbial “tip of the iceberg” of an exciting and important area of chromatin research. One of the key challenges for the future will be to devise chromatin remodeling assays that accurately reflect the specific functions of the factors in the cell. In this manner, the activities of different chromatin remodeling factors could be distinguished and characterized. It will also be a significant challenge to identify the biochemical and biological activities of yet-to-be-characterized proteins that are related to SWI2/SNF2. In this regard, genetic data will likely provide key insights. In addition, the purification of the native forms of the proteins will reveal whether or not they are present in a multisubunit complex (and the identification of other associated proteins, if they exist, would likely provide clues on the functions of the factors). From the mechanistic standpoint, it would be interesting to investigate whether chromatin remodeling factors function by the same basic mechanism in transcription, replication, repair, and recombination, or if different remodeling complexes possess unique activities that specifically facilitate each of the separate processes in the context of chromatin. Thus, in the future, these efforts should lead to the replacement of the somewhat vague activity that we currently call “chromatin remodeling” by more specific descriptive terms of the rich and diverse functions of these ATP-driven chromatin-reorganizing factors.

Phase separation drives heterochromatin domain formation

Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability, and silencing of transposon and gene expression. 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 DNA. 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 compartments. 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.

Dynamics of ATP-dependent chromatin assembly by ACF

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases. Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2–4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA. These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.

Chromatin assembly in vitro with purified recombinant ACF and NAP-1

Publisher Summary This chapter describes the methodology for the ATP-dependent assembly of periodic nucleosome arrays by using purified recombinant chromatin assembly factors. The assembly of DNA to chromatin is a critical step in the duplication and maintenance of the eukaryotic genome. Chromatin assembly is an ATP-dependent process and is mediated by the concerted action of core histone chaperones and ATP-dependent chromatin assembly machines. NAP-1, CAF-1, ASF-1, nucleoplasmin, and other histone chaperones play an essential role by escorting histones to the sites of nucleosome assembly. The reaction for chromatin assembly with plasmid DNA can be broken down in three steps: pre-binding of core histones with the chaperone, NAP-1; relaxation of the plasmid DNA (optional); and ATP-dependent nucleosome assembly. The assembled chromatin can be used directly in downstream assay reactions. Certain applications (for example, electron microscopy studies) may require further purification of the chromatin. The nucleosome arrays can be purified from chromatin assembly factors by sucrose gradient sedimentation or gel filtration. The assembly of chromatin with the linker histone H1 is discussed. H1 is efficiently incorporated in the nucleosome arrays, and these arrays possess structural and functional features of H1-containing native chromatin.

Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.

Drosophila H1 Regulates the Genetic Activity of Heterochromatin by Recruitment of Su(var)3-9

Silencing Transposons Eukaryotic DNA is packaged onto nucleosomes, which are composed of four core histones (H2A, H2B, H3, and H4). Chromatin also contains a fifth histone, H1, which binds to both the core particles and the “linker” DNA that joins adjacent nucleosomes, where it helps chromatin to fold into higher-order structures and generally silences gene expression. In Drosophila germline and somatic cells and tissues, Lu et al. (p. 78) found that the repressive function of H1 in vivo was directed toward transposable elements and independent of small RNA silencing pathways. Instead, H1 acted through direct recruitment of the histone methyltransferase Su(var)3–9, which methylates histone H3 lysine 9, a repressive histone mark. The “fifth” histone, H1, acts to recruit a histone-methylating enzyme to silence specific regions of the genome. Eukaryotic genomes harbor transposable elements and other repetitive sequences that must be silenced. Small RNA interference pathways play a major role in their repression. Here, we reveal another mechanism for silencing these sequences in Drosophila. Depleting the linker histone H1 in vivo leads to strong activation of these elements. H1-mediated silencing occurs in combination with the heterochromatin-specific histone H3 lysine 9 methyltransferase Su(var)3-9. H1 physically interacts with Su(var)3-9 and recruits it to chromatin in vitro, which promotes H3 methylation. We propose that H1 plays a key role in silencing by tethering Su(var)3-9 to heterochromatin. The tethering function of H1 adds to its established role as a regulator of chromatin compaction and accessibility.

Binding of Acf1 to DNA Involves a WAC Motif and Is Important for ACF-Mediated Chromatin Assembly

ABSTRACT ACF is a chromatin-remodeling complex that catalyzes the ATP-dependent assembly of periodic nucleosome arrays. This reaction utilizes the energy of ATP hydrolysis by ISWI, the smaller of the two subunits of ACF. Acf1, the large subunit of ACF, is essential for the full activity of the complex. We performed a systematic mutational analysis of Acf1 to elucidate the functions of specific subregions of the protein. These studies revealed DNA- and ISWI-binding regions that are important for the chromatin assembly and ATPase activities of ACF. The DNA-binding region of Acf1 includes a WAC motif, which is necessary for the efficient binding of ACF complex to DNA. The interaction of Acf1 with ISWI requires a DDT domain, which has been found in a variety of transcription and chromatin-remodeling factors. Chromatin assembly by ACF is also impaired upon mutation of an acidic region in Acf1, which may interact with histones during the deposition process. Lastly, we observed modest chromatin assembly defects on mutation of other conserved sequence motifs. Thus, Acf1 facilitates chromatin assembly via an N-terminal DNA-binding region with a WAC motif, a central ISWI-binding segment with a DDT domain, and a C-terminal region with an acidic stretch, a WAKZ motif, PHD fingers, and bromodomain.

The chromodomains of CHD1 are critical for enzymatic activity but less important for chromatin localization

The molecular motor protein CHD1 has been implicated in the regulation of transcription and in the transcription-independent genome-wide incorporation of H3.3 into paternal chromatin in Drosophila melanogaster. A key feature of CHD1 is the presence of two chromodomains, which can bind to histone H3 methylated at lysine 4 and thus might serve to recruit and/or maintain CHD1 at the chromatin. Here, we describe genetic and biochemical approaches to the study of the Drosophila CHD1 chromodomains. We found that overall localization of CHD1 on polytene chromosomes does not appreciably change in chromodomain-mutant flies. In contrast, the chromodomains are important for transcription-independent activities of CHD1 during early embryonic development as well as for transcriptional regulation of several heat shock genes. However, neither CHD1 nor its chromodomains are needed for RNA polymerase II localization and H3K4 methylation but loss of CHD1 decreases transcription-induced histone eviction at the Hsp70 gene in vivo. Chromodomain mutations negatively affect the chromatin assembly activities of CHD1 in vitro, and they appear to be involved in linking the ATP-dependent motor to the chromatin assembly function of CHD1.