Dmitry Fyodorov | Albert Einstein College of Medicine (original) (raw)
Papers by Dmitry Fyodorov
![Research paper thumbnail of Transcriptional Analysis of Acetylcholine Receptor [IMAGE]3 Gene Promoter Motifs That Bind Sp1 and AP2](https://attachments.academia-assets.com/49543246/thumbnails/1.jpg)
](Journal of Biological Chemistry, 1995
Journal of Neurobiology, 1997
elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three r... more elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis
Neuron, 2003
neuromodulatory capacity of the 5-HT system is further reflected in the numerous human behavioral... more neuromodulatory capacity of the 5-HT system is further reflected in the numerous human behavioral disorders that are thought to involve a dysfunction of the 5-HT system. A large body of data suggests that a deficiency of central serotonergic signaling is a major factor involved in the development of disorders such as aggres-1 Department of Neurosciences sion, impulsivity, anxiety, depression, suicide, and ob-2 Department of Psychiatry sessive-compulsive disorder (Davidson et al., 2000; School of Medicine Lucki, 1998; Mann et al., 2001; Nelson and Chiavegatto, Case Western Reserve University 2001). Recent evidence for an early postnatal role of Cleveland, Ohio 44106 forebrain 5-HT 1a receptors in the acquisition of normal 3 Division of Neuroscience adult anxiety-like behavior (Gross et al., 2002) supports Baylor College of Medicine the notion that a developmental deficit in 5-HT signaling Houston, Texas 77030 may predispose individuals to mood disorders. Despite the prominence of the 5-HT system in central neuromodulation and psychiatric disorders, the genetic mecha-Summary nisms governing the generation of 5-HT neurons are poorly understood and it is not known how these mecha-The central serotonin (5-HT) neurotransmitter system nisms are linked to eventual serotonergic control of beis an important modulator of diverse physiological prohavior in adults. cesses and behaviors; however, the transcriptional 5-HT immunoreactive cells first appear in the mantle mechanisms controlling its development are largely layer of the embryonic hindbrain adjacent to the floor unknown. The Pet-1 ETS factor is a precise marker of plate at about E13 in rat (Lidov and Molliver, 1982; Waldeveloping and adult 5-HT neurons and is expressed lace and Lauder, 1983). These neurons form the rostral shortly before 5-HT appears in the hindbrain. Here we domain of the developing 5-HT system, which is posishow that in mice lacking Pet-1, the majority of 5-HT tioned just caudal to the isthmus. The rostral domain neurons fail to differentiate. Remaining ones show
Journal of Neurobiology, 1997
elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three r... more elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis
European Journal of Pharmacology, 2000
Receptors assembled from the products of a neuronal b4a 3a 5 NAChR gene cluster depend on these g... more Receptors assembled from the products of a neuronal b4a 3a 5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the a 3 and b4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called b43 X . This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate b43 X -dependent transcription. The neuron-selective properties of b43 X and its location suggest that it is a component of the cis regulatory information required to control expression of the b4 and a 3 genes in specific populations of neurons. q
Journal of Neurobiology, 1998
We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from ad... more We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from adrenal chromaffin-derived PC12 cell RNA that b43, within the 3-untranslated exon of the neuronal encodes a novel ETS-domain factor, Pet-1. The denicotinic acetylcholine receptor (nAchR) b4 subunit duced primary structure of Pet-1 is composed of 340 gene. Similar to Pet-1, the b4 gene is also expressed amino acids and the encoded polypeptide has a prein PC12 cells. The presence of putative ETS-domain dicted molecular mass of 35.4 kD. The pattern of Petbinding sites in the b43 enhancer led us to hypothe-1 gene expression in the neonatal rat is highly resize that members of the ets gene family activate neustricted and suggests that Pet-1 functions primarily in ronal nAchR genes. Cotransfection assays show that the nervous system. Adrenal gland expresses the high-Pet-1 can activate reporter gene transcription in a est level of Pet-1 among the tissues examined. In situ b43 enhancer-dependent and cell type-dependent hybridization indicates that Pet-1 is expressed in the manner. Our results lead us to hypothesize that Petadrenal medulla but not the adrenal cortex. Slightly 1 acts as a transcriptional regulator of downstream weaker Pet-1 hybridization is detected in brain and target genes involved in cholinergic neurotransmislow levels are detectable in intestine and eye. Pet-1 sion. ᭧ 1998 John Wiley & Sons, Inc. J Neurobiol 34: 151-163, can bind specifically to a PEA3 ETS DNA-binding 1998 motif and can modulate transcription of synthetic pro-Correspondence to: E. Deneris cell phenotypes.
Journal of Neurobiology, 1998
We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from ad... more We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from adrenal chromaffin-derived PC12 cell RNA that b43, within the 3-untranslated exon of the neuronal encodes a novel ETS-domain factor, Pet-1. The denicotinic acetylcholine receptor (nAchR) b4 subunit duced primary structure of Pet-1 is composed of 340 gene. Similar to Pet-1, the b4 gene is also expressed amino acids and the encoded polypeptide has a prein PC12 cells. The presence of putative ETS-domain dicted molecular mass of 35.4 kD. The pattern of Petbinding sites in the b43 enhancer led us to hypothe-1 gene expression in the neonatal rat is highly resize that members of the ets gene family activate neustricted and suggests that Pet-1 functions primarily in ronal nAchR genes. Cotransfection assays show that the nervous system. Adrenal gland expresses the high-Pet-1 can activate reporter gene transcription in a est level of Pet-1 among the tissues examined. In situ b43 enhancer-dependent and cell type-dependent hybridization indicates that Pet-1 is expressed in the manner. Our results lead us to hypothesize that Petadrenal medulla but not the adrenal cortex. Slightly 1 acts as a transcriptional regulator of downstream weaker Pet-1 hybridization is detected in brain and target genes involved in cholinergic neurotransmislow levels are detectable in intestine and eye. Pet-1 sion. ᭧ 1998 John Wiley & Sons, Inc. J Neurobiol 34: 151-163, can bind specifically to a PEA3 ETS DNA-binding 1998 motif and can modulate transcription of synthetic pro-Correspondence to: E. Deneris cell phenotypes.
Journal of Biological Chemistry, 2010
ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor ... more ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked ␣-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.
Genes & Development, 2009
We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone ... more 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 ;20% of the level in wildtype 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.
Molecular and Cellular Biology, 2002
PLOS One, 2010
CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histo... more CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3 CENP-A , which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3 CID in Drosophila.
Nature, 2002
The assembly of DNA into chromatin is a critical step in the replication and repair of the eukary... more The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome 1-8 . It has been known for nearly 20 years that chromatin assembly is an ATPdependent process 9 . 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 10,11 . 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 12 . 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.
PLOS One, 2010
CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histo... more CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3 CENP-A , which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3 CID in Drosophila.
Genes & Development, 2004
Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin a... more 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.
Science, 2007
The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 i... more 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. 1 to 1394) CHD1 polypeptide. Arrowhead, wild-type CHD1 (250 kD); arrow, left, NAP-1 (loading control);
Cell, 2001
. It thus appears that there is a broad range of nontranscriptional functions of chromatin remode... more . It thus appears that there is a broad range of nontranscriptional functions of chromatin remodeling proteins ).
Genes & Development, 1999
The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI... more 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.
Methods in Enzymology, 2003
To study eukaryotic transcriptional mechanisms in vitro, it is important to analyze gene regulato... more To study eukaryotic transcriptional mechanisms in vitro, it is important to analyze gene regulatory sequences in the context of chromatin. Here we describe the ATP-dependent assembly of chromatin by using completely purified components. This system uses chromatin assembly factors ACF and NAP-1 in conjunction with purified core histones for the assembly of extended periodic arrays of nucleosomes. We additionally describe the assembly of chromatin that contains the linker histone H1. This histone H1-containing chromatin resembles bulk native chromatin in metazoans.
Journal of Biological Chemistry, 1995
Journal of Neurobiology, 1997
elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three r... more elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis
Neuron, 2003
neuromodulatory capacity of the 5-HT system is further reflected in the numerous human behavioral... more neuromodulatory capacity of the 5-HT system is further reflected in the numerous human behavioral disorders that are thought to involve a dysfunction of the 5-HT system. A large body of data suggests that a deficiency of central serotonergic signaling is a major factor involved in the development of disorders such as aggres-1 Department of Neurosciences sion, impulsivity, anxiety, depression, suicide, and ob-2 Department of Psychiatry sessive-compulsive disorder (Davidson et al., 2000; School of Medicine Lucki, 1998; Mann et al., 2001; Nelson and Chiavegatto, Case Western Reserve University 2001). Recent evidence for an early postnatal role of Cleveland, Ohio 44106 forebrain 5-HT 1a receptors in the acquisition of normal 3 Division of Neuroscience adult anxiety-like behavior (Gross et al., 2002) supports Baylor College of Medicine the notion that a developmental deficit in 5-HT signaling Houston, Texas 77030 may predispose individuals to mood disorders. Despite the prominence of the 5-HT system in central neuromodulation and psychiatric disorders, the genetic mecha-Summary nisms governing the generation of 5-HT neurons are poorly understood and it is not known how these mecha-The central serotonin (5-HT) neurotransmitter system nisms are linked to eventual serotonergic control of beis an important modulator of diverse physiological prohavior in adults. cesses and behaviors; however, the transcriptional 5-HT immunoreactive cells first appear in the mantle mechanisms controlling its development are largely layer of the embryonic hindbrain adjacent to the floor unknown. The Pet-1 ETS factor is a precise marker of plate at about E13 in rat (Lidov and Molliver, 1982; Waldeveloping and adult 5-HT neurons and is expressed lace and Lauder, 1983). These neurons form the rostral shortly before 5-HT appears in the hindbrain. Here we domain of the developing 5-HT system, which is posishow that in mice lacking Pet-1, the majority of 5-HT tioned just caudal to the isthmus. The rostral domain neurons fail to differentiate. Remaining ones show
Journal of Neurobiology, 1997
elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three r... more elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis
European Journal of Pharmacology, 2000
Receptors assembled from the products of a neuronal b4a 3a 5 NAChR gene cluster depend on these g... more Receptors assembled from the products of a neuronal b4a 3a 5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the a 3 and b4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called b43 X . This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate b43 X -dependent transcription. The neuron-selective properties of b43 X and its location suggest that it is a component of the cis regulatory information required to control expression of the b4 and a 3 genes in specific populations of neurons. q
Journal of Neurobiology, 1998
We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from ad... more We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from adrenal chromaffin-derived PC12 cell RNA that b43, within the 3-untranslated exon of the neuronal encodes a novel ETS-domain factor, Pet-1. The denicotinic acetylcholine receptor (nAchR) b4 subunit duced primary structure of Pet-1 is composed of 340 gene. Similar to Pet-1, the b4 gene is also expressed amino acids and the encoded polypeptide has a prein PC12 cells. The presence of putative ETS-domain dicted molecular mass of 35.4 kD. The pattern of Petbinding sites in the b43 enhancer led us to hypothe-1 gene expression in the neonatal rat is highly resize that members of the ets gene family activate neustricted and suggests that Pet-1 functions primarily in ronal nAchR genes. Cotransfection assays show that the nervous system. Adrenal gland expresses the high-Pet-1 can activate reporter gene transcription in a est level of Pet-1 among the tissues examined. In situ b43 enhancer-dependent and cell type-dependent hybridization indicates that Pet-1 is expressed in the manner. Our results lead us to hypothesize that Petadrenal medulla but not the adrenal cortex. Slightly 1 acts as a transcriptional regulator of downstream weaker Pet-1 hybridization is detected in brain and target genes involved in cholinergic neurotransmislow levels are detectable in intestine and eye. Pet-1 sion. ᭧ 1998 John Wiley & Sons, Inc. J Neurobiol 34: 151-163, can bind specifically to a PEA3 ETS DNA-binding 1998 motif and can modulate transcription of synthetic pro-Correspondence to: E. Deneris cell phenotypes.
Journal of Neurobiology, 1998
We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from ad... more We report a cDNA clone prepared recently identified a neural cell-type specific enhancer, from adrenal chromaffin-derived PC12 cell RNA that b43, within the 3-untranslated exon of the neuronal encodes a novel ETS-domain factor, Pet-1. The denicotinic acetylcholine receptor (nAchR) b4 subunit duced primary structure of Pet-1 is composed of 340 gene. Similar to Pet-1, the b4 gene is also expressed amino acids and the encoded polypeptide has a prein PC12 cells. The presence of putative ETS-domain dicted molecular mass of 35.4 kD. The pattern of Petbinding sites in the b43 enhancer led us to hypothe-1 gene expression in the neonatal rat is highly resize that members of the ets gene family activate neustricted and suggests that Pet-1 functions primarily in ronal nAchR genes. Cotransfection assays show that the nervous system. Adrenal gland expresses the high-Pet-1 can activate reporter gene transcription in a est level of Pet-1 among the tissues examined. In situ b43 enhancer-dependent and cell type-dependent hybridization indicates that Pet-1 is expressed in the manner. Our results lead us to hypothesize that Petadrenal medulla but not the adrenal cortex. Slightly 1 acts as a transcriptional regulator of downstream weaker Pet-1 hybridization is detected in brain and target genes involved in cholinergic neurotransmislow levels are detectable in intestine and eye. Pet-1 sion. ᭧ 1998 John Wiley & Sons, Inc. J Neurobiol 34: 151-163, can bind specifically to a PEA3 ETS DNA-binding 1998 motif and can modulate transcription of synthetic pro-Correspondence to: E. Deneris cell phenotypes.
Journal of Biological Chemistry, 2010
ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor ... more ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked ␣-thalassemia mental retardation (ATR-X) syndrome. Here we perform biochemical and genetic analyses of the Drosophila melanogaster ortholog of ATRX. The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity.
Genes & Development, 2009
We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone ... more 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 ;20% of the level in wildtype 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.
Molecular and Cellular Biology, 2002
PLOS One, 2010
CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histo... more CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3 CENP-A , which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3 CID in Drosophila.
Nature, 2002
The assembly of DNA into chromatin is a critical step in the replication and repair of the eukary... more The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome 1-8 . It has been known for nearly 20 years that chromatin assembly is an ATPdependent process 9 . 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 10,11 . 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 12 . 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.
PLOS One, 2010
CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histo... more CHD1 is a SNF2-related ATPase that is required for the genome-wide incorporation of variant histone H3.3 in the paternal pronucleus as well as in transcriptionally active nuclei in Drosophila embryos. The S. pombe and vertebrate orthologs of CHD1 have been implicated in the assembly of the centromeric histone H3 variant CenH3 CENP-A , which occurs in a DNA replication-independent manner. Here, we examined whether CHD1 participates in the assembly of CenH3 CID in Drosophila.
Genes & Development, 2004
Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin a... more 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.
Science, 2007
The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 i... more 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. 1 to 1394) CHD1 polypeptide. Arrowhead, wild-type CHD1 (250 kD); arrow, left, NAP-1 (loading control);
Cell, 2001
. It thus appears that there is a broad range of nontranscriptional functions of chromatin remode... more . It thus appears that there is a broad range of nontranscriptional functions of chromatin remodeling proteins ).
Genes & Development, 1999
The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI... more 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.
Methods in Enzymology, 2003
To study eukaryotic transcriptional mechanisms in vitro, it is important to analyze gene regulato... more To study eukaryotic transcriptional mechanisms in vitro, it is important to analyze gene regulatory sequences in the context of chromatin. Here we describe the ATP-dependent assembly of chromatin by using completely purified components. This system uses chromatin assembly factors ACF and NAP-1 in conjunction with purified core histones for the assembly of extended periodic arrays of nucleosomes. We additionally describe the assembly of chromatin that contains the linker histone H1. This histone H1-containing chromatin resembles bulk native chromatin in metazoans.