DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de... more
DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.
Research Interests: Molecular Biology, Biology, Medicine, Gene expression, Transgenic Mice, and 14 moreBiological Sciences, Humans, Mice, Methylation, Animals, DNA methylation, Base Sequence, CpG island, Embryos, Tissue Specificity, Molecular Sequence Data, Restriction Mapping, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains that undergo DNA replication asynchronously during S phase. Here we show, using both fluorescence in situ hybridization... more
Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains that undergo DNA replication asynchronously during S phase. Here we show, using both fluorescence in situ hybridization analysis and S-phase fractionation techniques, that differential replication timing is associated with imprinted genes in a variety of cell types, and is already present in the pre-implantation embryo soon after fertilization. This pattern is erased before meiosis in the germ line, and parent-specific replication timing is then reset in late gametogenesis in both the male and female. Thus, asynchronous replication timing is established in the gametes and maintained throughout development, indicating that it may function as a primary epigenetic marker for distinguishing between the parental alleles.
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The development of mature B cells involves a series of molecular decisions which culminate in the expression of a single light-chain and heavy-chain antigen receptor on the cell surface. There are two alleles for each receptor locus, so... more
The development of mature B cells involves a series of molecular decisions which culminate in the expression of a single light-chain and heavy-chain antigen receptor on the cell surface. There are two alleles for each receptor locus, so the ultimate choice of one receptor type must involve a process of allelic exclusion. One way to do this is with a feedback mechanism that downregulates rearrangement after the generation of a productive receptor molecule, but recent work suggests that monoallelic epigenetic changes may also take place even before rearrangement. To better understand the basis for distinguishing between alleles, we have analysed DNA replication timing. Here we show that all of the B-cell-receptor loci (mu, kappa and lambda) and the TCRbeta locus replicate asynchronously. This pattern, which is established randomly in each cell early in development and maintained by cloning, represents an epigenetic mark for allelic exclusion, because it is almost always the early-replicating allele which is initially selected to undergo rearrangement in B cells. These results indicate that allelic exclusion in the immune system may be very similar to the process of X chromosome inactivation.
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After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid),... more
After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not necessary for generating the overall bimodal methylation pattern but does appear to be involved in resetting methylation patterns during somatic-cell reprogramming.
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CpG island-like sequences are commonly thought to provide the sole signals for designating constitutively unmethylated regions in the genome, thus generating open chromatin domains within a sea of global repression. Using a new database... more
CpG island-like sequences are commonly thought to provide the sole signals for designating constitutively unmethylated regions in the genome, thus generating open chromatin domains within a sea of global repression. Using a new database obtained from comprehensive microarray analysis, we show that unmethylated regions (UMRs) seem to be formed during early embryogenesis, not as a result of CpG-ness, but rather through the recognition of specific sequence motifs closely associated with transcription start sites. This same system probably brings about the resetting of pluripotency genes during somatic cell reprogramming. The data also reveal a new class of nonpromoter UMRs that become de novo methylated in a tissue-specific manner during development, and this process may be involved in gene regulation. In short, we show that UMRs are an important aspect of genome structure that have a dynamic role in development.
Research Interests: Genetics, Algorithms, Reprogramming, Gene regulation, Biology, and 15 moreMedicine, Biological Sciences, Humans, DNA methylation, CpG islands, Genome, Gene, Chromatin, CHEMICAL SCIENCES, Human Genome, Embryonic Development, Microarray Analysis, CpG island, Pluripotent Stem Cells, and Medical and Health Sciences
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The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This... more
The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.
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Research Interests: Genetics, Biology, Silence, DNA replication, Medicine, and 5 moreGene Silencing, Animals, DNA methylation, Gene, and Chromatin
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Research Interests: Reprogramming, Biology, Cell Biology, Epigenetics, Medicine, and 15 moreBiological Sciences, Cell Differentiation, Mice, Methylation, Animals, DNA methylation, Histones, Embryonic Development, Chromatin Immunoprecipitation, Acetylation, Histone, DNA binding proteins, Heterochromatin, fibroblasts, and Medical and Health Sciences
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In animal cells, the process of DNA replication takes place in a programmed manner, with each gene region designated to replicate at a fixed time slot in S phase. Housekeeping genes undergo replication in the first half of S phase in all... more
In animal cells, the process of DNA replication takes place in a programmed manner, with each gene region designated to replicate at a fixed time slot in S phase. Housekeeping genes undergo replication in the first half of S phase in all cell types, whereas the replication of many tissue specific genes is developmentally controlled, being late in most tissues but early in the tissue of expression. Here we employ nuclear DNA injection as an experimental system to test whether this phenomenon is due to differences in the ability to set up transcriptional competence during S phase. Our results show that, regardless of sequence, exogenous genes are a better template for transcription when injected into nuclei of cells in early as opposed to late S phase, and this expression state, once initiated, is preserved after cell division. DNA injected in late S phase is apparently repressed because it is packaged into chromatin containing deacetylated histones, and the same is true for late replicating chromosomal DNA. These findings suggest a mechanistic connection between replication timing and gene expression that might help to explain how epigenetic states can be maintained in vivo.
Research Interests: Biology, DNA replication, Epigenetics, Medicine, Gene expression, and 15 moreMultidisciplinary, Gene Silencing, Cell Division, DNA, Cell line, Animals, Gene, Chromatin, Histones, Acetylation, Chromosomes, Hydroxamic Acids, Gene Expression Regulation, Histone deacetylase inhibitors, and Histone deacetylases
Research Interests: Genetics, Molecular Biology, Biology, Medicine, Gene expression, and 15 moreMultidisciplinary, Nature, Cell Culture, Concentration, Cell Differentiation, Mice, Animals, Embryonic Stem Cell, Gene, Major histocompatibility complex, Antigen, Gene Expression Regulation, Differential expression, Embryonal carcinoma, and endoderm
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Although acetylcholine is a major neurotransmitter in Aplysia, labeling studies with methionine and serine showed that little choline was synthesized by nervous tissue and indicated that the choline required for the synthesis of... more
Although acetylcholine is a major neurotransmitter in Aplysia, labeling studies with methionine and serine showed that little choline was synthesized by nervous tissue and indicated that the choline required for the synthesis of acetylcholine must be derived exogenously. Aanglia in the central nervous system (abdominal, cerebral, and pleuropedals) all took up about 0.5 nmol of choline per hour at 9 muM, the concentration of choline we found in hemolymph. This rate was more than two orders of magnitude greater than that of synthesis from the labeled precursors. Ganglia accumulated choline by a process which has two kinetic components, one with a Michaelis constant between 2-8 muM. The other component was not saturated at 420 muM. Presumably the process with the high affinity functions to supply choline for synthesis of transmitter, since the efficiency of conversion to acetylcholine was maximal in the range of external concentrations found in hemolymph.
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Serotonin and dopamine, both likely transmitter substances in Aplysia, stimulated formation of adenosine-3',5' monophosphate (cAMP) in ganglia, connectives, and identified nerve cell bodies. This widespread distribution suggests... more
Serotonin and dopamine, both likely transmitter substances in Aplysia, stimulated formation of adenosine-3',5' monophosphate (cAMP) in ganglia, connectives, and identified nerve cell bodies. This widespread distribution suggests that receptors for the response are localized throughout the nervous system, as is adenyl cyclase. Both synthesis of cAMP-3H from precursor previously labeled in incubations with adenine-3H and total content of cAMP were stimulated up to 15-fold. The acetylcholine analogue carbachol, glutamate, norepinephrine, and histamine were inactive. Full stimulation occurred within 2–4 min of applying serotonin; the extent of the effect was half maximal at 6µ serotonin. Even in the continued presence of serotonin, the increased cAMP diminished with time. When serotonin was removed, tissue remained refractory for 15–20 min; sensitivity returned after 25 min. Serotonin stimulated cAMP after removal of extracellular Na, K, or Cl and in isotonic sucrose, with all e...
Research Interests: Endocrinology, Biology, Medicine, Dopamine, Adenosine, and 15 moreInternal Medicine, cyclic AMP, Animals, Acetylcholine, Article, Mollusca, Medical Physiology, Neurons, Nervous System, Action Potentials, Adenine, General Physiology, Adenosine Triphosphate, Aplysia californica, and Electric stimulation
Although most mammalian genes are expressed from both alleles, there is a small group of special genes which are imprinted so that only one of the parental alleles is actually expressed in target cells. This epigenetic process involves... more
Although most mammalian genes are expressed from both alleles, there is a small group of special genes which are imprinted so that only one of the parental alleles is actually expressed in target cells. This epigenetic process involves regulation at a number of different stages of development and is very complex. In principle, imprinted gene regions must be marked in cis in the gametes using epigenetic features capable of being maintained through cell division and able to direct multigenic monoallelic expression in differentiated cells of the mature organism. The difference between alleles must be erased during early gametogenesis to allow the imprint to be reset in the mature gametes. In this review we will summarize what is currently known about the molecular mechanisms which mediate these steps. J. Cell. Biochem. 88: 400–407, 2003. © 2002 Wiley‐Liss, Inc.
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Research Interests: Genetics, Immunology, Biology, Epigenetics, Medicine, and 6 moreGene Silencing, Animals, Chromatin, Allele, Allelic Exclusion, and alleles
We studied the molecular mechanism of demethylation and its role in kappa chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a... more
We studied the molecular mechanism of demethylation and its role in kappa chain gene regulation. Following transfection into B cell cultures, this gene undergoes regional demethylation in a process that is developmentally regulated in a lineage- and stage-specific manner. Although a germline V kappa promoter is not required for the demodification activity, a fragment containing the intronic kappa chain transcriptional enhancer and the nearby matrix attachment region is essential. In its natural location downstream to the J kappa 5 sequence, this element induces bidirectional demodification of plasmid constructs in a distance- and orientation-independent manner. When this enhancer is placed in an upstream position, however, the kappa gene remains modified and transcriptionally inactive, demonstrating that demethylation is required for kappa chain activation. These studies suggest that the kappa enhancer plays a dual role in regulating B cell differentiation by inducing demethylation and by promoting tissue-specific transcription.
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Research Interests: Biochemistry, Computer Science, Chemistry, Kinetics, Medicine, and 15 moreDNA, Icon, Escherichia coli, Animals, Nucleic acid hybridization, Proteins, Citation, Genes, Chromatin, Ducks, ICON, Biochemistry and cell biology, Adenosine Triphosphate, Polyribosomes, and Medical biochemistry and metabolomics
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Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell... more
Stochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-al...
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Significance We have studied the dynamics of DNA methylation in pancreatic α- and β-cells and reached surprising insights into the establishment of islet cell identity. Different islet cell types share lack of methylation in... more
Significance We have studied the dynamics of DNA methylation in pancreatic α- and β-cells and reached surprising insights into the establishment of islet cell identity. Different islet cell types share lack of methylation in cell-type–specific gene promoters, while DNA methylation differences between islet cell types are concentrated in enhancer regions. The findings support the fundamental role of enhancer methylation in determining cell identity, and have implications for the understanding of islet cell plasticity in diabetes.
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Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring... more
Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation o...
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The degree of methylation of active regions of the chromosome has been investigated by several techniques. DNase I (deoxyribonuclease I, EC 3.1.21.1) was used to introduce nicks in the active regions of the nucleus and thereby... more
The degree of methylation of active regions of the chromosome has been investigated by several techniques. DNase I (deoxyribonuclease I, EC 3.1.21.1) was used to introduce nicks in the active regions of the nucleus and thereby specifically label these areas. By using the methylation-specific restriction enzymes Hpa II and Hha I it could be shown that active genes are more sensitive to these probes than are other parts of the genome. In order to measure the amount of methylation at all CpG residues, DNA was nick-translated in the presence of [alpha-32P]dGTP as the sole nucleotide source and the methylated cytosine was detected by the standard nearest-neighbor analysis. Using this assay, we found that about 70% of all CpG sequences in animal cell DNA are methylated. In active nuclear regions that are sensitive to DNase I, only 30-40% of the CpG residues are methylated. This method was also employed to study the gene sequences that are complementary to cellular RNA. By this criterion e...
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Replication timing is an important developmentally regulated regional property that is correlated with chromosome structure and gene expression, but little is known about the establishment and maintenance of these patterns. Here we... more
Replication timing is an important developmentally regulated regional property that is correlated with chromosome structure and gene expression, but little is known about the establishment and maintenance of these patterns. Here we followed the fate of replication timing patterns in cells that undergo reprogramming either through somatic-cell nuclear transplantation or by the generation of induced pluripotential stem cells. We have investigated three different paradigms, stage-specific replication timing, parental allele-specific asynchrony (imprinted regions), and random allelic asynchronous replication. In all cases, somatic replication timing patterns were reset exactly at the appropriate stage in early development and could be properly established upon re-differentiation. Taken together, these results suggest that, unlike DNA methylation, the molecular mechanisms governing replication timing are not only stable but can also be easily reprogrammed.
Research Interests: Genetics, Biology, Genomic Imprinting, DNA replication, Cellular Reprogramming, and 15 moreCell Biology, Epigenetics, Medicine, Gene expression, Biological Sciences, Cell Differentiation, Female, Animals, Male, Embryonic Stem Cells, Differentiation, Embryonic Development, alleles, Medical and Health Sciences, and Cell culture techniques
Chicken erythrocyte nuclei have been labeled in the active regions of the chromosome by using the nick translation reaction. In this procedure, accessible areas of the genome are preferentially nicked by the action of pancreatic DNase I... more
Chicken erythrocyte nuclei have been labeled in the active regions of the chromosome by using the nick translation reaction. In this procedure, accessible areas of the genome are preferentially nicked by the action of pancreatic DNase I and subsequently labeled by using DNA polymerase I from Escherichia coli. These nuclei were employed as a substrate for studying the factors responsible for maintaining the special chromatin conformation of the overall population of active genes. Treatment of nuclei with 0.35 M NaCl resulted in the loss of DNase I sensitivity in the active genes, but this sensitivity could be restored when nuclei were reconstituted with the NaCl eluate. Further purification of the released factors revealed that the HMG (high-mobility group) proteins HMG-14 and HMG-17 are involved in maintaining the conformation of the active regions. These factors are not tissue specific and seem to be involved in the chromosomal structure of most of the active genes.
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DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On... more
DNA-mediated gene transfer was used to investigate the mode of inheritance of 5-methylcytosine in mouse L cells. Unmethylated phi X174 replicative form DNA remains unmethylated after its introduction and integration into these cells. On the other hand, phi X174 replicative form DNA that was methylated in vitro at its C-C-G-G residues retains these methylations as shown by restriction enzyme analysis with Hpa II and Msp I to detect methylation at this specific site. Although these unselected methylated vectors are prone to lose 30-40% of their methyl moieties upon transfection, this demethylation appears to be random. Once established, the resulting methylation pattern is stable for at least 100 cell generations. In order to examine the specificity of methylation inheritance, fully hemimethylated duplex phi X174 DNA was synthesized in vitro from primed single-strand phi X174 DNA by using 5-methyl deoxycytidine 5'-triphosphate. This molecule was inserted into mouse L cells by cotr...
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The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was... more
The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low fre...
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The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the... more
The herpesvirus thymidine kinase gene has been used to introduce foreign DNA sequences into mouse L cells by DNA-mediated gene transfer. These inserted genes were then assayed for methylation at the specific sequence C-C-G-G by using the restriction enzyme isoschizomers Hpa II and Msp I. Despite the fact that 70% of the cellular C-C-G-G sites are methylated, herpesvirus sequences, plasmid DNA, and growth hormone gene DNA were found to remain unmethylated in 90% of the clones that contain these genes. DNA that had been methylated in vitro with Hpa II methylase was also inserted into L cells. The presence of this modification in the vector DNA did not, however, guarantee that these sequences remained methylated in the recipient clones. Only 10% of all transformed clones were found to contain methylated C-C-G-G sequences in the vector DNA, and these modifications were stable for 25-50 generations. Hha I and Mbo I were used to probe for methyl groups at these restriction sites, but none...
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We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification... more
We have introduced [alpha-32P]dGTP into permeabilized cells and measured the degree of methylation at CpG sites by nearest-neighbor analysis. This method reveals a lag of approximately 1 min between DNA synthesis and the modification event. When methylation is inhibited by the addition of S-adenosyl-L-homocysteine in the presence of continued DNA synthesis, the resulting hemimethylated sites are methylated immediately after the release of inhibition. The results suggest that the methylase activity in the cell allows immediate methylation but conditions at the replication fork bring about a short delay in the onset of the modification reaction.
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The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA... more
The proteins of chromatin serve to restrict the transcription of DNA. The relevance of these findings to the control of gene expression is contingent upon the demonstration that this restriction is specific and mirrors the patterns of RNA synthesis observed in vivo . In this study we demonstrate by RNA-DNA hybridization that the vast majority of the chromatin-directed RNA is synthesized from the unique regions of the reticulocyte genome. Furthermore, by use of the DNA complement of globin mRNA as a probe in annealing reactions, de novo synthesis of globin RNA was detected in RNA transcripts from duck reticulocyte chromatin. No globin sequences were detected in similar preparations of RNA in vitro either from liver chromatin or from DNA freed of protein. These results show that the proteins of chromatin serve to restrict transcription in a very specific manner and provide convincing evidence for the existence of transcriptional control factors in eukaryotes.