Liver Chromatin Research Articles

Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver

The alpha-fetoprotein (AFP) gene is highly activated in fetal liver but is dramatically repressed shortly after birth. The mechanisms that underlie AFP transcriptional repression in postpartum liver are not well understood. AFP enhancer, repressor region, and promoter are implicated to be involved in AFP postnatal repression, but the major transcriptional repressor remains undefined. We previously identified a zinc finger protein gene ZBTB20. To determine its physiological functions in vivo, we have generated hepatocyte-specific ZBTB20 knockout mice by the Cre/loxP approach and demonstrated here that ZBTB20 ablation in liver led to dramatic derepression of the AFP gene in entire liver throughout adult life, although the hepatocytes were normally under nonproliferating status. Furthermore, we found that ZBTB20 was a transcriptional repressor capable of specifically inhibiting AFP promoter-driven transcriptional activity. Liver chromatin immunoprecipitation and mobility shift assays showed that ZBTB20 bound to AFP promoter directly. ZBTB20 was developmentally activated in liver after birth and inversely correlated with its AFP gene expression, suggesting that activated ZBTB20 expression in liver mediated AFP gene repression. Our data point to ZBTB20 as a key regulator governing AFP gene transcription and postulate a new model for the postnatal gene repression of AFP in liver.

Thematic Review Series: Sphingolipids. Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed.

Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: The role of a gene enhancer in the liver chromatin

The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D 3-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity [Gachon, F, Olela, FF, Schaad, O, Descombes, P and Schibler, U, 2006. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. 4, 25–36.; McElwee, JJ, Schuster, E, Blanc, E, Thomas, JH and Gems, D, 2004. Shared Transcriptional Signature in Caenorhabditis elegans Dauer Larvae and Long-lived daf-2 Mutants Implicates Detoxification System in Longevity Assurance. J. Biol. Chem., 279, 44533–43.]. Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month- and 20-month-old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D 3 and the catatoxic synthetic steroid PCN (pregnenolone-16α-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-α (retinoid X receptor) and PXR in mice injected with D 3 or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.

MacroH2A1 Histone Variants Are Depleted on Active Genes but Concentrated on the Inactive X Chromosome

Using a novel thiol affinity chromatography approach to purify macroH2A1-containing chromatin fragments, we examined the distribution of macroH2A1 histone variants in mouse liver chromatin. We found that macroH2A1 was depleted on the transcribed regions of active genes. This depletion was observed on all of the 20 active genes that we probed, with only one site showing a small amount of enrichment. In contrast, macroH2A1 was concentrated on the inactive X chromosome, consistent with our previous immunofluorescence studies. This preferential localization was seen on genes that are active in liver, genes that are inactive in liver, and intergenic regions but was absent from four regions that escape X inactivation. These results support the hypothesis that macroH2As function as transcriptional repressors. Also consistent with this hypothesis is our finding that the heterochromatin protein HP1beta copurifies with the macroH2A1-containing chromatin fragments. This study presents the first detailed examination of the distribution of macroH2A1 variants on specific sequences. Our results indicate that macroH2As have complex distribution patterns that are influenced by both local factors and long-range mechanisms.

Aspects of large-scale chromatin structures in mouse liver nuclei can be predicted from the DNA sequence

The large amount of non-coding DNA present in mammalian genomes suggests that some of it may play a structural or functional role. We provide evidence that it is possible to predict computationally, from the DNA sequence, loci in mouse liver nuclei that possess distinctive nucleosome arrays. We tested the hypothesis that a 100 kb region of DNA possessing a strong, in-phase, dinucleosome period oscillation in the motif period-10 non-T, A/T, G, should generate a nucleosome array with a nucleosome repeat that is one-half of the dinucleosome oscillation period value, as computed by Fourier analysis of the sequence. Ten loci with short repeats, that would be readily distinguishable from the pervasive bulk repeat, were predicted computationally and then tested experimentally. We estimated experimentally that less than 20% of the chromatin in mouse liver nuclei has a nucleosome repeat length that is 15 bp, or more, shorter than the bulk repeat value of 195 ± bp. All 10 computational predictions were confirmed experimentally with high statistical significance. Nucleosome repeats as short as 172 ± 5 bp were observed for the first time in mouse liver chromatin. These findings may be useful for identifying distinctive chromatin structures computationally from the DNA sequence.

Stability and evolution of transcriptional regulatory networks

Stability and evolution of transcriptional regulatory networks

The Human Alpha Hemoglobin Stabilizing Protein (AHSP) Gene Locus in EKLF-Deficient Erythroid Cells.

AHSP is an erythroid-specific protein that complexes with free α-hemoglobin, protecting it from precipitation. AHSP has been proposed as a modifier gene in β thalassemia and as a candidate gene for unexplained Heinz body anemias, thus understanding its regulation may lead to novel therapies for these disorders. Identified as an erythroid-specific, GATA-1 inducible gene, decreased AHSP mRNA has been found in the fetal livers of mice deficient in the erythroid transcription factor EKLF by both microarray and RNA subtraction analysis. In fetal livers from d13.5 EKLF-deficient mice, AHSP/α-globin mRNA ratios were decreased to 11–16% of wild type by RT-PCR and RPA. In the same fetal livers, no AHSP protein was detected on Western blots with a MoAB against AHSP. EKLF interacts with the proximal CACCC box of the β-globin gene promoter, establishing local chromatin structure and directing high-level β-globin transcription. We hypothesized that chromatin across the AHSP locus would be perturbed in erythroid cells from EKLF-deficient mice. We performed DNase I hypersensitive site (HS) mapping and chromatin immunoprecipitation (ChIP) analysis using wild type and EKLF deficient fetal liver cells. A strong HS was identified in the AHSP 5′ flanking DNA in the core promoter region, that was absent in day 13.5 fetal liver DNA from EKLF-deficient mice. Fine mapping placed this 5′ HS over a CACCC site in the core AHSP promoter. ChIP across the entire AHSP locus with d13.5 fetal liver chromatin identified 2 regions of hyperacetylation of histones H3 and H4 in wild type mice, one corresponding to the 5′ HS and the other 3′ to the AHSP coding sequence. Both of these hyperacetylated regions were hypoacetylated in EKLF-deficient fetal liver cells. ChIP across the AHSP locus with chromatin obtained from mice with an HA tag knocked into the 3′ end of the EKLF gene identified a peak of EKLF binding extending from the 5′HS to intron one, peaking over the core promoter CACCC site. The sequence of this region (ACCCACCCT) has a single mismatch compared to the EKLF consensus site (CCNCNCCCN). Using the AHSP CACCC site as probe in mobility shift assays with rEKLF protein yielded a complex that migrated at the same mobility as a complex obtained with a control β-globin promoter CACCC site probe. Both AHSP and control β-globin complexes were effectively competed by an excess of unlabeled AHSP probe, unlabeled β-globin probe, or ELKF antiserum. Mutant AHSP CACCC probes did not form DNA-protein complexes nor did they effectively displace wild type AHSP CACCC or β-globin CACCC probes in competition assays. Probes with the AHSP CACCC site mutated to the β-globin sequence (A to C) or the other 2 possibilities (A to G, A to T) yielded complexes comparable to wild type AHSP and control β-globin CACCC probes. In transfection assays in K562 cells, an AHSP promoter-luciferase reporter plasmid was transactivated by an EKLF expression plasmid to a degree comparable to a β-globin promoter-luciferase plasmid. These results support the hypotheses that the hemolytic anemia in EKLF-deficient mice is exacerbated by decreased AHSP expression and that EKLF acts as a transcription factor and a chromatin modulator for genes other than β-globin. Our data also support the hypothesis that AHSP and EKLF may be modifier genes for the β-thalassemia syndromes.

Changes in Composition of Acid Soluble Proteins and DNA in Chromatin of Rat Liver and Brain Bound and Not Bound to Nuclear Envelope as a Function of Age and under the Influence of Antioxidant Ionol

In two-day rat pups, the histone H1 content in the brain chromatin was higher than in the liver chromatin, as compared to histone of the nucleosome core. The H1 content in the brain chromatin decreased with the age, while in the liver chromatin it increased. At the same time, in the adult brain chromatin bound to the nuclear envelope, a high level of H1 characteristic of chromatin of the newborn rats was preserved, while in a similar chromatin of the adult liver, the H1 content increased, but still remained less than in the chromatin not bound to the nuclear envelope. In both organs, the composition and quantitation of H1 subfractions were different in chromatins bound and not bound to the nuclear envelope. The chromatin from the liver and brain bound to the nuclear envelope differed also in the composition and quantitation of minor acid soluble proteins. In the presence of the antioxidant ionol, the 5-methylcytosine content in DNA of chromatin of the rat liver bound to the nuclear envelope increased while in the chromatin not bound to the nuclear envelope, it remained unchanged. Thus the chromatins bound and not bound to the nuclear envelope differ in the composition and mount of acid soluble proteins, including histone H1, the contents of these proteins in bound and not bound chromatin are different and change with the age in different ways. The antioxidant ionol affects differently the methylation of bound and not bound chromatin.

Excision of apurinic sites from DNA with enzymes isolated from rat-liver chromatin.

Apurinic sites were excised from phi X174 RF DNA with two enzymes isolated from rat liver chromatin: an apurinic/apyrimidinic endodeoxyribonuclease and a 5'-3'-exonuclease; the resulting gap was filled with DNA polymerase beta also prepared from rat liver chromatin and the repair was fully terminated with T4 ligase.

Long-range oscillation in a periodic DNA sequence motif may influence nucleosome array formation

We have experimentally examined the characteristics of nucleosome array formation in different regions of mouse liver chromatin, and have computationally analyzed the corresponding genomic DNA sequences. We have shown that the mouse adenosine deaminase (MADA) gene locus is packaged into an exceptionally regular nucleosome array with a shortened repeat, consistent with our computational prediction based on the DNA sequence. A survey of the mouse genome indicates that <10% of 70 kb windows possess a nucleosome-ordering signal, consisting of regular long-range oscillations in the period-10 triplet motif non-T, A/T, G (VWG), which is as strong as the signal in the MADA locus. A strong signal in the center of this locus, confirmed by in vitro chromatin assembly experiments, appears to cooperate with weaker, in-phase signals throughout the locus. In contrast, the mouse odorant receptor (MOR) locus, which lacks locus-wide signals, was representative of ∼40% of the mouse genomic DNA surveyed. Within this locus, nucleosome arrays were similar to those of bulk chromatin. Genomic DNA sequences which were computationally similar to MADA or MOR resulted in MADA- or MOR-like nucleosome ladders experimentally. Overall, we provide evidence that computationally predictable information in the DNA sequence may affect nucleosome array formation in animal tissue.

Binding of (MTR) 2Zn 2+ complex to chromatin: a comparison with (MTR) 2Mg 2+ complex

Mithramycin (MTR), a member of aureolic group of anticancer antibiotic, binds reversibly to double stranded DNA via minor groove with (G.C) base specificity. It leads to inhibition of replication and transcription. Results from different laboratories have shown that at and above physiological pH, Mg 2+ is an obligatory factor for the DNA binding and subsequent transcription inhibitory property of mithramycin. Zn 2+ is another physiologically important bivalent cation. Its coordination property leads to its important role as a cofactor in different enzymes and nucleosomal DNA binding proteins. Characterization of the complex between mithramycin and Zn 2+ using spectroscopic methods shows that the drug forms single type of complex with Zn 2+ in the mole ratio of 2:1 in terms of antibiotic: Zn 2+. DNA binding properties of the (MTR) 2Zn 2+ complex has been studied using calf thymus DNA, rat liver chromatin and nucleosome core. (MTR) 2Zn 2+ complex binds to calf thymus DNA with affinity higher than the corresponding dimer complex with Mg 2+ ion. The presence of histone proteins in chromatin and nucleosome reduces the accessibility and hence binding potential of (MTR) 2Zn 2+ complex to nucleosomal DNA. We have also examined the effect of (MTR) 2Zn 2+ complex upon the stability of nucleosome core particle. The complex disassembles nucleosome structure leading to the release of nucleosomal DNA. Significance of the results to understand the molecular basis of the action of the drug in vivo is discussed.

Polynucleosomal Synthesis of Poly(ADP‐ribose) Causes Chromatin Unfolding as Determined by Micrococcal Nuclease Digestion

We have evaluated the influence of protein poly(ADP-ribosyl)ation in the relaxation of chromatin by exposing a rat liver polynucleosomal extract to micrococcal nuclease (MNase) digestion. The kinetic susceptibility of polynucleosomes to endonuclease digestion was determined as a function of the time of incubation as well as endonuclease concentration. To validate our assay, we also ran control experiments with protein-free calf thymus DNA as the opposite of polynucleosomal DNA. Rat liver chromatin was also incubated in the absence or presence of exogenously added 200 microM betaNAD(+), the poly(ADP-ribosyl)ation substrate, before MNase digestion. For incubations in the presence of betaNAD(+), the synthesis of polynucleosomal poly(ADP-ribose) was stopped with 1 mM benzamide. After addition of MNase, endonuclease digestion was blocked with EDTA to chelate the Mg(2+) ions needed for enzymatic activation, and the samples were subjected to electrophoresis through 1.5% agarose gels. As expected, a faster degradation of chromatin into oligonucleosomal DNA ladders was observed upon protein poly(ADP-ribosyl)ation when the chromatin extract was preincubated with 200 microM betaNAD(+). Thus, our results are consistent with the conclusion that the covalent poly(ADP-ribosyl)ation of polynucleosomal proteins favors a more "relaxed" or "open" structure, which renders chromatin more susceptible to MNase digestion.

Defining the Active Chromatin Domain of the Erythroid Ankyrin Promoter.

Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p<0.001; χ2=9.0, p<0.01 respectively), indicating boundary function. We have previously shown that mutations at positions −153 and −108 in 5′HS associated with Hereditary Spherocytosis cause position dependent and variegated expression of the γ-globin transgene in mice. When the −153/−108 sequence was used to flank the β-globin/EGFP transgene gene silencing was observed in 5/10 K562 cell lines (χ2=5.7,p<0.02). Because boundary elements are sites of high levels of histone acetylase (HA) activity we hypothesized that the −153/−108 mutations disrupted histone acetylation in ankR. EMSA analysis showed that the −153/−108 mutations disrupted the binding of a complex that includes both CTCF (a zinc finger protein associated with boundary elements in the β-globin cluster) and Brg-1 (the ATP dependent subunit of the HA complex). Chomatin Immune Precipitation analysis of transfected K562 cells showed that histone acetylation in sequences containing the −153/−108 mutations was 4-fold less than in wild type sequences (p<0.02). These studies have defined the active chromatin domain of the ankR and have demonstrated that the −153/−108 mutations cause Hereditary Spherocytosis by inhibiting histone acetylation in ankR.

Multiple Defects in Erythroid Gene Expression in Erythroid Krupple-Like Factor (EKLF) Target Genes in EKLF-Deficient Mice.

EKLF is the founding member of the KLF family of transcription factors. EKLF-deficient (−/−) mice die at d14-15 of gestation of severe anemia, attributed to decreased expression of the β-globin gene. Morphology of fetal-liver derived circulating erythroid cells in EKLF −/− mice does not mimic that seen in thalassemia, and mice expressing high levels of γ-globin bred onto an EKLF −/− background do not correct the anemia or rescue the −/− mice. These data suggest additional red cell defects are present in EKLF −/− mice contributing to the anemia. To address this hypothesis, we performed subtractive hybridization and microarray analyses with RNA isolated from d13.5 fetal livers of wild type (WT,+/+) and EKLF-deficient mice. In subtractive hybridization, WT (1X) was the tester population and EKLF −/− (30X) was the driver. >200 differentially expressed clones were sequenced. 122 clones were β-globin; 21 alpha hemoglobin stabilizing protein (AHSP); and the remainder were RBC membrane proteins including band 3 and β-spectrin. Microarray analyses were performed with Affymetrix GeneChip Mouse Genome 430 2.0 arrays; 3 independent EKLF +/+ and −/− RNA samples were analyzed. AHSP had the most significantly decreased expression in −/− samples, reduced to 5% of WT (p<0.0001). Other significantly down regulated genes, in addition to those identified by subtractive hybridization, included pyruvate kinase and ankyrin. Reduced expression in EKLF −/− RNA was confirmed using ribonuclease protection assay and/or real-time RT-PCR. AHSP mRNA was decreased by 75–90%; band 3 and β-spectrin were decreased by ~40%; ankyrin by 45%, and pyruvate kinase 15%. Flow cytometry of +/+ and −/ − fetal liver cells using TER119 revealed a TER119Hi population of cells absent in −/ − fetal liver cells, suggesting a block in differentiation to more mature erythroid progenitors. To ensure that the potential EKLF target genes were expressed in the TER119Lo population and were not identified because they were present only in the TER119Hi, the expression of each selected gene was analyzed in TER119Lo and TER119Hi cells by real time PCR. Target gene/α-globin ratios indicated the selected genes were expressed at levels >2 fold higher in TER119Lo than TER119Hi cells. Studies of the AHSP locus identified a strong DNaseI hypersensitive site (HS) in WT fetal liver nuclei between 5′ of the AHSP promoter, that was absent in −/ − chromatin. Chromatin immunoprecipitation analysis of WT fetal liver chromatin spanning 3.5kb of the AHSP locus using 13 primer pairs (~300bp intervals from >1kb 5′ and 3′) demonstrated 2 regions of hyperacetylation of histones H3 and H4. The 5′ region corresponded to the DNaseI HS, and the other region maps 3′ of the AHSP polyA signal. Histones H3 and H4 were also acetylated in the interval between these hyperacetylated regions, while the chromatin upstream and downstream of these regions were hypoacetylated. In chromatin from −/ − fetal liver cells, all sites were hypoacetylated, correlating with the severe reduction in AHSP gene expression. These results support the hypothesis that the anemia in EKLF −/ − mice is due to both decreased expression of the β-globin gene and other erythroid genes including those involved in membrane integrity, stabilization of α-globin protein, and glycolysis leading to defects in erythrocyte structure, function, and metabolism. They also suggest that EKLF may act as a transcription factor and a chromatin modulator for genes other than β-globin.

Chromatin Remodeling of the Mouse AHSP Gene Requires EKLF.

Alpha-Hemoglobin Stabilizing Protein (AHSP) is an erythroid-specific protein that binds to α-globin, preventing precipitation of α-hemoglobin tetramers. Our interest has focused on how the AHSP gene is specifically expressed in erythroid cells, and we have investigated the roles of cis acting DNA sequences, the transcription factor EKLF and chromatin structure on AHSP gene expression. We have previously shown that the AHSP gene has a single mRNA initiation site followed by a non-coding exon. Putative promoter sequences from −904, −479 or −170 were active in luciferase reporter assays only when the constructs contained the downstream 269 bp containing exon 1 and intron 1. The −904/+269, −479/+269 and −170/+269 constructs gave 53.3+2.0 to 122.1+8.8 -fold increased levels of luciferase expression in K562 cells compared to plasmids without exon 1 and intron 1 (p<0.001; Gallagher et al., BLOOD 102, 267a, 2003). In vitro DNase I footprinting and EMSA assays revealed two regions (−75 to −67 and +153 to +164) that bound GATA-1. Analysis of 5 lines of transgenic mice with between 1 and 11 copies of the −170/+269 promoter fused to the human γ-globin gene demonstrated position independent expression (5/5 lines express) of γ-globin mRNA, at levels that were 4.6% the level of mouse α-globin mRNA per transgene copy. There was no correlation between transgene copy number and the level of γ-globin mRNA and 3/5 lines exhibited variegated expression. We concluded that sequences upstream of −170 or downstream of +269 are required for authentic expression from the AHSP promoter. To examine the role of EKLF in AHSP expression, we used subtractive hybridization, microarray and RNase protection analysis to compare AHSP mRNA levels in fetal liver cells from wild type and EKLF−/− mice. EKLF −/− fetal liver cells had 9 -fold less AHSP mRNA than wild type fetal liver cells, which we propose would exacerbate the moderate β- thalassemia described in EKLF −/− mice. Based on the observation that EKLF associates with an erythroid chromatin remodeling complex (Armstrong et al. Cell 95, 93–104, 1998), we hypothesized that EKLF was involved in chromatin remodeling at the AHSP locus. We assayed for DNase I Hypersensitive sites (HS) in chromatin from 13.5 day wild type and EKLF−/− mouse fetal liver nuclei. We demonstrated a strong DNase I HS between −200 and −400 of the AHSP gene, just upstream of the minimal −170 promoter, that was not present in chromatin from EKLF−/− fetal liver cells. To examine histone acetylation across the 3.5 kb AHSP locus we performed Chromatin Immune Precipitation analysis on wild type and EKLF −/− fetal liver chromatin using 13 different primer pairs (~300 bp intervals). In wild type chromatin there are two regions where histones H3 and H4 were hyperacetylated relative to a control region from the mouse α-globin gene promoter. The 5′ region corresponded to the DNase I HS at −400 to −200, while the second region maps 3′ to the AATAAA signal in the AHSP gene. Histones H3 and H4 were also acetylated in the interval between the hyperacetylated regions, while the chromatin upstream and downstream (~1 kb in each direction) of these regions was hypoacetylated. In contrast, all sites were hypoacetylated in chromatin from EKLF−/− fetal liver cells, correlating with the severe reduction in AHSP gene expression. We conclude that EKLF is required for remodeling the chromatin of the AHSP locus and that EKLF could be a modifier gene for the thalassemia syndromes.

Plasmalogens in rat liver chromatin: new molecules involved in cell proliferation.

A minor component of chromatin, the phospholipid fraction, changes during cell cycle as result of the activation of intranuclear lipid metabolism enzymes including phosphatidylcholine-dependent phospholipase C activity. It is known that this enzyme may be activated by phosphatidylcholine plasmalogen (Plg). Until now, there has been little evidences for the presence of Plgs inside the nucleus. The aim of our study is to ascertain if they are present in the nucleus and are responsible of the activation of phosphatidylcholine-dependent phospholipase C during cell proliferation and apoptosis. Therefore, we have analysed the Plg composition of the whole homogenate, cytosol, nuclei and chromatin of hepatocytes. The phosphatidylcholine-dependent phospholipase C activity was assayed using both phosphatidylcholine and plasmalogenyl-phosphatidylcholine as substrates. Our results show, for the first time, that Plgs are present in chromatin and the plasmalogenyl-phosphatidylcholine stimulates the phosphatidylcholine-dependent phospholipase C activity more than phosphatidylcholine. Finally, in order to verify the possible role of these molecules during cell proliferation and apoptosis, we used liver of rats fed with ciprofibrate which stimulates hepatocytes proliferation during the treatment and, after withdrawal, apoptosis. After 3 days of ciprofibrate treatment, the chromatin plasmalogenyl-phosphatidylcholine increases as well as the phosphatidylcholine-dependent phospholipase C activity. After drug withdrawal, when the hepatocytes undergo to apoptosis, the plasmalogenyl-phosphatidylcholine content together with phosphatidylcholine-dependent phospholipase C activity decreases. Therefore, it can be concluded that plamalogens are present in the chromatin, and probably may have a function both in regulating phosphatidylcholine dependent phospholipase C and cell cycle.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and hepatocyte nuclear factor 4gamma (HNF-4gamma) and HNF-4alpha regulate the bovine growth hormone receptor 1A promoter through a common DNA element.

The growth hormone receptor (GHR) 1A promoter is responsible for transcription of the liver-specific GHR mRNA variant 1A in several mammalian species. We previously found that the region between nucleotide -218 and nucleotide -151 (relative to the major transcription start site) of the bovine GHR 1A promoter contained a binding site between -196 and -178 for the liver-enriched transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) and that the same region might also interact with additional transcription factors in the liver. Using the -218/-151 region as bait to screen a bovine liver cDNA library in a yeast one-hybrid analysis, we identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and HNF-4gamma, as well as HNF-4alpha, as binding proteins to the -218/-151 region. These binding proteins were also identified in a second yeast one-hybrid analysis using the -196/-178 region as bait, suggesting that COUP-TFII, HNF-4gamma and HNF-4alpha all bind to the -196/-178 region. Electrophoretic mobility shift assays confirmed the ability of these three transcription factors in bovine liver nuclear extracts to interact with the -196/-178 region. These interactions also appeared to exist in vivo, as the GHR 1A promoter containing the -196/-178 region could be recovered by immunoprecipitation of the bovine liver chromatin with antibody against COUP-TFII, HNF-4gamma or HNF-4alpha. In co-transfection analyses, each of these three transcription factors could activate GHR 1A promoter and the activation was dependent on the -196/-178 region. These results together identify COUP-TFII, HNF-4gamma and HNF-4alpha as transcription factors regulating GHR 1A promoter activity through binding to a common DNA element.

Association of chromatin with anticancer antibiotics, mithramycin and chromomycin A3

Mithramycin and chromomycin A3 are two anticancer antibiotics, which inhibit protein biosynthesis via transcription inhibition. They bind reversibly to DNA with (G.C) base specificity. At and above physiological pH in the absence of DNA, they form two types of complexes with Mg2+, complex I (1:1 in terms of antibiotic: Mg2+) and complex II (2:1 in terms of antibiotic: Mg2+). These are the DNA binding ligands. In vivo, the antibiotics interact with chromatin, a protein–DNA complex. In order to understand the mode of action of these antibiotics at molecular level, we have carried out spectroscopic, gel electrophoretic and UV melting studies of complex I of these antibiotics with rat liver chromatin, nucleosome core particle and DNA stripped of all chromosomal proteins. Analysis of the results has led us to propose that the antibiotic: Mg2+ complex binds to both nucleosomal and linker DNA in native chromatin. Histone proteins reduce the binding potential and accessibility of the complexes to the minor groove of (G.C) rich regions of chromosomal DNA. The antibiotic: Mg2+ complex stabilizes DNA duplex and histone- DNA contacts in chromatin fiber. It also leads to the aggregation of chromatin fibers. From a comparison of the association of the antibiotic: Mg2+ complexes with different levels of chromatin structure and their effects upon the structure, we suggest that the sugar moieties of the antibiotics play a role in the binding process. Significance of these results to understand the molecular basis of the transcription inhibition potential of the antibiotics in eukaryotes is discussed.

Sequential Chromatin Immunoprecipitation from Animal Tissues

This chapter presents two different protocols for formaldehyde crosslinking of mouse tissue chromatin, followed by methods for chromatin solubilization, purification, fragmentation, and immunoprecipitation; some of these steps require special adaptations for animal tissues. The chromatin immunoprecipitation (ChIP) technique for identifying transcription factors and histones bound to specific DNA sites in cells has revolutionized the analysis of gene regulation. In this procedure, cells or nuclei are exposed to formaldehyde, which crosslinks proteins bound to each other and to DNA in native chromatin. The crosslinked chromatin is isolated and fractionated, the proteins are immunoprecipitated and assessed by a quantitative polymerase chain reaction (PCR) to determine the relative extent to which the antigen is associated with the DNA. Although the methods presented in the chapter were developed for liver chromatin, these methods represent a good starting point for working with other tissue types. Despite the technical obstacles regarding the handling of animal tissues for ChIP assays, a growing number of laboratories have made advances in the area.