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Departments of Genetics and Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Nancy E. Cooke, 752b Clinical Research Building, University of Pennsylvania, 415 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: necooke{at}mail.med.upenn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The hGH multigene cluster represents a tightly controlled and robustly expressed model system for analysis of developmental controls. This cluster, located on chromosome 17q22–24, is composed of five structurally conserved genes: hGH-N, human chorionic somatomammatropin (hCS)-L, hCS-A, hGH-V, and hCS-B (Fig. 1A
). These genes evolved subsequent to the rodent/primate divergence via successive local duplications of a precursor GH gene (9, 10). The five genes display distinct, mutually exclusive tissue specificities despite their close juxtaposition and strong structural conservation. hGH-N is expressed in somatotrope cells of the anterior pituitary; the remaining four genes are expressed in the syncytiotrophoblast (STB) cellular layer that lines the fetal-maternal interface of the placental villi. The four placental genes further differ in levels of expression. hCS-A and hCS-B are robustly transcribed in the mid- to late-gestation placenta. hGH-V mRNA is expressed at 100- to 1000-fold lower levels (9, 11), but its expression is sufficient to encode the major GH in maternal serum (12). hCS-L activity, although quite low, is difficult to accurately quantify because most of its transcripts are shunted along nonproductive splicing pathways (13). The B lymphocyte-specific CD79b gene immediately flanks the hGH cluster at its 5'-end and encodes Igß, one of the two B cell receptor subunits. The striated muscle-specific SCN4A gene, encoding a sodium channel component, is located further 5'. The testis-specific TCAM gene flanks the hGH cluster at its 3'-end (Fig. 1A). This close packing of eight genes expressed in five different tissues presents a complex and potentially informative model of gene regulation.
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). The sets of HS formed in the chromatin of pituitary somatotrope cells and placental STB cells are partially overlapping. HSV and HSIII are found in both cell types, HSIV is placenta specific, and HSI and -II are pituitary-specific. When linked to the full LCR, the hGH multigene cluster is expressed in transgenic mice in a copy number-dependent and site-of-insertion-independent manner; hGH-N is expressed specifically in the pituitary, and the hCS/hGH-V genes are selectively expressed in the placenta (14, 15).
Distinct patterns of chromatin modification are associated with activation of the hGH cluster in the pituitary and the placenta. Pituitary somatotropes expressing hGH-N establish a 32-kb domain of acetylated chromatin. This domain encompasses the entire LCR and contiguous hGH-N promoter but excludes the four placental genes (16). The formation of this acetylated domain is dependent upon HSI, which is pituitary specific and located in the center of the acetylated domain. Selective deletion of HSI results in loss of acetylation throughout the region and loss of hGH-N expression in transgenic pituitaries (17). In placental chromatin, acetylation 5' of the cluster is limited to the region encompassing HSIII–HSV (18). Regions internal to the multigene cluster are selectively acetylated in placental chromatin. One strongly acetylated region within the cluster encompasses the placental P element. The P elements are a set of conserved sequences situated 2 kb upstream from each of the four placental genes that have been implicated in the control of placental gene expression (Fig. 1). Whether the P element enhances gene expression in the placenta and/or represses gene expression in pituitary remain major unresolved questions (18, 19).
Establishing the profile of histone modifications at a locus can yield insights into epigenetic pathways leading to mechanisms of gene activation. This has been the case for the hGH-N gene in the pituitary (16, 17). It may be equally informative to define the patterns of epigenetic modification in the placenta and contrast them to those in the pituitary. However, prior attempts to establish a comprehensive picture of chromatin modification within the GH cluster were impeded by structural similarities among its five gene units and were limited in the repertoire of histone modifications studied (18, 20). In the present report we have substantially increased the density and resolution of the epigenetic map at the hGH multigene locus in human placental STB chromatin. The patterns of histone acetylation and methylation suggested that these modifications play distinct roles in selective activation of the placental genes from the hGH cluster.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and Table 1). PCR amplimers located 5' and 3' to the cluster corresponded to unique genomic elements. The cluster itself is comprised of five repeated domains reflecting duplications of an initial precursor gene in primate species. The initial duplication resulted in GH and CS precursor genes. The 3'-flanking regions of these two genes diverged significantly, and the GH and CS units then underwent duplications to result in two GH repeated domains encompassing GH-N and GH-V and three CS repeated domains encompassing CS-A, CS-B, and CS-L (Fig. 1A). Thus amplimers located within the multigene cluster detect sites represented in three to five of these repeated domains (Fig. 1, A and B). The level of histone modification at each site was calculated as the ratio of DNA in the antibody-bound chromatin to that in the input sample. This ratio was normalized to the corresponding ratio for a constitutively positive promoter--glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, defined as 100. Parallel analysis of normal human fibroblast chromatin provided an assessment of tissue specificity. Each PCR signal was documented to be in the linear range by analysis of serial dilutions of each template. A representative set of studies scanning the entire hGH locus for acetylated histone H3 is shown in Fig. 2A. Compilations of multiple studies of histone H3 and H4 acetylation across the locus are represented in Fig. 2, panels B and C, respectively.
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, A and B, light bars). Thus, histone H3 acetylation in placental chromatin was prominently detected at HSV and HSIII of the LCR and throughout the hGH cluster. The acetylated LCR and gene cluster were separated by a 28-kb gap of minimally modified chromatin.
Histone H4 acetylation at the hGH locus.
The levels of H4 acetylation at the hGH locus were less robust than H3 acetylation, but the pattern was generally conserved; there were maximal acetylation at the placental LCR and at the hGH cluster with marginal levels of acetylation in the intervening 28 kb (compare panels B and C of Fig. 2, black bars). In contrast to the H3 acetylation study, HSIV was strongly H4-acetylated when compared with the GAPDH promoter control. Fibroblast chromatin was H4 acetylated at low and variable levels throughout the 98-kb region with highest levels at HSI, II and Pe8 (Fig. 2C, light bars). Thus, as was the case for H3 histones, the acetylation of H4 histones in placental chromatin was most prominent at the placental LCR and within the hGH cluster.
The hGH Cluster Is Encompassed in a Continuous Domain of Acetylated Chromatin
ChIP analyses within the hGH gene cluster revealed significant levels of H3 and H4 acetylation at all sites (Fig. 2). However, each of the amplimers within the cluster is represented in three to five repeated domains. Therefore, the data in Fig. 2 do not establish whether the modification of the cluster is continuous or is limited to specific repeated domains. To address this, each of the four P elements and each of the five gene units were individually assessed for H3 and H4 acetylation (Figs. 3 and 4, respectively).
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). Signals corresponding to each of the four individual P element determinants were then identified and quantified by sequential hybridization of the amplified fragment with oligoprobes corresponding to each of the four P elements (PL, PA, PV, and PB) (Fig. 3). This targeted detection revealed that each of the P elements was acetylated on histones H3 and H4 at levels equal to or greater than the GAPDH control. Levels of histone H4 acetylation were roughly equivalent at each of the P elements, whereas histone H3 acetylation varied by as much as 2-fold in pairwise comparisons (i.e. PV vs. PA).
The placental genes are selectively acetylated at histone H3.
Modifications at each of the five gene units were detected by amplification with a primer pair common to the 5'-terminus of all five genes (amplimer 233GH/CS; Fig. 1B). This was followed by digestion of the amplified DNA with two partially overlapping sets of restriction enzymes that recognize rare sequence differences specific to each of the five genes (Fig. 4A). The 159-bp hCS-L fragment, common to both sets of digestions, was used to normalize the two data sets (Fig. 4A, linked arrows). This assay revealed robust acetylation of histone H3 at each of the four placental genes and a significantly lower level of modification at hGH-N (Fig. 4B). Analysis of histone H4 acetylation demonstrated acetylation at all five genes at levels equivalent to the active gene (GAPDH) promoter (Fig. 4C).
The combined ChIP studies (Figs. 2, 3, and 4) showed that the four placental genes in the hGH cluster are encompassed within a continuous domain of acetylated chromatin. This domain does not form in fibroblast (Fig. 2) or pituitary chromatin (16). The acetylated H4 domain extends further 5' in the cluster than the acetylated H3 domain, as indicated by the higher level of modification at the hGH-N gene (compare panels B and C of Fig. 4). LCR determinants HSV and HSIII, common to pituitary and placenta, are acetylated on histones H3 and H4, whereas the placenta-specific HSIV is more strongly modified at histone H4 than H3. The placental LCR is separated from the hGH cluster by a 28-kb intervening region that is only minimally acetylated.
Analysis of Histone H3K4 Methylation at the hGH Locus
Histone acetylation and methylation can play distinct roles in epigenetic pathways (8, 21). Di- and trimethylation of histone H3 at lysine 4 (H3K4-me2 and H3K4-me3) appear to be of particular importance in gene activation and/or prevention of gene silencing; di- and trimethylation at H3K4 may mediate distinct functions and/or reflect recruitment of distinct coactivator complexes (22, 23, 24). Therefore, ChIP studies were carried out on placental chromatin to specifically assess the levels and pattern of di- and trimethylation at H3K4. Remarkably, H3K4-me2 modification was essentially confined to the repeated domains of the gene cluster; there was minimal evidence of tissue-specific modification 5' to the hGH cluster (Fig. 5, A and B). In this region only HSIII was dimethylated at levels that exceeded the flanking SCN4A and TCAM genes. However, HSIII was also modified in fibroblast chromatin. Thus the placental LCR lacked significant or specific H3K4-me2 modification.
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, A and B). Of note, sites immediately 3' of the hGH-N and hGH-V genes (amplimer 3'GH) were unmodified. There was no significant modification in the fibroblast chromatin. As defined by histone H3K4-me2 modifications, these data indicated that the hGH gene cluster in placental chromatin is organized as a series of discontinuous domains encompassing individual gene units and their immediate flanking sequences.
ChIP was next carried out with antibodies to the H3K4-me3. HSV was robustly modified at levels comparable to the GAPDH promoter (Fig. 5C, black bars). HSIV and HSIII showed much lower, but still significant, levels of H3K4-me3 modification. Levels in the region extending from HSIII through the hGH-N promoter (hGHNPr amplimer) were quite low, but appeared to be significant because they remained above that at the flanking SCN4A locus (SCNex13) and above the matched fibroblast controls.
H3K4-me3 modification was detected at most sites within the hGH cluster and extended further 5' and 3' than the dimethylated region. The levels of H3K4-me3 modification within this region appeared to increase gradually from the 5'-end of the repeated domains toward the center. The most prominent modification was at the structural genes (hGH/CS amplimer) and immediately 3' to the hCS genes (amplimer 3'CS1), and the least modified of any site in the cluster corresponded to the P element core (Pe2). There was no appreciable H3K4-me3 modification at any site within the hGH locus in fibroblast chromatin (Fig. 5C, white bars).
H3K4 Methylation in the hGH Cluster in STB Chromatin Is Restricted to the Placental Genes
The di- and trimethylation at H3K4 within the hGH cluster might encompass each of the five repeated domains or be more limited in distribution. To further define this epigenetic profile, the gene units were individually analyzed (Fig. 6). H3K4-me2 modification levels varied dramatically among the five structural genes. When compared with the controls, the modification at hGH-N was limited to background levels (6% of GAPDH). In contrast, hCS-B was modified at 20% of the GAPDH control, hCS-A was modified at 38%, and hGH-V and hCS-L regions were the most strongly modified at 65–72%. The analysis of trimethylation at H3K4 gave a more uniform result (Fig. 6C); all four placental genes in the cluster were robustly modified, ranging from 106–162% of the positive control. In contrast, the hGH-N gene was modified to a far lower extent (39% GAPDH).
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and 6) showed a modification pattern that was distinct from the acetylation pattern. Dimethylation of H3K4 defined four distinct subdomains in the hGH cluster in human placental chromatin corresponding to the four placentally expressed genes. Trimethylation at H3K4 encompassed the entire placental LCR (HSIII-HSV) with robust modification at HSV. It also defined the same four subdomains in the hGH cluster encompassing each placental gene unit. However, the trimethylated subdomains in the cluster extended more 5' and 3' than the dimethylated subdomains. The P element core was remarkable in that analyses of both di- and trimethylation at H3K4 demonstrated only minimal modifications at this site. A summary of the histone acetylation and methylation patterns at the hGH cluster in placental chromatin is presented in diagrammatic format in Fig. 7A.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). This HAT activity spreads to create a continuous 32-kb domain of modified chromatin that encompasses the hGH-N promoter, facilitating local trans-factor binding, and triggering gene transcription (17). In the present study, a distinct pattern of chromatin modifications is defined at the hGH locus in placental chromatin. This epigenetic profile is based on ChIP analyses of 73 sites spanning the locus. The resultant data support and extend a model in which activation of the hGH cluster in the pituitary and placenta are mediated by two distinct pathways of chromatin modification.
A Distinct Pattern of Histone Acetylation and Methylation at the hGH Locus in Placental Chromatin
A comparison of the epigenetic profiles in pituitary and placenta suggest that activation of the placental genes from the hGH cluster relies on a more complex pathway. There are several notable differences between these epigenetic profiles. The acetylated and methylated chromatin domains encompassing the LCR and the gene cluster in placental chromatin are separated by a 28-kb region of minimally modified chromatin (Fig. 7A). This contrasts significantly with the continuous, robustly acetylated domain extending from LCR to hGH-N in pituitary chromatin (Fig. 7B). The placental LCR is further removed from its target genes than the pituitary LCR. The hGH-N gene is maintained in a silent state in placenta despite its location between the LCR and activated placental genes. Finally, the placental genes themselves are encompassed in discrete chromatin subdomains, and they are activated to different magnitudes. Thus, clear differences distinguish the epigenetic pathways used by the partially shared LCR in placental and pituitary genes.
Aspects of the placental gene activation pathway may be inferred by considering the pattern of epigenetic modification. The gene cluster in placental chromatin is encompassed in a continuously acetylated domain, but is subdivided into four subdomains defined by H3K4 di- and trimethylation (Fig. 7A). The continuously acetylated domain suggests that HAT activity, once recruited to the cluster, may spread throughout the cluster. This model is reminiscent of HAT targeting to HSI in pituitary chromatin with its subsequent establishment of an expansive domain encompassing the hGH LCR (Fig. 7B). However, an alternative model based on multisite targeting and coalescence of subdomains may be more directly suited to the placental chromatin. Such multisite targeting could be mediated by determinants repeated in each of the four placental gene units (Fig. 1B). Although it should be possible to distinguish these two models by selective interruption of candidate targeting sites, this approach will be technically challenging due to the conserved nature of the hGH genes and their surrounding sequences.
The sites that recruit HAT coactivators to the hGH cluster in STB nuclei remain to be identified. A role for the P elements in this pathway is supported by its strong evolutionary conservation, its linkage to each of the four placental genes, its robustly acetylated state, and its ability to independently activate placental gene expression in transgenic models (14, 18). Prior work from our laboratory using a mixture of antibodies to acetylated forms of histones H3 and H4 indicated that the P element is robustly acetylated in placental chromatin (18). By using separate antibodies to acetyl-H3 and acetyl-H4, and monitoring at multiple sites throughout the cluster, the present study supported these findings and further revealed that the P element is embedded within an uninterrupted acetylated domain. A less extensive study of histone H4 acetylation by others is consistent with this observation (20). Studies from our laboratory and from others have failed to identify binding factors in human term placental nuclear extracts that might mediate P element activity (Ref.19 and our unpublished studies). More detailed studies in developmentally dynamic settings will be needed to determine whether the P element is a HAT recruitment site and to identify the underlying cis-trans interactions. Generation of a continuous acetylated domain by multisite HAT recruitment with subsequent fusion of acetylated subdomains would represent a novel pathway of epigenetic modification.
The domain of acetylated chromatin that encompasses the hGH gene cluster in STB nuclei is subdivided into smaller subdomains defined by H3K4 methylation (Figs. 5, 6, and 7A). The H3K4 dimethylated subdomains extend from approximately 1 kb 5' to the transcription start sites of each of the placental genes (boundary between Pe4 and Pe6) to approximately 3.8 kb 3' to the CS genes (boundary between 3'CS1 and 3'CS2). These dimethylated subdomains encompass the individual placental gene units with their respective promoters and are separated from each other by a segment of chromatin in which dimethylation is at marginal levels (Pe0 to Pe4). The H3K4 trimethylated subdomains overlap the dimethylated subdomains and extend further 5' and, most markedly, further 3'. Remarkably, H3K4 di- and trimethylation domains both effectively exclude the P element core (Pe2) (Figs. 5 and 7A). These data suggest that H3K4 methylation is most likely mediated by multisite recruitment of histone methyltransferase (HMT) coactivator complexes and that any role(s) of the P element in HAT recruitment is divorced from recruitment of HMT complexes.
The relationship between chromatin acetylation and H3K4 methylation has been studied in a number of systems. The literature supports a model in which HMT recruitment and histone acetylation are linked processes and/or occur in parallel (25, 26, 27, 28, 29, 30), although certain exceptions have been reported [e.g. the mouse ß-globin locus (31)]. Certain studies support a cooperative, one-step recruitment of HAT and HMT (32, 33), whereas others support sequential recruitment (34, 35). The pattern of di- and trimethylation at the hGH cluster is most consistent with four independent targeting events (Fig. 7A). The sites of HMT recruitment within the cluster might correspond to the robustly methylated peaks at the hGH/hCS gene units and the 3'CS1 regions (Fig. 5, B and C). The peak at the gene unit may reflect a linkage between PolII elongation and HMT activity, and the methylated chromatin domain may serve as a memory module or reinforcing structural alteration (36). The 3'CS1 site is located immediately 3' to a putative hCS enhancer element known to be active after transient cell transfections (37); its in vivo epigenetic modification may relate to its enhancer activity. However, in vivo function of this putative enhancer element has not been demonstrated. Thus, the current data suggest that the domain of histone acetylation and the subdomains of histone H3K4 methylation at the hGH locus in the placenta are established by independent sets of determinants and reflect distinct facets of the placental gene activation pathway.
The low level of chromatin modification in the region between the placental LCR and the gene cluster may be of particular interest. In pituitary chromatin this region is encompassed in a continuous and robustly acetylated domain (Fig. 7B). The levels of histone H3 and H4 acetylation and H3K4 methylation in this region in placental chromatin are low but significant when compared with fibroblast chromatin or to modifications at the flanking SCN4A and TCAM. Studies in the literature suggest that certain modifications, such as H3K4-me3, may be generated during initial gene activation and persist for considerable time after the activation events have occurred (38, 39). It is possible that the low-level trimethylation and acetylation in the region between the LCR and the gene cluster may reflect residual modifications imparted by a prior chromatin activation event in placental cells. Establishing models in which the developmental process of hGH cluster activation can be followed temporally will allow this model to be tested.
Activation of the hGH Genes in Pituitary and Placenta
The hGH LCR is necessary for full activation of the multigene cluster in both pituitary and placenta (14 ,15). In pituitary, a tissue-specific transcription factor, Pit-1, binds to an array of sites at HSI and recruits HAT activity to establish the broad histone acetylation domain that is essential to hGH-N transcription (Fig. 7B and Refs.16 ,17 , and 40). This spreading of histone acetylation conforms to the tracking model for LCR action (41, 42). In contrast, the substantial separation of the placental LCR determinants (HSV-HSIII) from the target genes is more consistent with activation via a looping mechanism (Fig. 7A). Evidence that looping actually occurs in vivo has been recently provided in the mouse ß-globin locus where the ß-globin LCR is separated from the ß-globin target genes by 30 kb of unmodified intervening sequences (43, 44, 45, 46, 47). Surprisingly, deletion of the ß-globin LCR, although decreasing ß-globin expression, does not diminish the levels of histone acetylation at the active ß-globin genes (44). These data have been interpreted to indicate that the direct contact of the ß-globin LCR with the target ß-globin promoters regulates the rate of transcription but does not play an active role in establishment of the acetylated domains (48). Thus, analysis of the ß-globin cluster supports a looping interaction between its LCR and target promoters and suggests that the epigenetic imprint at the locus is established before this interaction.
The epigenetic structures of the hGH locus in pituitary and placental chromatin support a model in which looping and tracking pathways may be selectively used in a tissue-specific manner (Fig. 7). In contrast to the findings in the ß-globin cluster, the continuous domain of acetylation in pituitary somatotropes is wholly dependent on LCR function; inactivation of HSI results in loss of this acetylated chromatin domain as well as loss of hGH-N expression (17). The general epigenetic structure of the cluster in the placenta is more closely parallel to that of the ß-globin locus with a spacer of minimally modified chromatin separating the LCR determinants from the target promoters (45). HSIII–HSV regions and the gene cluster regions may recruit histone modification complexes independently. A looping mechanism may then bring the two activated regions into physical proximity with resultant assembly of promoter initiation complexes and initiation of gene transcription (Fig. 7A). Looping of the LCR to the hGH cluster could be selectively directed to each of the placental gene promoters, yet effectively exclude the hGH-N gene. The question of whether the four subdomains of the hGH cluster are established and function as independent entities is now open to study. Furthermore, the model of independent targeting of modifiers to the LCR and cluster and selective activation of the four placental genes via a looping mechanism can also now be directly tested.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Purification of Nuclei from Cultured Cells
The human skin fibroblast cell line, CCDsk-25 (American Type Culture Collection, Manassas, VA), was cultured at 37 C with 5% CO2 in MEM (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Invitrogen Life Technologies). Cells were washed with PBS and lysed with Nonidet P-40 (NP-40) lysis buffer [10 mM Tris-HCl, pH 7.5; 10 mM NaCl; 3 mM MgCl2; 0.5% NP-40; and proteinase inhibitor cocktail (Roche Molecular Biochemicals)] containing 10 mM sodium butyrate on ice. The lysate was incubated on ice for 10 min, and then nuclei were collected by centrifugation. The nuclei were washed with NP-40 lysis buffer twice and stored in glycerol storage buffer containing 20 mM sodium butyrate before analysis.
Chromatin Immunoprecipitation Assay of Unfixed Chromatin
The ChIP assay was conducted as previously described with minor modifications (16). Digestions used 0.6 mg nuclei in 1 ml digestion buffer [50 mM NaCl; 20 mM Tris-HCl, pH 7.5; 3 mM MgCl2; 1 mM CaCl2; 10 mM sodium butyrate; 0.1 mM phenylmethylsulfonyl fluoride (PMSF)]. These were digested with 20 U of micrococcal nuclease (Amersham Biosciences, Piscataway, NJ) at 37 C for 6 min. Digestions were terminated by addition of EDTA to 0.5 mM, and samples was centrifuged at 12,000 x g for 10 min at 4 C to generate supernatant S1. The pellet was resuspended in 0.6 ml of low-salt lysis buffer [10 mM Tris-HCl, pH 7.5; 10 mM sodium butyrate; 0.25 mM EDTA; 0.1 mM PMSF], incubated on ice for 10 min, and then centrifuged as before. Repeating this step generated supernatants S2 and S3. The three supernatants were combined and concentrated with Amicon Microcon centrifugal filters (Millipore Corp., Bedford, MA). Fifteen percent of the resulting soluble chromatin was kept as input, and the rest was separated into two fractions, each adjusted to 0.5 ml with immunoprecipitation (IP) buffer (50 mM NaCl; 20 mM Tris-HCl, pH 7.5; 5 mM EDTA; 10 mM sodium butyrate). The immunoprecipitation reactions were performed on these fractions by adding 10 µl of antibody and incubating the samples overnight at 4 C with gentle rotation. The antibodies used were: antiacetyl histone H3 (catalog no. 06–599, Upstate Biotechnology, Inc., Lake Placid, NY), antiacetyl histone H4, ChIP Grade (catalog no. 06–866, Upstate Biotechnology, Inc.), antidimethyl histone H3 (Lys4) (catalog no. 07–030, Upstate Biotechnology, Inc.), and antitrimethyl histone H3 (Lys4) (catalog no. ab8580, Abcam, Cambridge, UK). The chromatin samples were added to 50 mg protein-A sepharose beads (Amersham Biosciences), prewashed with immunoprecipitation buffer, and incubated at 4 C for 3 h with gentle rotation. The beads were washed three times with 10 ml buffer A (50 mM Tris-HCl, pH 7.5; 10 mM EDTA; 10 mM sodium butyrate; 0.1 mM PMSF) containing increasing amounts of NaCl to 150 mM. Bound fractions were eluted twice by incubating the beads in 0.3 ml buffer A containing 1% sodium dodecyl sulfate (SDS) at room temperature for 20 min with constant inversion. DNA was isolated from the input and the bound fractions by phenol/chloroform extraction and ethanol precipitation. The precipitates were dissolved in water; aliquots containing 0.16%, 0.08%, and 0.04% of each sample were analyzed by PCR to confirm that each assay was within the linear range of amplification.
PCRs were performed as follows: 94 C for 2 min, 21–28 cycles of 94 C for 30 sec, 55–66 C for 30 sec, 72 C for 2 min, and 72 C for 7 min. The reaction mixtures (25 µl) contained 0.5 µl DNA samples, 1x PCR buffer, 0.2 mM deoxynucleotide triphosphate mixture, 0.5 µM paired primers, and 0.6 U of Ampli-Taq (PE Applied Biosystems, Norwalk, CT). The primers used in this study are listed in Table 1, and positions of their amplimers are indicated in Fig. 1. The amplified products were fractionated on 0.9% agarose gels and then transferred to Hybond-N+ nylon membrane (Amersham Biosciences) using 0.4 M NaOH as transfer buffer. The blots were hybridized at 65 C for 16 h in 0.5 M sodium phosphate, pH 7.2; 5% SDS; 1% BSA; 0.1 mM EDTA; and 0.1 mg/ml denatured salmon sperm DNA with 32P-labeled probes. The probes were amplified by PCR using the hGH/P1 clone (15) or human genomic DNA as template and were subsequently isolated from agarose gels after electrophoresis. The membranes were washed with 0.1x sodium chloride/sodium citrate (SSC), 0.1% SDS at 65 C. Signal intensities were quantified by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) using ImageQuant software. Ratios between bound and input fractions were generated using a linear PCR amplification range. Each ratio was normalized to the comparable signal detected at the ubiquitously expressed GAPDH promoter (defined as 100).
ChIP Assay to Distinguish the Four P Elements
The ChIP assay was performed as described above except that a lower amount of micrococcal nuclease (2.5 U) was used to prepare longer segments of chromatin (average length, 1.2 kb). The bound fraction together with the input fraction were subjected to PCR amplification using a common primer pair (5'-TGGAGAGGTGAGA GGAGACACTC-3' and 5'-ATGGGGTGCCTTGTGGAAACCTC-3') to generate the 930P amplimer (Fig. 1B) that anneals to identical sequences in all four P elements. The PCR was performed in 25 µl as follows: 94 C for 2 min followed by 23 cycles of 94 C for 30 sec, 66 C for 30 sec, 72 C for 2 min, and 72 C for 7 min. Portions (5 µl) of the products were applied to each lane of 0.9% agarose gels that were electrophoresed and Southern blotted. The DNA blots were hybridized with each specific oligoprobe at 42 C for 16 h in 5x SSC, 50 mM sodium acetate, pH 8.0; 0.1% SDS; 10x Denhardt’s solution; 0.01% sodium pyrophosphate; and 0.1 mg/ml denatured salmon sperm DNA. The sequences of the oligoprobes are shown in Fig. 3A. The oligoprobes were 5'-end labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [32P]
ATP at 37 C for 60 min. The membranes were washed with 0.1x SSC, 0.1% SDS at 42 C, and the specific signals were detected as above.
PCR and Restriction Enzyme Digestions that Distinguish the Five hGH/hCS Genes
A common primer pair (5'-AGCATGTGTGGGAGGAGCTT-3' and 5'-GATTTTAGGGGCGCTTACCT-3') was identified that amplifies the region located between 146 bp upstream and 87 bp downstream of the transcription initiation site of each GH-related gene (amplimer 233GH/CS; Fig. 1B). After 5'-end labeling the sense primer with [32P]ATP as described previously, PCR was carried out as follows: 94 C for 2 min followed by 25 cycles of 94 C for 30 sec, 57 C for 30 sec, 72 C for 2 min, and 72 C for 7 min. The products were digested with restriction enzymes HinfI, DraIII, and PstI and divided into two portions. One portion was further digested with SmlI, the other with MscI. The digestion products were separated on a 6% polyacrylamide, 7 M urea gels, and bands were detected by autoradiography. Based upon specific fragment sizes on the gel compared with the known DNA sequence of the region, individual bands were assigned to each of the five genes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: ChIP, Chromatin immunoprecipitation; CS, chorionic somatomammotropin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HAT, histone acetyltransferase; HMT, histone methyltransferase; HS, hypersensitive site; LCR, locus control region; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecyl sulfate; SSC, sodium chloride/sodium citrate; STB, syncytiotrophoblast.
Received for publication December 3, 2003. Accepted for publication December 31, 2003.
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A. P. Kimura, D. Sizova, S. Handwerger, N. E. Cooke, and S. A. Liebhaber Epigenetic Activation of the Human Growth Hormone Gene Cluster during Placental Cytotrophoblast Differentiation Mol. Cell. Biol., September 15, 2007; 27(18): 6555 - 6568. [Abstract] [Full Text] [PDF]
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M. A. Trujillo, M. Sakagashira, and N. L. Eberhardt The Human Growth Hormone Gene Contains a Silencer Embedded within an Alu Repeat in the 3'-Flanking Region Mol. Endocrinol., October 1, 2006; 20(10): 2559 - 2575. [Abstract] [Full Text] [PDF]
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E. J. Yoo, I. Cajiao, J.-S. Kim, A. P. Kimura, A. Zhang, N. E. Cooke, and S. A. Liebhaber Tissue-Specific Chromatin Modifications at a Multigene Locus Generate Asymmetric Transcriptional Interactions Mol. Cell. Biol., August 1, 2006; 26(15): 5569 - 5579. [Abstract] [Full Text] [PDF]
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 X. Q. Xu, B. S. Emerald, E. L. K. Goh, N. Kannan, L. D. Miller, P. D. Gluckman, E. T. Liu, and P. E. Lobie Gene Expression Profiling to Identify Oncogenic Determinants of Autocrine Human Growth Hormone in Human Mammary Carcinoma J. Biol. Chem., June 24, 2005; 280(25): 23987 - 24003. [Abstract] [Full Text] [PDF]
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M. Bulger Hyperacetylated Chromatin Domains: Lessons from Heterochromatin J. Biol. Chem., June 10, 2005; 280(23): 21689 - 21692. [Abstract] [Full Text] [PDF]
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H. Szutorisz, C. Canzonetta, A. Georgiou, C.-M. Chow, L. Tora, and N. Dillon Formation of an Active Tissue-Specific Chromatin Domain Initiated by Epigenetic Marking at the Embryonic Stem Cell Stage Mol. Cell. Biol., March 1, 2005; 25(5): 1804 - 1820. [Abstract] [Full Text] [PDF]
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