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We have investigated the role of DNA methylation in the initiation and maintenance of silenced chromatin in somatic mammalian cells. We found that a mutated transgene, in which all the CpG dinucleotides have been eliminated, underwent transcriptional silencing to the same extent as the unmodified transgene. These observations demonstrate that DNA methylation is not required for silencing. The silenced CpG-free transgene exhibited all the features of heterochromatin, including silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, lack of H3-K4 methylation, and enrichment in tri-methyl-H3-K9. In contrast, when we tested for transgene reactivation using a Cre recombinase-mediated inversion assay, we observed a marked difference between a CpG-free and an unmodified transgene: the CpG-free transgene resumed transcription and did not exhibit markers of heterochromatin whereas the unmodified transgene remained silenced. These data indicate that methylation of CpG residues conferred epigenetic memory in this system. These results also suggest that replication delay, lack of histone H3 and H4 acetylation, H3-K4 methylation, and enrichment in tri-methyl-H3-K9 are not sufficient to confer epigenetic memory. We propose that DNA methylation within transgenes serves as an intrinsic epigenetic memory to permanently silence transgenes and prevent their reactivation.
Regulation of gene expression during development and differentiation occurs at many levels including transcriptional gene silencing and activation. Understanding the mechanism of gene silencing has practical importance because genes inserted in the genome for gene therapy are often silenced, decreasing the efficiency and increasing the risk associated with these procedures. In this report, the authors created a gene that cannot be methylated by mutating all of its CpG dinucleotides and examined in detail the role of DNA methylation and other epigenetic modifications in the establishment and maintenance of gene silencing. The authors found that DNA methylation is not required for the establishment or the maintenance of the silent chromatin state, but that this covalent DNA modification was unique among the epigenetic marks tested because it conferred to the chromatin structure a long-term intrinsic epigenetic memory that prevents gene reactivation.
Citation: Feng Y-Q, Desprat R, Fu H, Olivier E, Lin CM, Lobell A, et al. (2006) DNA Methylation Supports Intrinsic Epigenetic Memory in Mammalian Cells. PLoS Genet 2(4): e65. https://doi.org/10.1371/journal.pgen.0020065
Editor: Wolf Reik, The Babraham Institute, United Kingdom
Received: September 9, 2005; Accepted: March 17, 2006; Published: April 28, 2006
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: EEB is supported by National Institutes of Health (NIH) grants DK56845, HL55435, and DK061799. Research in MIA's laboratory was supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, and NIH. SNG was supported by the HHMI/Montgomery County fellowship program.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ChIP, chromatin immuno-preciptation; Dnmt, DNA methyltransferase; EGFP, enhanced-green fluorescent protein; FACS, fluorescence-activated cell sorter; HYTK, hygromycin-thymidine kinase; LCR, locus control region; MEL, mouse erythroleukemia; RMCE, recombinase-mediated cassette exchange
Silencing of transgenes, viruses, and transposons is a general phenomenon that has been documented from yeast to mammals [1] and is believed to be part of a genome defense mechanism against mobile genetic elements [2]. Understanding the epigenetic mechanisms underlying transgene silencing should provide insights into this important nuclear process and also have practical importance: for instance, designing safer gene therapy cassettes. Numerous studies have shown that gene expression in general, and transgene expression in particular, is regulated in part by DNA methylation, post-translational histone modifications, and timing of replication, three of the major epigenetic factors controlling chromatin structure.
DNA methylation is associated with transgene silencing, X chromosome inactivation, genomic imprinting, silencing of mobile genetic elements, and the developmental regulation of some tissue-specific genes [3]. In mammalian cells, DNA methylation is maintained and established by three DNA methyltransferases (Dnmt) [4]. Dnmt3a and Dnmt3b are particularly important for de novo methylation, while Dnmt1 is critical for the maintenance of DNA methylation after DNA replication [5]. The greater affinity of Dnmt1 for hemi-methylated DNA than for unmethylated DNA has been suggested as the mechanism for the maintenance of methylation patterns after each cell division (reviewed in [6]).
Histone modifications have emerged as major mediators of the formation of repressive chromatin structures [7]. Deacetylation of lysines in the tails of histone H3 and H4 is associated with silent chromatin, while acetylation of the same lysine residues is associated with gene activation [8,9]. Methylation of lysine 4 of histone H3 (H3-K4-Me) is associated with active chromatin, while methylation of H3-K9 and H3-K27 has been shown to be important for the formation of constitutive and facultative heterochromatin [10–14].
The maintenance of histone modifications after each DNA replication is not fully understood. Once certain silent chromatin structures are established, they can be stably transmitted through cell division in the absence of the initiating signal [15]. This epigenetic memory is best understood for genes regulated by the Polycomb proteins in Drosophila and by the Sir proteins in yeast [15–17]. A similar phenomenon has been elegantly demonstrated in mammalian cells by the Rauscher lab [10]. In that report, the memory was conferred to the transgenes by the recruitment of HP1.
Models for the inheritance of histone modifications are based on the observations that the effectors of silencing that bind post-translationally modified histones can also recruit the enzymes that modify the histones. After DNA replication, the newly assembled nucleosomes are believed to be made of a random mixture of new unmodified histones and of the old modified histones. Binding of effector proteins to the old histones would recruit histone-modifying enzymes and lead to the re-establishment of the pattern of histone modifications that existed before DNA replication [16,18].
In mammals, multiple histone-modifying enzymes and chromodomain proteins are known to be important for silencing, but the interactions between DNA methylation and histone modifications are complex. The Dnmts have been shown to interact with at least 15 proteins including HP1, histone methyltransferases, and histone deacetylases [19–21]. Whether histone modifications and DNA methylation are redundant, cooperative, or independent pathways of gene silencing remain unclear.
Timing of replication during S phase is likely to be an important factor for gene silencing since the epigenetic information must be replicated after each cell cycle [22]. This was suggested more than 15 years ago by the observation that repressed genes tend to replicate later in S phase than active genes. More recently, Simon et al. [23] provided support to the hypothesis that timing of replication affects the maintenance of epigenetic information by showing that different histone modifications were added to transgenes introduced early or late in S phase. Importantly, we recently demonstrated that the timing of replication of transgenes inserted into reference integration sites in mammalian chromosomes correlates with expression and the condensation state of chromatin, as manifested by histone modifications and methylation on CpG residues [24]. However, whether the timing of replication is a cause or a consequence of silencing, and whether it contributes to the maintenance of the silenced or active states, is still unclear.
In this study, we aimed to assess the relative contribution of CpG methylation, timing of replication, and histone modifications in the establishment and maintenance of transcriptionally silent, late replicating, and condensed chromatin domains. We found that silencing of transcriptional activity, delayed DNA replication, lack of histone H3 and H4 acetylation, and lack of H3-K4 methylation occurred in the absence of DNA methylation. We demonstrate that DNA methylation within the transgene serves as an intrinsic epigenetic memory to prevent transgene reactivation. We also show that delayed DNA replication, combined with the lack of histone H3 and H4 acetylation and with H3-K9 methylation, are not sufficient to confer epigenetic memory in this system.
Experimental System
We previously developed an experimental system to study expression and silencing of globin transgenes in mouse erythroleukemia cells (MEL). This system is based on the use of recombinase-mediated cassette exchange (RMCE), a method that utilizes the Cre recombinase to integrate transgenes at predefined chromosomal sites at high efficiency [25,26]. The principle of our experimental system is to create in the genome reference integration sites that can be re-used to study transgenes in a defined chromatin context (Figure 1A). To study silencing of globin reporter transgenes, we thus created in MEL cells three reference sites of integration (termed RL4, RL5, and RL6) by selection of clones containing single copies of a randomly integrated hygromycin-thymidine kinase fusion selectable marker (HYTK) flanked by two inverted lox sites. Using RMCE, we then replaced the HYTK gene with a series of reporter cassettes driven by various globin regulatory elements [27,28].
(A) Experimental system: reference integration sites are created by insertion in MEL cells of the HYTK gene flanked by two inverted L1Lox sites (black triangles). The HYTK gene can then be exchanged for any other cassettes by transfection of a Cre expression plasmid and of plasmids containing the cassette of interest. Clones with the cassettes in both orientations are obtained. Inserted cassettes can be inverted in vivo by re-expression of Cre.
(B) Southern blots demonstrating insertion of cassettes 234-β-EGFP, 23-β-EGFP, and CpG-free 23-β-EGFP. For each cassette, multiple clones in each orientation are shown.
(C) FACS analysis of typical clones in the P and the N orientations performed 6 wk post-integration. x-axis, forward-scatter; y-axis, EGFP fluorescence. All three cassettes are rapidly silenced in the N orientation, demonstrating that DNA methylation is not necessary for silencing at the RL4 locus.
(D) Quantitative analysis of expression. Top histograms: Plots of normalized mean EGFP fluorescence (± SD) of three to five clones analyzed 2-mo post-integration in the P and N orientations. EGFP fluorescence of all cassettes is normalized to expression of cassette 234-β-EGFP in the P orientation at RL4. The CpG-free cassette in the P orientation is expressed at about 5% of the efficiency of the CpG-containing 23-β-EGFP cassette and 2% of the efficiency of the 234-β-EGFP cassette. Bottom histograms, percent of cells expressing EGFP, 1- and 2-mo post-integration. The cut-off between expressing and non-expressing cells is placed using untransfected MEL cells as controls.
(E) Quantitative RT-PCR analysis of CpG-free cassette 23-β-EGFP and CpG-containing cassette 234-β-EGFP at RL4. Results are normalized to levels of β-2-micro-globulin. At the RNA level, the difference in expression between the CpG-containing and CpG-free cassettes is less than 3-fold.
N, non-permissive orientation; P, permissive orientation.
A particularity of our RMCE system is that insertion of the reporter cassettes at sites RL4, RL5, and RL6 occurs in both orientations because the Lox sites flanking the HYTK reporter are inverted [26]. This particularity turned out to be critical because analysis of these reporters revealed that expression depends on the orientation of the cassette relative to the flanking sequences at all three reference sites. For instance, expression of cassette 234-β-EGFP, which contains DnaseI hypersensitive sites 2, 3, and 4 of the human β-globin locus control region (LCR) and the human β-globin promoter driving the enhanced-green fluorescent protein (EGFP) reporter, is high when the cassette is integrated in one orientation at site RL4, but is rapidly silenced when the cassette is integrated in the opposite orientation. We term the orientation, in which the cassette expresses, the permissive orientation, the other, the non-permissive orientation. A similar phenomenon can be observed at site RL6, demonstrating that this silencing process is not idiosyncratic for one site of integration [27].
Because silencing at site RL4 is faster than at RL6 (6 wk versus 12 wk), we focused on this site to try to understand the mechanisms of initiation and maintenance of repressed chromatin structure, and in particular, the role of DNA methylation.
We previously reported that silencing at RL4 is associated with DNA methylation of the EGFP coding sequence, the promoter, and HS2 of the LCR [27]. In a separate study, we also reported that globin transgenes, pre-methylated in vitro at all their CpGs and subsequently inserted by RMCE at sites RL5 and RL6, were silenced regardless of their orientations. These studies demonstrated that DNA methylation was sufficient to cause silencing in MEL cells [29]. Similarly, Dalle et al. [30] have recently reported that silencing of reporter genes in mice and cell culture were dependent on CpG dinucleotides.
In the present study, we have created a cassette that cannot be methylated because it is totally devoid of CpG dinucleotides, and we have used this cassette to determine if methylation is necessary for silencing.
The 4,866 bases CpG-free 23-β-EGFP cassette was created by mutating 92 CpG dinucleotides in hypersensitive sites 2 and 3 of the β-globin LCR, the β-globin promoter, the coding sequence of the EGFP gene, and an SV-40-derived poly-adenylation signal. HS4 was deleted from the original cassette to minimize the efforts involved in mutating the cassette. To control for the effect of this deletion, we have also produced a CpG-containing 23-β-EGFP cassette.
Transient transfection experiments in MEL cells demonstrated that the CpG-free cassette was functional and produced the EGFP protein, albeit at a lower level than a CpG-containing 23-β-EGFP cassette (unpublished data).
To determine if the absence of CpG dinucleotides would prevent silencing, the CpG-free 23-β-EGFP cassette and two different CpG-containing control cassettes were inserted at integration site RL4 by RMCE (Figure 1B). Multiple clones with the cassette in either orientation were identified by Southern blot hybridization, and expression was then monitored by fluorescence-activated cell sorter (FACS) for several months.
CpG Methylation Is Not Necessary for Silencing
As expected, the control CpG-containing cassette 234-β-EGFP was expressed at high levels when it was integrated in the permissive orientation but was completely silenced 4–6 wk after integration in the non-permissive orientation (Figure 1B, 1C, and 1D). The control CpG-containing 23-β-EGFP cassette behaved similarly: It was stably expressed (at about 40% of the level of cassette 234-β-EGFP) when integrated in the permissive orientation, and was completely silenced 4–6 wk after integration in the non-permissive orientation (Figure 1B, 1C, and 1D), demonstrating that deletion of HS4 has no effect on silencing.
Interestingly, the CpG-free 23-β-EGFP cassette behaved as the control cassettes. It was stably expressed when integrated in the permissive orientation and silenced within 6 wk in the non-permissive orientation. This result clearly demonstrates that at this site of integration, CpG methylation of the β-globin promoter and the LCR was not necessary for silencing.
The flow cytometry results also revealed that in the permissive orientation, the CpG-free cassette was expressed at only 5%–10% of the level of the control 23-β-EGFP cassette. To determine whether this was due to sub-optimal codon usage in the CpG-free EGFP coding sequence or to a decreased transcriptional activity of the CpG-free mini-LCR and promoter, we performed quantitative RT-PCR on total RNAs extracted from cells containing the CpG-containing and CpG-free cassettes. This revealed that at the RNA level, expression of the CpG-free 23-β-EGFP cassette was only two to three times lower than the control 234-β-EGFP (Figure 1E). We therefore conclude that the major cause of the low level of expression of the CpG-free cassette in the permissive orientation was post-transcriptional.
Because variations of gene expression can stem from a variety of biochemical mechanisms, it was essential to establish whether the silencing observed with the CpG-free cassette at the non-permissive orientation shared some or all of the characteristics of gene silencing that we previously observed with the CpG-containing cassettes. Two important features of the orientation-dependent silencing are the replication delay and the appearance of histone post-translational modifications associated with condensed chromatin. To determine which histone modifications are associated with silencing of the CpG-free transgenes, we used the chromatin immuno-precipitation assay (ChIP) to measure the levels of acetylation and methylation of histones H3 and H4 in chromatin derived from cells containing the CpG-free 23-β-EGFP cassette in either orientation. We used primers for the EGFP coding sequence and the β-globin promoter, both contained within the transgene, to assess the abundance of transgene sequences in chromatin enriched for specific histone modifications. As controls, we also used primers for the mouse β-major globin and amylase genes which are known to be associated with condensed and decondensed chromatin in these cells, respectively.
When the CpG-free cassette was integrated in the permissive orientation at RL4, sequences from the human β-globin promoter and within the coding sequence of EGFP were abundant in chromatin enriched for acetylated histones H3 and H4 and for histone H3 methylated at K4 (Figure 2A). By contrast, when the CpG-free cassette was in the non-permissive orientation, the EGFP coding sequence (and to a lesser extent the β-globin promoter) were not enriched in chromatin containing acetylated H3 and H4 and methylated H3-K4. These results are similar to our previous observations on the CpG-containing cassettes [24] and demonstrate that the changes in histone modifications associated with silencing at RL4 occur independently from DNA methylation.
(A) Histone modification analysis by ChIP assays. The histograms summarize the results of quantitative PCR measurements of abundance of the EGFP coding sequence, the hβ-globin promoter, the mouse β-major promoter region (mBmaj), and a fragment of the mouse amylase coding sequence (mAmylase) in chromatin from cells containing cassette 23-β-EGFP at RL4 precipitated with polyclonal antibodies against acetylated H3-tails (left), acetylated H4-tails (middle), and methylated H3-K4-tails. The enrichment of specific sequences in modified chromatin compared to total chromatin was calculated as described in Materials and Methods. Both the EGFP coding sequence and the β-globin promoter of the CpG-free 23-β-EGFP cassette are enriched when acetylated on H3 and H4 and H3-K4 methylation and when they are in the P orientation, but not when they are in the N orientation.
(B) ChIP analysis of the CpG-containing cassette inserted at RL5 in the N or P orientation. The two orientations are similar, and, as at RL4, the enrichment is higher for the EGFP coding sequences than for the promoter.
(C) Timing of replication analysis: The histograms summarize the results of quantitative PCR measurements of the abundance of the CpG-free and CpG-containing EGFP coding sequence in FACS-separated, BrdU-immunoprecipitated newly replicated DNA fragments from cells containing the CpG-containing (left) or the CpG-free (right) LCR-β-EGFP cassettes. Both cassettes replicate early when they are in the P orientation and late when they are in the N orientation. Controls were as in Figure 3.
N, non-permissive orientation; P, permissive orientation.
(A) Bisulfite sequencing analysis was performed on cells with CpG-containing cassette 234-β-EGFP at RL4 after N to P inversion. There were 11 molecules sequenced. Most of the CpGs were methylated (see text).
(B) Histograms illustrating H3 acetylation and H3-K4 methylation results on the CpG-containing cassettes. The analysis was performed as in Figure 2. Mouse β-major globin (mBmaj) is a positive control for H3 and H4 acetylation and a negative control for H3-K9 tri-methylation. The α subunit of the guanine nucleotide-binding protein (mGnasx) and the mouse amylase genes are a positive control for H3-K9 tri-methylation and negative controls for H3 acetylation. The CpG-containing cassette is enriched in H3 acetylation and H3-K4 methylation in the P orientation but poor in acetylated H3 and H3-K4 methylation. After N to P inversion, the cassette remains poor in H3 acetylation and H3-K4 methylation.
(C) Plots illustrating replication timing analysis of the CpG-containing cassette after N to P inversion. The analysis was performed as in Figure 2. Mouse β-major globin and amylase are early and late replicating controls. After N to P inversion, the CpG-containing cassette replicate late in S phase.
N, non-permissive orientation; P, permissive orientation.
(D) Histograms illustrating H3-K9 tri-methylation results on the CpG-containing cassettes. The analysis was performed as in Figure 2. The controls are as in Figure 3B. The enrichment is low when the cassette is in the P orientation and high in the N orientation. The enrichment remains high after N to P inversion.
These studies also revealed that the enrichment in acetylated histone H3 and methylated H3-K4 was higher on the coding sequence than on the promoter. To determine if this was specific to the RL4 site of integration, we performed ChIP experiments on MEL cells with the CpG-containing cassette integrated at RL5, a site of integration similar to RL4, but where silencing does not occur. These experiments revealed that, as at RL4, the enrichment was higher in the coding sequence than in the promoter region (Figure 2B), perhaps indicating that the promoter region is partly depleted in nucleosomes.
We then assessed the timing of replication of the CpG-free cassette by measuring the abundance of sequences from the CpG-free cassette in BrdU-labeled, newly replicated DNA from asynchronous cells fractionated by FACS according to their positions in the cell cycle. As controls, we determined the timing of replication of sequences in the murine β-globin and amylase loci. Figure 2C shows that sequences from the CpG-free cassette were abundant in the early S-phase fractions when the cassette was in the permissive orientation and abundant in the late S-phase fraction when the cassette was in the non-permissive orientation. This demonstrates that the changes in replication timing associated with silencing at RL4 occur independently from DNA methylation. Again, these results are similar to those obtained with the control CpG-containing cassettes.
CpG Methylation Confers Epigenetic Memory
Because transgenes integrated at RL4 are flanked by inverted Lox sites, they can be inverted in vivo by re-expression of Cre in the cells (Figure 1A). We have taken advantage of this property to examine the maintenance of the epigenetic marks of the open and closed chromatin structures that form at RL4. We previously reported that inversion of a CpG-containing cassette from the permissive to the non-permissive orientation leads to its rapid silencing, but that inversion of a CpG-containing silenced cassette from the non-permissive to the permissive orientation does not lead to its full reactivation [31]. Instead, expression of the inverted cassette occurred at low levels in a small number of cells. This demonstrated that silencing leaves a permanent epigenetic mark on the transgene, since the inverted cassette retained a memory of having been silenced. We termed this memory phenomenon "somatic imprinting." In the same report, we also observed that the somatically imprinted cassettes (inverted from the non-permissive to the permissive orientation) retained their DNA methylation at the promoter and in the coding sequence of EGFP using methylation-sensitive restriction nucleases [32]. To extend these results to the individual CpG level, we have performed a DNA methylation analysis by bisulfite sequencing on clones with a non-permissive to permissive inversion that had been in culture for more than 3 mo. As shown in Figure 3A, this analysis revealed that the seven CpG dinucleotides analyzed were 90%–100% methylated; demonstrating that methylation of the β-globin promoter was very stable in the inverted cassette.
To further investigate the physical support of this epigenetic memory, we performed ChIP on the inverted CpG-containing cassette using primers located in the β-globin promoter and in the coding sequence of EGFP. This revealed that after non-permissive to permissive inversion, the 234-β-EGFP cassette is poor in histone H3 acetylation and H3-K4 methylation (Figure 3B). As for the CpG-free cassette, the enrichments observed were generally higher for the coding sequence than for the promoter.
Since previous analyses had demonstrated striking differences of replication timing of the CpG-containing cassette in the non-permissive and permissive orientation, we determined the timing of replication of the CpG-containing cassette after non-permissive to permissive inversion (Figure 3C). This revealed that the non-permissive- to permissive-inverted CpG-containing cassette replicated late in S phase, and therefore the timing of replication was not changed by the inversion.
Since tri-methylation of Lys 9 of histone H3 has been associated with epigenetic memory through recruitment of HP1 [10], we also performed ChIP analysis using antibodies specific for this histone modification. As described in Figure 3D, when the CpG-containing cassette was inserted in the permissive orientation it was not enriched in tri-methyl-H3-K9 histones. In contrast, when the same cassette was inserted in the non-permissive orientation and therefore silenced, it was highly enriched for this modification. As expected, control PCR revealed that the mouse β-major promoter (expressed) was poor in tri-methyl-H3-K9 while the mouse major satellite and the α subunit of the guanine nucleotide-binding protein (mGnasx, an imprinted gene) were highly enriched in tri-methyl-H3-K9. We conclude from these experiments that the silenced chromatin that forms when cassette 234-β-EGFP is inserted in the non-permissive orientation at RL4 is associated with tri-methylation of H3-K9. ChIP analysis after inversion of the silenced CpG-containing cassette back toward the permissive orientation revealed that tri-methylation of H3 was retained.
Together, these experiments revealed that the inverted CpG-containing cassette had retained the epigenetic marks that it had acquired on its promoters and coding sequences when it was silenced in the non-permissive orientation. Based on these results we hypothesized that the epigenetic memory that imprints the cassette after its inversion could be CpG methylation, a histone post-translational modification, or replication in late S phase.
To differentiate between these possibilities, we have inverted the CpG-free 23-β-EGFP cassette that had been inserted at RL4 in the non-permissive orientation for 3 mo and was therefore completely silenced. Multiple individually isolated clones containing the CpG-free 23-β-EGFP were transfected with a Cre expression plasmid and 1 mo after the transfection, sub-clones with the inversion were identified by Southern blots (Figure 4A). Expression levels were then determined by FACS and compared with CpG-containing and CpG-free controls (Figure 4B). In sharp contrast with the results obtained with the CpG-containing cassette, this analysis revealed that inversion of the CpG-free cassette from the non-permissive to the permissive orientation led to its rapid reactivation. Therefore, in the absence of CpG methylation, the cassette loses its epigenetic memory. Quantitative RT-PCR analysis on total RNA confirmed these results (Figure 4C) and demonstrated that the silencing occurs primarily at the transcriptional level. We conclude from these experiments that DNA methylation contributes to the permanent mark that confers memory to these cassettes.
(A) Southern blots demonstrating inversion of the 23-β-EGFP cassette from the N to the P orientation. N to P lanes, Hind III digested genomic DNA from three N to P inverted sub-clones isolated after transfection of cells with cassette CpG-free 23-β-EGFP in the N orientation with a Cre recombinase expression plasmid. N and P, control sub-clones.
(B) FACS analysis of representative clones with the CpG-free and CpG-containing cassettes in the P or N orientation or after N to P inversion. x-axis, forward scatter; y-axis, EGFP fluorescence. N to P inversion leads to the reactivation of the CpG-free but not the CpG-containing cassette.
(C) Quantitative RT-PCR analysis of EGFP expression on total RNA extracted from two to five sub-clones with the CpG-containing or the CpG-free cassette in the P or N orientation and after N to P in vivo inversion. Results are expressed as percent of β-2-micro-globulin expression (100 × number of EGFP transcripts/number of B2M transcripts). This analysis confirms the FACS data and demonstrates that silencing and reactivation of the CpG-free cassette occurs primarily at the transcriptional level.
(D) Plots illustrating replication timing analysis of the CpG-free cassette after N to P inversion. The graphs and controls are as in Figure 3C. After N to P inversion, the CpG-free cassette replicates early in S phase.
(E) ChIP analysis of the CpG-free 23-β-EGFP cassette. The graphs and controls are as in Figure 3B. The results show that the CpG-free cassette is enriched in H3 acetylation and poor in H3-K9 tri-methylation in the P orientation, but that it is poor in H3 acetylation and rich in H3-K9 tri-methylation in the N orientation. After N to P inversion, the CpG-free cassette becomes enriched in H3 acetylation but poor in H3-K9 tri-methylation.
N, non-permissive orientation; P, permissive orientation.
Replication timing analyses revealed that the timing of replication of the CpG-free cassette that had been inverted from the non-permissive to the permissive orientation had reverted to early S phase (Figure 4D). Therefore, the replication delay that persisted in the inverted CpG-containing cassette did not persist in the absence of CpG methylation. ChIP analyses revealed that sequences derived from the inverted CpG-free cassette were enriched in chromatin-containing acetylated histone H3 (Figure 4E), again contrasting with the results obtained with the inverted CpG-containing cassette, which maintained the deacetylated chromatin state. We therefore conclude that CpG methylation is required to maintain the delayed replication and the deacetylated histone phenotypes when the cassettes are inverted toward the permissive orientation.
ChIP analysis revealed that insertion of the CpG-free cassette in the permissive orientation is associated with low levels of enrichment for tri-methyl-H3-K9, and that insertion in the non-permissive orientation is associated with high levels of enrichment for tri-methyl-H3-K9 (Figure 4E), suggesting that establishment of this histone modification does not require DNA methylation. Importantly, inversion of the CpG-free cassette from the non-permissive to the permissive orientation revealed a complete loss of enrichment for this modification. As for histone acetylation, we therefore conclude that CpG methylation is required to maintain the tri-methylation of histone when the cassettes are inverted toward the permissive orientation.
Similar experiments using an anti-tri-methyl H3-K27 antibody did not reveal any enrichment for this modification in these cassettes in either orientations or in several control DNA segments, suggesting that this modification is not commonly present in single-copy active or inactive genes in MEL cells, and that it does not seem to play a role in silencing of these transgenes (unpublished data).
Cre Recombination Per Se Does Not Alter Expression
One possible interpretation of some of these results was that the changes in chromatin structure that we observe after inversion are caused by the recombination process itself, since recombination involves the creation of double-stranded breaks that might alter the topological conformation of the chromatin or recruit repair proteins that would in turn recruit chromatin- modifying proteins. Since the CpG-containing 234-β-EGFP cassette inverted from the non-permissive to the permissive orientation at RL4 retains its chromatin structure, we can eliminate the possibility that the inactive chromatin structure is altered by the recombination process when DNA methylation is present. To test the possibility that the active chromatin structure is altered by the recombination process, we inverted cassette 234-β-EGFP after it was inserted at RL5, a site at which expression occurs in both orientations (Figure 5) (albeit about 50% lower in one of the two orientations). These experiments revealed that inversions at RL5, regardless of the starting orientation, did not lead to silencing; demonstrating that the recombination process itself does not lead to the collapse of the active chromatin structure. These experiments do not exclude that the recombination event could have subtler effects on the chromatin structure.
Cells containing cassette 234-β-EGFP at RL5 were transfected with the Cre recombinase, and inverted sub-clones were identified using primer pairs p1/p2 (specific for the P orientation) and p1/p3 (specific for the N orientation).
(A) Location of the primers used to identify the inverted clones.
(B) P to N inversion: The histogram on the left illustrates expression profile of a clone with insertion in the P orientation before inversion. The histogram on the right illustrates expression 8 wk post-inversion. The ethidium bromide stain illustrates the PCR analysis demonstrating the inversion.
(C) Same as (B) but for the N to P inversion. This analysis clearly demonstrates that Cre recombination per se does not lead to silencing (see text). As reported previously, levels of expression in the N orientation at RL5 are lower than in the P orientation [32].
N, non-permissive orientation; P, permissive orientation.
Gautsch and Wilson [33], more than 20 years ago, suggested that DNA methylation might be necessary for maintenance of transgene repression but not for its initiation in early embryos. Since then, several other chromatin features have been associated with gene repression, and we now know that transgene silencing is generally associated with CpG methylation, delayed replication, low levels of H3-K4 methylation, H3 and H4 acetylation, and high levels of tri-methyl-H3-K9 or K27 [34]. While these associations have been clearly demonstrated, the relationships between these chromatin marks are complex and difficult to decompose. Knockout and over-expression studies have provided profound insights regarding the roles of DNA methylation and some of the histone modifications, but redundancies can mask some functions. In addition, complete elimination of DNA methylation or of histone modifications have cell-wide consequences, complicating interpretation, since the observed phenotypes are potentially indirect. In this study, we have taken advantage of the RMCE method and of a CpG-free cassette to dissect the role of CpG methylation in relation to other known epigenetic modifiers in the control of expression of a single-copy gene using silencing at RL4 as a model. The mechanism of orientation-dependent silencing at this site of integration remains incompletely understood. However, we have recently provided evidence that the triggering event for silencing might be transcriptional interference induced by the presence of the β-globin mini-LCR [35].
We report here that at site RL4 transcriptional silencing, delayed replication, enrichment in tri-methyl-H3-K9, and lack of histone acetylation and H3-K4 methylation occurs independently of CpG methylation.
In most experiments, enrichment in the ChIP experiments was higher for the EGFP coding sequence than for the promoter: whether this reflects the fact that the promoter is depleted in nucleosomes is unclear. In any case, these experiments show that CpG methylation is not necessary for the initiation and maintenance of the heterochromatic structure that forms at RL4, and that histone modifications and delayed replication are sufficient to prevent expression in the non-permissive orientation at this site of integration. A previous report has shown that DNA methylation is sufficient to initiate transgene silencing [29] without requiring delayed replication. More recently, Mutzkov et al. [34] have suggested that methylation of H3-K9 was not the primary causative event for silencing of transgenes in chicken erythroid cells.
Together these data suggest that a silent heterochromatic structure can be initiated either by CpG methylation or by histone modifications and delayed replication, and that these features can be acquired in more than one order, maybe because of the multiple interactions that are known to exist between Dnmt and histone-modifying enzymes. This raises the question of whether these chromatin features have unique functions. As discussed below, the cassette inversion experiments suggest that CpG methylation differs from the other chromatin marks studied above because it confers epigenetic memory.
Epigenetic Memory
The chromatin structure of silenced transgenes is very stable at RL4: clones containing silenced cassettes in the non-permissive orientation can be kept in culture for more that 1 y and will never reactivate (unpublished data). The epigenetic information is therefore faithfully replicated through hundreds of cell divisions suggesting the existence of an epigenetic memory. This epigenetic memory can theoretically result from two types of mechanisms: the silent chromatin structure could be maintained through cell division simply because the factors required for its establishment remain present in the cell at all times. Alternatively, after initiation of silencing, the chromatin might acquire some inherent characteristics that allow the propagation of the epigenetic information even when the initiating factors are no longer present. We propose that there are two types of epigenetic memory. The first, termed apparent memory would require the continued presence of an initiating signal. The second, termed intrinsic memory would persist even in the absence of the initiating signal. We also propose that both intrinsic and apparent epigenetic memories have important but different biological roles in mammalian cells. Intrinsic epigenetic memory could be particularly useful to permanently silence retroviruses and transposons throughout development and differentiation, or oncogenes in cell types where they can cause transformation. Apparent epigenetic memory could be more appropriate to temporarily silence genes that need to be re-expressed at a later time during differentiation or in response to induction by environmental agents.
We have observed here that the effects of in vivo inversion of a silent heterochromatic cassette depended on the presence of CpG dinucleotides: If the cassette contained CpG dinucleotides, the chromatin remained condensed and expression continued to be silenced. If the cassette did not contain CpGs, the chromatin converted to a decondensed configuration and expression was reactivated.
These experiments unambiguously demonstrate that CpG methylation is necessary for the persistence of the epigenetic information acquired in the non-permissive orientation. We conclude that DNA methylation confers intrinsic epigenetic memory since the silent-state persists after a major perturbation of the chromatin structure that eliminates the initiating silencing event.
In the absence of CpG methylation, all of the marks of silent chromatin that we tested were erased. We have therefore failed to detect any evidence that histone modifications or delayed replication confer an intrinsic epigenetic memory, independently of DNA methylation. This result can be interpreted either by the hypothesis that these chromatin marks are not self-replicating and are unable to confer intrinsic epigenetic memory or by the hypothesis that except for DNA methylation, a covalent modification, all of the chromatin marks tested were erased by the Cre-mediated inversion event itself. The latter hypothesis is not supported by the experiments in Figure 5 which show that at locus RL5, Cre-mediated inversions had no effects on expression; or by reports by Ghirlando et al. [36] and Boeger et al. [37] that in vivo excision of chromatin fragments with restriction nucleases or with the Cre recombinase do not dramatically perturb the chromatin structure.
In any case, we have demonstrated that among the five chromatin characteristics tested, methylation of CpG dinucleotides is unique in its ability to confer an intrinsic epigenetic memory that persists after a DNA rearrangement. It remains to be determined if in mammalian cells, histone-code based, intrinsic memory systems independent of DNA methylation exist, and if they do, whether they are active at all developmental times or restricted to early development. Conferring intrinsic epigenetic memory might be a critical, non-redundant function of DNA methylation that plays an important role in the protection of genome integrity and accounts for its conservation in many organisms.
Materials and Methods
The CpG-free EGFP coding sequence was constructed by mixing five overlapping 200 bases synthetic oligonucleotides in a single PCR reaction, resulting in the self-assembly of the CpG-free cDNA. Sub-cloning and sequencing of this CpG-free cDNA revealed the presence of five unwanted substitutions that were corrected by conventional recombinant PCR. The CpG-free β-globin promoter was then added to the CpG-free cDNA by a three-way ligation of PCR products generated with long primers that contained mutations that eliminated all CpG dinucleotides from the promoter. Finally the LCR part of the construct was added as a 1.1-kb synthetic fragment synthesized by a commercial supplier. The entire plasmid was sequenced. A total of 92 CpG dinucleotides were eliminated: four CpGs in HS3, 11 CpGs in HS2, and three CpGs in the promoter were replaced by nucleotides found in the homologous sequences of primates or mice. In the coding sequences, 61 CpGs were replaced by nucleotides that yield synonymous codons without dramatically altering codon usage [38]. Finally, 13 CpGs that were located in remnants of multiple cloning sites between the various cis-elements of the cassette were deleted. Plasmid 234-β-EGFP has been described previously [31]. Plasmid 23-β-EGFP is identical to 234-β-EGFP except for the deletion of HS4. Details of the construction and sequences of all plasmids are available on request. Tissue cultures were performed as in [27,31].
RMCE and cassette inversions.
These experiments were performed as previously described [26,31]. Briefly, 2–4 million MEL cells containing the HYTK cassette and under-hygromycin selection were electroporated with 200 μg of exchange cassette and 50 μg of Cre expression plasmid and selected in gancyclovir for 10 d. Sub-clones were then picked and analyzed by Southern blots. At least three sub-clones with the cassettes in each orientation were identified and subsequently analyzed. Inversions were performed, either by electroporating 2–4 million cells with 50 μg of Cre expression plasmid or by transfection of 100,000 cells with 1μg of Cre expression plasmid using Fugene (Stratagene, La Jolla, California, United States). All Southern blots were probed with the EGFP coding sequence. The sequence of primers p1, p2, and p3 is available on request.
Expression analysis.
FACS analyses were performed as described earlier [27]. Quantitative RT-PCR on total RNAs were performed using the Light-Cycler (Roche, Basel, Switzerland) and the Qiagen (Valencia, California, United States) Quantitech kit according to the manufacturer instructions. The primers and probes used are available on request. All quantifications were performed at least in duplicate using at least three independently derived clones. To estimate the number of molecules present in each sample, standard curves were created using purified PCR products. Expression results were normalized to the number of β-2-microglobulin molecules present in each sample.
Replication timing analyses.
These experiments were performed as described previously [24]. Briefly, asynchronously growing cells were labeled with the thymidine analog BrdU for 90 min and fractionated based on DNA content after Hoechst 33342 labeling using a Vantage FACS. Newly replicated, BrdU-substituted DNA was isolated by immunoprecipitation. After two rounds of immunoprecipitation and DNA isolation, mitochondrial DNA sequences (mMT primers) were used to verify that each fraction contained similar quantities of amplifiable DNA strands. Samples containing 8 ng of DNA were analyzed by real-time PCR on an ABI 7900 (Columbia, Maryland, United States). The sequences of primers and probes are available on request. The abundance of an amplified sequence was calculated using genomic DNA standards with a standard curve ranging from 0.005 ng–50 ng per reaction. Each measurement was performed in triplicate. The relative abundance for each probe/primer combination was calculated as the ratio of the number of molecules amplified from a specific cell-cycle fraction to the number of molecules amplified from the cell-cycle fraction where amplification was maximal. Each experiment was repeated twice on one or two independently derived clones.
ChIP assays.
These assays were carried out as described previously [24]. MEL cells were fixed for 5 min at room temperature by the addition of 1% formaldehyde to the growth medium. After a series of washes, chromatin was shredded by sonication and subjected to immunoprecipitation with antibodies against modified histones. Antibodies used included anti-acetyl-histone H3 (06–599), anti-acetyl-histone H4 (06–866) and anti-dimethyl-histone H3 (Lys 4) (07–030), anti-tri-methyl-histone H3 (Lys 9) (07–442), and anti-trimethyl-histone H3 (Lys 27) (05–851), all from Upstate Biotechnology (Lake Placid, New York, United States). DNA was released by heating followed by treatment with RNAse and proteinase K. DNA concentrations of the samples were determined by Pico green fluorescence (Molecular Probes, Eugene, Oregon, United States). Real-time PCR was used to amplify same amount of ChIP-enriched DNA and input genomic DNA. The enrichment in the abundance of the transgenes or host genes in acetylated chromatin was calculated as the ratio between the abundance of the amplified sequences in immunoprecipitated DNA and the abundance of the amplified sequences in total genomic DNA. Each experiment was repeated twice with independently derived clones. The sequences of primers and probes are available on request. Because of sequence differences between the CpG-containing and CpG-free cassettes, the sequences of the primers and probes used to assay these two transgenes were not identical. We therefore limited our interpretations of the difference between the CpG-free and CpG-containing cassette to ratios between enrichment values rather than to the absolute values.
Real-time PCR primers and probes.
All real-time PCR reactions for the replication timing and ChIP analysis were performed on an ABI Prism 7900HT SDS. The sequences of all the primers and probes used in this study are available on request.
We thank Dr. R. Hardison (Pennsylvania State University, College Station, Pennsylvania, United States) for generously sharing the unpublished sequences of the RL5 integration site. We thank Dr. Marc Lalande (University of Connecticut Health Center, Farmington, Connecticut, United States) for helpful discussions.
Author Contributions
YQF, RD, HF, EO, CML, AL, MIA, and EEB conceived and designed the experiments. YQF, RD, HF, EO, CML, AL, SNG, and MIA performed the experiments. YQF, RD, HF, EO, CML, AL, MIA, and EEB analyzed the data. MIA and EEB wrote the paper.
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text:
By Charles Odum, AP Sports Writer
ATLANTA The Atlanta Falcons' new rookie coach already is sounding like the veteran who took the team to its only Super Bowl. Mike Smith, the former Jacksonville defensive coordinator hired by the Falcons on Wednesday night, could have been reading from former Atlanta coach Dan Reeves' script.
"My goal is to build a well-disciplined, hard-nosed football team that will be able to run the football on offense and stop the run on defense," Smith said. "I've always believed that's what makes championship teams."
Reeves used that formula to take the Falcons to the NFC championship in 1998. Reeves' successor, Jim Mora, also stressed the run in his three years. Then came Bobby Petrino, who lasted only 13 games.
In announcing his first hire as general manager, Thomas Dimitroff said Smith "possesses all of the key qualities we were looking for in a head coach."
Dimitroff said the 48-year-old Smith, a 26-year coaching veteran and the Jaguars' defensive coordinator since 2003, "has strong experience with winning teams, a track record of success, a solid, smart approach to the game, and high character and integrity."
Smith had his second interview with the Falcons on Friday.
Falcons quarterback Byron Leftwich, who was with Smith in Jacksonville for four years, applauded the hire.
"I've played against his defense more than anybody in the world," Leftwich said. "I did it every day in practice for four years. I think he's a great guy for the job.
"A lot of people might not know his name, but I've seen the work he puts in every game, how guys were so prepared on Sundays that they knew exactly what teams were going to do."
Smith, a former defensive assistant with Baltimore, had the league's No. 12 defense with Jacksonville this season after ranking second in 2006 and sixth in 2005.
Leftwich said Smith would never receive enough credit in Jacksonville because many assumed Jaguars coach Jack Del Rio, a former defensive coordinator, was the real mastermind of the defense.
Jaguars defensive end Marcellus Wiley confirmed that opinion when he said on ESPN Wednesday that Smith "was just a guy who stood at the front of the room when Jack Del Rio was leading the defense."
Jacksonville defensive end Paul Spicer disagreed, saying the Falcons "are getting a gem."
"He is very competitive," Spicer said of Smith. "He wants the best out of you. He demands the best of you. Mike Smith gained the trust of a lot of Jaguars players over the years. He has definitely gained my trust."
Jaguars defensive tackle Marcus Stroud said Smith is "definitely a deserving coach."
"He will bring stability and a tough-minded approach," Stroud said. "He's a leader and will do a great job in Atlanta."
Leftwich said he was aware of Wiley's comments but said of Smith: "I was with him for four years. Somebody had to be calling those plays in Jacksonville. It was him.
"We always said Smitty would be a great head coach because the time he put in and the way he interacted with players and how seriously he took his job."
The Falcons were mostly woeful in their 4-12 season, but most of the few bright spots were on defense, where Smith will find several established or rising stars: John Abraham, Rod Coleman, Keith Brooking, DeAngelo Hall, Lawyer Milloy and Michael Boley.
The Falcons may try to build a strong defense around those established players while rebuilding an offense devastated by the exit of Michael Vick, the star quarterback who was the face of the franchise before his guilty plea to federal dogfighting charges.
Vick is serving a 23-month sentence. He also is under an indefinite suspension by NFL commissioner Roger Goodell.
A key early decision by Smith and Dimitroff will be whether to choose a new quarterback with the Falcons' first pick, as high as No. 3 overall, in the April draft.
Leftwich, Joey Harrington and Chris Redman shared the position in 2007.
The loss of Vick was too much for Petrino to overcome. Petrino quit with a 3-10 record to take a job at Arkansas, leaving secondary coach Emmitt Thomas to finish out the 4-12 season as the interim head coach.
Smith will be Atlanta's sixth coach since December 2003. Before Petrino quit, Reeves and Mora were fired. Wade Phillips and Thomas worked three games each as interim replacements.
Other candidates included Minnesota Vikings defensive coordinator Leslie Frazier and former Ravens defensive coordinator Rex Ryan.
The team did not wait on an interview with New York Giants defensive coordinator Steve Spagnuolo. The Falcons were denied permission to interview Spagnuolo while the Giants, preparing for the Super Bowl, are in the postseason.
Copyright 2008 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.

subset: commoncrawl


text:
Monday, October 19, 2009
Meet The Bean
I have been totally holding out on you. Like, shamelessly. I have been pregnant for THIRTEEN WHOLE WEEKS (and change) and I haven't made one little peep about it. How did I manage such a stunning feat of self control? Well, I'll tell you: I've been writing about it THE WHOLE FREAKING TIME and saving the resulting bits in DRAFT MODE rather than posting them. Heh!
So, without further ado, an oversharing account of my pregnancy thus far including such tasty little morsels as:
How I Found Out!
The Vomitiousness That Is Orange Juice!
- and -
Really, Boobs?! Are You Freaking Serious?!
Saturday, August 1: Boobs killing me. Attend performance of Spamalot whilst Busting! Out! Of! My! Dress!
Wednesday, August 5: Veggie burgers! Veggie burgers are the best food ever! I COULD EAT NOTHING BUT VEGGIE BURGERS ALL WEEK and then I'd die happy. Cannot wait for our Disneyland vacation because THEY HAVE THE BEST VEGGIE BURGERS THERE! Consume 2 boxes in a span of 3 days. Make special mid-week trip to the store for additional veggie burgers.
Thursday, August 6: As boobs still killing me, buy no wire bras for the express purpose of SLEEPING IN.
Sunday, August 9: Bitter chocolate!!! 85% dark!!! NOM! Assume PMS. Buy maxi pads.
Monday, August 10: Period is due on the 13th. If I take a test now, there is an 83% chance it will be accurate. Pee on stick. Stick says no. Cry. Refuse to go see bands that night as we'd planned. Spend $40 online at website called 'Baby Hopes'. Go to bed early.
Tuesday, August 11: Explicitly tell my mother that I am not pregnant.
Thursday, August 13: GUYS! Miscalculated date period due. DUE FRIDAY NOT THURSDAY. Meaning tested in the 58% accuracy zone instead of 83%. Very silly mistake to have made, but obviously am not pregnant. Buy a bottle of wine on the way home.
- Later that evening -
Pee on stick so that I can drink said wine with a clear conscience. Sit on bed with husband looking at books while stick develops. BASICALLY IGNORE STICK COMPLETELY FOR FIVE MINUTES. Pick up stick, almost as an afterthought. Wonder why there are three letters in the word on stick's digital display. Dumb stick misspelled "no". Heh . . . wait. Why is there a plus sign on stick? Commence flapping wildly. Husband totally confused. Thrust peed-on stick in his general direction. His words: "Shut. Up. Go pee on another."
Manage a pitiful amount in a cup. Put every test we own (three [3]) in cup. All positive.
Line tests up on bathroom counter. Photograph. Check every hour or so to make sure all are still positive. Shout updates to husband for remainder of evening.
File under things they do not tell you (although in this case I think D-crazy DID attempt a warning at one point): So you think because you are pregnant you'll escape your monthly pain and suffering for a while? PFT! You will have cramps FOR THE WHOLE NINE MONTHS (I am assuming, based on 2 weeks experience). Dismal and unexpected development.
Oh, hai, headaches! And constant peeing! Happy 5 weeks to us!
I am 5 weeks, 3 days pregnant and currently wearing my pants unbuttoned. ALREADY. Not "showing" per-se just So! Freaking! Bloated! At this rate it will be a miracle if I can keep the news to myself for 7 more weeks. And if my belly doesn't give it away, my boobs will. They will need their own ZIP CODE if they get any bigger.
I have reached the stage where no food whatsoever sounds appealing to me. I went thorough a thing last week where all I wanted was orange juice. Polished off a half gallon in a weekend. Bought TWO more half gallons at Trader Joes. By the time I got the new OJ home, I had decided it was Liquid Satan. I could picture it sloshing around in my belly all acid-y and REVOLTING. I HAVE TO STOP WRITING ABOUT IT BEFORE I PUKE.
Quite like pineapple though. For the moment.
Here's the thing about morning sickness (for me) (so far): It isn't so much that I'm going to puke as it is that NOTHING sounds edible. In the process of trying to figure out just one item that I *could* conceivably eat, I have to consider and reject at least 15 options of variable unsuitability. That process . . . is GAGWORTHY.
Lunchtime is a minefield. I could get a burrito? GAG. Or a salad? RALPH. Maybe a nice piece of pizza? VOMIT. And so on and so forth until I finally decide on ONE! miraculous food that I think I could stomach (i.e.; peas and brown rice, an entire bag of Baked Lays potato chips, etc.). By the time I actually get to eat anything I'm feeling weak and frail from the parade of revoltingness I've just played out in my head.
Not that these food aversions have done anything whatsoever for the fit of my pants. I'm expanding at quite a nice rate, having just purchased Gap jeans in a size 6, where heretofore I've worn 2's.
My husband just told me that I'm still a 2, it is just that the baby is a 4.
Internet, don't we love him?
Naps! Naps are freaking awesome!
Tuesday (Sept. 22) was a BIG DAY. I had my First! Doctor! Appointment! Up until the appointment, nausea, sore boobs and weight gain notwithstanding, I could've *possibly* been making this whole pregnancy thing up. Which, I know, seriously, is the LEAST RATIONAL thought ever to have flitted through my head. But, really? Early pregnancy is WEIRD. You pee on a stick, get all excited and aside from a smattering of weird symptoms you're more or less on your own until that first doctor visit, which, for me, didn't happen until I was NINE WEEKS AND THREE DAYS PREGNANT. Granted, the day I found out I was already at 3 1/2 weeks but the subsequent six weeks of waiting were HORRIFIC and all the while I waited feeling like a fraud.
Not rational. I know. But still.
Within minutes of arriving at the doctor's office I got hooked up to an ultrasound machine and we confirmed that I was VERY MUCH NOT FAKING and INDEED 100% PREGNANT. My doctor's office is pretty high tech . . . they had a huge plasma TV up on the wall for me to watch while the ultrasound tech did her thing. I had Googled photos of 9 week ultrasounds and had figured we'd see a little bean shaped blob, but we got really lucky with the bean's position and we were able to see his* beating heart and his adorable little arms. The technician flipped a switch and a fast paced whooshing noise filled the room. I sat there stunned for a few seconds and finally asked "IS THAT THE BABY?!?!" (aka: "The dumbest question in the history of ultrasounds"). I can't even describe how incredible (and, ok, a little weird) it was to hear that heartbeat and see those little bean arms. There is a person! In my belly! Small and bean shaped and curled up on his side like a little hamster. I love him. Lots.
Which is all well and good because it made the next bit a little easier. I've been dreading the blood test (first of many, I know!) planned for my first visit. I have a *thing* about people touching the insides of my elbows (or the backs of my knees for that matter) so the thought of someone sticking a needle in there and SUCKING OUT MY BLOOD freaked me RIGHT THE FUCK OUT. I'd been building this whole fiasco up in my head for Six! Weeks! and all I can say is that I'm thrilled they did the ultrasound first because by the time the nurse came at me with a needle I'd made peace with the whole procedure as something Bean needed me to do. It really wasn't all THAT bad in the end either. I had my husband there to hold my hand and the nurse was great and got me talking. I chattered away about baby names for the minute or so I spent with an needle in my arm and before I knew it she was slapping on a band-aid.
Leaving Boy and I free to go home and gape at the printouts of our Bean.
* I'm going to be saying "his" until I know for sure. I suspect he's a boy and I refuse to refer to Bean as "it".
Maternity pants get such a bad rap, but, you know? They're comfortable. They fit. They don't make me feel like I'm bisecting my Bean ever time I sit down or, you know, BREATHE. The waistbands are pretty ugly on their own, but I can always WEAR A SHIRT OVER THEM. It isn't like I have to TUCK THINGS IN, omg.
My other options are a) Wear the pants I always used to wear, but with a hair tie looped through the button hole and a Bella Band holding the whole lot up. This works pretty well, if I'm going to be mainly standing. Unfortunately, those of us with a penchant for ultra skinny, super low rise denim like me will likely find that sitting in ultra low rise, super skinny jeans rigged up with a hair tie and a Bella Band results in plumbers crack the likes of which would BLOW YOUR FREAKING MIND.
So! For days when I'm not exclusively standing, another option is b) regular pants that are a size too big. These make me look like a hesher circa 1992. So, basically, NO.
Which leaves me with the aforementioned "Not That Bad" maternity pants. I have been cleaning out the clearance section of Gap.com and Oldnavy.com on a pretty regular basis and I've scored quite a few pairs of super cheap, demi panel pants. These in particular were a really great buy (I wear them tucked into tall boots).
In the tops department, I've discovered that anything with side ruching looks totally adorable over my growing bump and makes me feel more PREGNANT than FOND OF DONUTS (<- always nice to be pregnant and not fat)
In short: Maternity clothes are the most comfortable and flattering things in my closet right now. For nights out I still make do with rigging my pants and wearing flowy non-maternity tops. After a few long hard looks in the mirror though, I've decided that for work and for everyday, the maternity stuff really is more flattering. More comfortable and practical to boot. No shame: YEA FOR MATERNITY PANTS!
Boy and I went to a dinner party last night and about an hour in the general volume level in the room went up a notch or two and I started wondering why everyone seemed so . . . happy. OH YEAH. Because they'd all got a glass of wine in them.
We ended up the night at a show in Echo Park, where more drinking was done by people other than me, and where I made a pretty startling discovery. I always thought I talked a lot of shit and was generally crap in social situations BECAUSE of the drink or two I'd have to loosen up and relax. It turns out, I talk a lot of shit even whilst sober! I just AM crap in social situations! Hallelujah for deeper self knowledge.
Handy tip of the week: A great way to get your husband to do the dishes every night is to drape yourself dramatically over the arm of the couch after dinner and moan "I'm sooooo preeegggnannnnnttttt". Invariably, mine will send me off to (read in) (and eat candy in) bed while he cleans up the kitchen.
Thus endeth this extra long, reader's digest version of my first three months. Boy and I are beside ourselves with excitement and I'm thrilled to finally be able to tell y'all about Bean (aka: Beanie Bug, Bam-Bean-O and "He Who Makes Mama Barfy"). I'm especially thankful that I'll be able to tap the collective knowledge of the blogoverse throughout this adventure (For starters: Guys, really, give it to me straight: are my boobs going to keep growing? I ALREADY LOOK LIKE AN EXTRA ON BAYWATCH, HERE.)
23 comments:
- BethanyWD said...
Yah! So happy for you! Loved the "recap".
(And, yes, they keep growing. Just wait 'til there's MILK in there._
- October 19, 2009
- Meaghan said...
OMG I am so happy for you! Congrats and I can't wait to (kind of) go through this with you!
- October 19, 2009
- Wendy, Los Angeles said...
congratulations !!!
- October 19, 2009
- Nutsy Coco said...
OMG, congratulations!! I'm hoping to be there any day myself so I'm really going to be stalking your blog for updates. Unless of course I can't get prego in which case I might have to avoid all tihngs baby related. I'm sure you understand.
- October 19, 2009
- HollyLynne said...
Thanks, guys!
Nutsy, I didn't know you were trying! I'll send you good baby vibes :)
- October 19, 2009
- Rhiannon said...
HURRAY! SO exciting!
- October 19, 2009
Congratulations Holly! So excited for you and your DH! Your mom told me last week after you'd given the "all clear" to reveal (but I'd already had a dream the week before that you were pregnant so I guess I sort of already knew!). Best wishes for an easy pregnancy!
- Kimberly
- October 19, 2009
- merrymishaps said...
Congratulations! I'm going to have to make more of an effort to visit the Working Closet more often to watch Bean grow :)
- October 19, 2009
I HATE you.
- October 19, 2009
OMG! What an awful comment! I, for what it is worth, think you rock. I love, love, love your three month update and cannot wait to read more. Congrats again! Soooo excited for you! Xoxoxoxoxoxoxoox
- October 20, 2009
- HollyLynne said...
Thanks guys!!!
Maura, you just made me tear up. Not hard to do lately, but still :) I love my blog friends!
- October 20, 2009
- leenie said...
happy happy new baby!
i work with pregnant woman and still could read about pregnancy all day long and not get tired of it. it's so so incredible and crazy and scary and wonderful. i'm a longtime reader and wanted to finally comment with a huge congrats!
- October 20, 2009
- Brenda said...
Congratulations! My youngest daughter had her baby 2 1/2 months early..... but both are okay. I'm excited to hear your adventure!
- October 20, 2009
- Domesticrazy said...
Dude, I had to leave my gushing on my blog's comments because I couldn't get to your comments last night. I am so happy for you! YAY YAY YAY!!!!!
- October 21, 2009
- HollyLynne said...
Thanks, guys! Bean and I had a check up this morning and all is still well :)
- October 21, 2009
I have been reading your blog for a while and haven't ever commented but I couldn't not say CONGRATS!!!! Loved reading about your first three months of pregnancy. Can't wait to read more.
- October 21, 2009
- She Likes Purple said...
So happy for you, lady! Your kiddo is going to be stylish, one way or the other!
- October 21, 2009
- ReFabulous said...
EEEEEEEEEEEE! (You probably heard that for real!) Congrats, Holly! Soooo excited! Oh, just imagine all the baby craftiness.....!
- October 22, 2009
- ReFabulous said...
Oh, and about maternity pants... my favorites are Old Navy's, because they have that HUGE, but thin, stretchy band that can be rolled down, folded over, stretched up, whatever. They'll last you through the whole pregnancy, and the pants will actually STAY UP. (Um, yeah, I've had a pair FALL DOWN before. Not pretty.) Just my 2 cents.
- October 22, 2009
- HollyLynne said...
Refabulous, I have a pair like that from Gap that I LOVE! I wear a Be Band from Target over them now and they stay up beautifully. I just went and cleaned out their maternity clearance section of anything with full panels. At first, I though the full panels would be uncomfortable but you're so right . . .they stay up MUCH better!
- October 22, 2009
- Camels & Chocolate said...
Ahhhh so I'm really just like a week late. Not shabby, HA.
Yay, Baby Bean!!!
- October 27, 2009
- TamaliMama said...
Congrats, you have a very fun and interesting 7 months ahead of you!
After all the puking and uncomfortablness (near the end) it's all worth it!
I am waiting (not so patiently) for monday when I am going to have my c-section, our second bean! Ha that's what I called them too!
Welcome to Motherhood!
- October 28, 2009
- HollyLynne said...
They really just look like little beans! :D We're going to have a hard time calling the baby by it's actual name when it comes out, I think :)
- October 28, 2009


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The Lake District National Park Authority (LDNPA) took over the upkeep of the paths from Cumbria County Council 24 years ago.
But it said it can no longer afford the cost of path repairs and if it does not get cash help it will have to hand path responsibility back to the council.
The council said it would consider the request at a meeting on 20 December.
A spokesman for the county council said: "We have been aware for some time that the LDNPA wants the council to, either pay them for the upkeep of certain rights of way which they volunteered to maintain, or to carry out the work ourselves.
"Their request is one of many calls on us for funding which will be considered at a cabinet meeting later this month.
Economic benefits
"We have yet to receive an indication from the government about what the county council funding will be for 2006/7."
The LDNPA said it is facing a £600,000 overspend in the next financial year and spends almost £1m a year on the upkeep of paths.
A spokesman said: "A first-class rights of way system is regarded as vital in attracting walkers and ramblers to the area and the associated economic benefits they bring to the tourist industry.
"But faced with its current financial problems, LDNPA members have decided to ask the council to make a worthwhile contribution to that annual rights of way bill."


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QF 12-pr
When QF guns were first introduced in 1880 the Royal Navy was the first customer. In 1881 the RN saw the need for a light QF gun to combat fast motor torpedo boats (MTBs), and two years later adopted the Hotchkiss 6-pr for the purpose.
In 1885 the Royal Artillery adopted the Nordenfelt QF 6-pr for coast artillery, also to combat MTBs. As both Hotchkiss and Nordenfelt had performed equally well during trials both had been accepted into the services. Navy preferred the former, Army the latter.
As MTBs became bigger, faster, and more powerful a better gun was needed to deal with them. Thus the QF 12-pr was introduced in 1894 with Army also adopting it for coast defence.
Four QF 12-pr equipments as shown in Fig. 1 were received in New Zealand in 1900, and in 1901 were installed, two at Fort Cautley, Devonport, and two at Fort Ballance, Wellington (later moved to Fort Dorset, Seatoun). It seems at this stage the Government did not believe South Island ports were worth defending.
The gun was of the usual built-up construction (see Fig. 2), but possessed a unlque feature: the breech mechanism did not have extractors! A member of the detachment was provided with a hook-like implement with which he jerked the fired case from the chamber, thus performing extraction and ejection in one 'go' as it were. Apparently it worked reasonably well for no one ever requested extractors. However, they were fitted near the outbreak of World War 2.
About the same time as the 12-pr 12-cwt was introduced, a 12-pr 8-cwt was proposed as a ship, boat or field marine gun, ie for the support of Naval landing parties. However, the gun was mounted on a field carriage typical of those then in use by the Royal Artillery. The recoil system was not efficient and drag shoes had to be used under the wheels to keep the carriage steady in action. It is understood the Navy were not happy with the equipment and demanded a 'less powerful gun'.
The introduction of 'torpedo-boat destroyers;' later simply known as 'destroyers', led in 1903 to the production of a number of 12-prs of 18-cwt. These were similar to the 12-cwt guns but were 10 calibres longer. See Fig. 2.
The 12-pr on a high-angle mounting was adapted to the anti-aircraft role during World War 1, after which it reverted to the coast role. Some were 'resurrected' during World War 2 for use on defensively armed merchant ships (DEMS). See Fig. 6.
The 12-pr remained in service until the abolition of coast artillery in 1956 (1959 in New Zealand).
How a 12-pr came to be preserved in the Army Museum is interesting. As Gunners serving during the 1950s well remember the Government sold all coast artillery equipment as scrap meta1 - including near-new guns, e.g. BL 9.2 and 6-inch Mark 21 and 24s. Army made no attempt to retain any item for historical purposes.
Fig 6: QF 12-pr 12-cwt in anti-aircraft role on travelling carriage. The same equipment - less the carriage - was used on defensively equipped merchant ships (DEMS) during World War 2.
Determined to rescue something, members of 9 Coast Cadre, then stationed at the Artillery Yard, Devonport, dismounted one of the 12-prs then concealed it under a heap of rubble. The scrap merchant came and went but the gun remained. After allowing a suitable length of time to elapse, the Cadre 'resurrected' the gun and mounted it at Narrow Neck Camp. Eventually it found its way to the Museum. Unfortunately while the equipment lay unguarded thieves stole some of the brass fittings, e.g. the pistol grip, but the bulk survives.
ADDITIONAL DATA FOR 12-cwt EQUIPMENT:
WL Ruffell
Guns at Waiouru | Guns around New Zealand | More equipments | Home


text:
REDLANDS >> Valley Preparatory School received a hands-on lesson from area golf pros on Thursday afternoon.
Laughter and high-fives flooded the campus' grassy field where dozens participated in three activities centered around the sport with representatives from the Professional Golfers' Association of Southern California.
It was the second time the organization stopped by the school.
"The program is part of our 'Neighborhood Golf,' and what we do is try to bring golf to the communities off the course," said Alex Tegels, director of youth development for the golf organization. "What we hope to do is expose children when they are young and get them excited about the game. It's all about really exposing them to the game that is important to us."
In addition to teaching youth about the sport, the organization stresses the importance of honesty and respect. Since golf is a solo sport, the honor system is in play, Tegels said.
Valley Prep first grader Prubgeet "Pruby" Singh had a smile on his face as he waited his turn on the PGA's obstacle course.
While he did not have much experience with the game, the 7-year-old said it brought back memories of playing the sport with his dad.
"One time we went on vacation, and I got to go to a practice range and hit some balls," he said. "I like playing golf because I like swinging and hitting stuff."
The PGA traditionally attends community-related events.
"We don't do this very often at schools, but we usually do over 50 events a year," Tegels said.
To learn more, contact the school at 909-793-3063 or visit valleyprepredlands.org.


text:
10 Favorite Soup & Sandwich Shops in Greater Phoenix
Essence Bakery Cafe
Folks here in the Valley might not experience a true winter chill, but that doesn't mean we don't like to snuggle up with a hearty sandwich and a comforting bowl of soup when we need that extra bit of warmth in our souls.
See also: The 10 Best Things I Ate in 2012 10 Favorite Spots for Pie & Coffee in Greater Phoenix
Here are 10 of our favorites places to get both. Stop by one and curl up with some satisfying eats.
Essence Bakery Cafe This low-key Tempe eatery may be best known for Eugenia Theodosopoulos' luscious French macaróns, but its chef/owner boasts a top-notch sandwich and soup menu as well. Don't miss daily combo specials like a chicken, cornichons, and grain mustard sandwich with smokey ham and split pea soup or a tuna melt with cheddar cheese and cucumber served with a cup of organic yam with a touch of cream.
Postino Winecafe Sure, the casual-cool Postino's (with three Valley locations) is good to go for vino and bruschetta, but it does a fine job with sandwiches and soup-of-the-moment offerings, too. Don't miss the Select Two special ($9.75), in which half a panini sandwich (think a veggie with white bean hummus or The Nine Iron with bacon and grilled chicken) can be had with a fresh-made soup like cheddar potato, tomato, and roasted pepper and Gorgonzola.
DeFalco's At this first-rate Italian deli and grocery in Scottsdale, you're not just eating the ingredients; chances are you're sitting right next to them. Choose from about 40 kinds of monstrous hot or cold sandwiches made with top-notch ingredients. A soup of the day ($4.25/bowl) comes with hot focaccia bread and might be anything from minestrone to Italian wedding soup.
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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
I know hands on offenders are never eligible for parole but he isnt being charged with that. He's charged with downloading and sharing pictues / videos through a P2P network. He received a reasonable offer from the DA (assuming he's able to get out before as early as possible). Should he worry about not being eligible for parole based on his crime?
He should speak to his attorney.
Joseph A. Lo Piccolo, Esq.
Immediate Past President, Criminal Courts Bar Association 11'-12'
Hession Bekoff & Lo Piccolo
1103 Stewart Ave, Suite 200
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New York no longer has parole. NY has post release supervision.
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The Online Application for the Individual Membership
The Online Application for the Institutional Membership
Please join the Chinese Economic Society if you are interested in our activities. The CES memberships include: individual membership and institutional membership. The individual membership includes regular membership, family member membership, student membership, and lifetime membership. The institutional membership includes non-profit membership and business membership.
As a CES member, you will be eligible to the following benefits under CES bylaw (based on Amendment to the CES Membership, approved by the CES BOD on Sept 10, 2011):
The individual members of the Chinese Economist Society (CES) receive available logistic subsidies at the CES annual conference, register for the CES annual conference at discounted early-bird registration rate, propose paper presentations or discussion panels for CES events at ASSA/AEA annual meetings without a submission fee, have submission fee to China Economic Review (CER) waived (pending on amendment of CES-CER Agreement) and are eligible to apply for CES-Chow Short-term Teaching Program and be nominated for Gregory Chow Best Paper Award, or join other CES activities for members. All members have voting and election rights defined in the CES bylaws and will be included in the CES Email list.
The institutional members are entitled to post job advertisements and event announcements without charges on the CES web site and CES Email net (CES-L) with the approval of the CES Office, to make one free presentation slot at CES business meeting at ASSA/AEA annual meetings with the approval of the president, to receive two complementary registrations for the CES annual conference, to be listed in the CES annual conference program and to be included in the CES Email list.
Membership Dues:
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Student membership: $15 per year. Student status must be certified by a faculty adviser or school registrar with written verification, or copy of Student ID.
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Lifetime membership: $1,000 lump sum. For senior members (have served on the Board of Directors), the lifetime membership is $300 lump sum.
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Non-profit institution membership: $1,200 per year.
Business institution membership: $10,000 per year.
To apply for individual membership, you can submit your application online by clicking here.
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