Evaluation of site-specific methylation of the CMV promoter and its role in CHO cell productivity of a recombinant monoclonal antibody

We previously demonstrated that increased monoclonal antibody productivity in dihydrofolate reductase (DHFR)-amplified CHO cells correlates with phosphorylated transcription factor-cytomegalovirus (CMV) promoter interactions. In this article, we extend the characterization to include CMV promoter methylation and its influence on NFκB and CREB1 transcription factor binding to the CMV promoter in two families of DHFR-amplified CHO cell lines. CMV promoter methylation was determined using bisulfite sequencing. To overcome Sanger-sequencing limitations due to high CG bias and multiple transgenes copies, pyrosequencing was used to determine the frequency of methylated cytosines in regions proximal to and containing the NFκB and CREB1 transcription-factor consensus binding sites. Chromatin immunoprecipitation was performed to interrogate transcription factor-DNA interactions. Antibodies to CREB1 and NFκB were used to immunoprecipitate formaldehyde-crosslinked protein-DNA fractions, followed by reverse transcription quantitative real-time polymerase chain reaction to quantitate the number of copies of CMV-promoter DNA bound to the various transcription factors. The relative unmethylated fraction at the CREB1 and NFκB consensus binding sites determined by pyrosequencing was correlated with transcription factor binding as determined by chromatin immunoprecipitation. Azacytidine treatment reduced methylation in all treated samples, though not at all methylation sites, while increasing transcription. Distinct promoter methylation patterns arise upon clonal selection in different families of cell lines. In both cell line families, increased methylation was observed upon amplification. In one family, the NFκB binding-site methylation was accompanied by increased CREB1 interaction with the promoter. In the other cell line family, lower methylation frequency at the NFκB consensus binding site was accompanied by more NFκB recruitment to the promoter region

Effects of maternal folic acid supplementation during the second and third trimesters of pregnancy on neurocognitive development in the child:an 11-year follow-up from a randomised controlled trial

Background: Maternal folic acid (FA) supplementation before and in early pregnancy prevents neural tube defects (NTD), but it is uncertain whether continuing FA after the first trimester has benefits on offspring health. We aimed to evaluate the effect of FA supplementation throughout pregnancy on cognitive performance and brain function in the child. Methods: Follow-up investigation of 11-year-old children, residing in Northern Ireland, whose mothers had participated in a randomised trial of Folic Acid Supplementation in the Second and Third Trimesters (FASSTT) in pregnancy and received 400 μg/day FA or placebo from the 14th gestational week. Cognitive performance (Full Scale Intelligence Quotient, Verbal Comprehension, Working Memory, Perceptual Reasoning, and Processing Speed) was assessed using the Wechsler Intelligence Scale for Children. Neuronal function was assessed using magnetoencephalographic (MEG) brain imaging. Results: Of 119 mother-child pairs in the FASSTT trial, 68 children were assessed for neurocognitive performance at 11-year follow-up (Dec 2017 to Nov 2018). Children of mothers randomised to FA compared with placebo scored significantly higher in two Processing Speed tests, i.e. symbol search (mean difference 2.9 points, 95% CI 0.3 to 5.5, p = 0.03) and cancellation (11.3 points, 2.5 to 20.1, p = 0.04), whereas the positive effect on Verbal Comprehension was significant in girls only (6.5 points, 1.2 to 11.8, p = 0.03). MEG assessment of neuronal responses to a language task showed increased power at the Beta (13–30 Hz, p = 0.01) and High Gamma (49–70 Hz, p = 0.04) bands in children from FA-supplemented mothers, suggesting more efficient semantic processing of language. Conclusions: Continued FA supplementation in pregnancy beyond the early period currently recommended to prevent NTD can benefit neurocognitive development of the child. MEG provides a non-invasive tool in paediatric research to objectively assess functional brain activity in response to nutrition and other interventions. Trial registration: ISRCTN ISRCTN19917787. Registered on 15 May 2013

Folic acid intervention during pregnancy alters DNA methylation, affecting neural target genes through two distinct mechanisms

Background We previously showed that continued folic acid (FA) supplementation beyond the first trimester of pregnancy appears to have beneficial effects on neurocognitive performance in children followed for up to 11 years, but the biological mechanism for this effect has remained unclear. Using samples from our randomized controlled trial of folic acid supplementation in second and third trimester (FASSTT), where significant improvements in cognitive and psychosocial performance were demonstrated in children from mothers supplemented in pregnancy with 400 µg/day FA compared with placebo, we examined methylation patterns from cord blood (CB) using the EPIC array which covers approximately 850,000 cytosine–guanine (CG) sites across the genome. Genes showing significant differences were verified using pyrosequencing and mechanistic approaches used in vitro to determine effects on transcription. Results FA supplementation resulted in significant differences in methylation, particularly at brain-related genes. Further analysis showed these genes split into two groups. In one group, which included the CES1 gene, methylation changes at the promoters were important for regulating transcription. We also identified a second group which had a characteristic bimodal profile, with low promoter and high gene body (GB) methylation. In the latter, loss of methylation in the GB is linked to decreases in transcription: this group included the PRKAR1B/HEATR2 genes and the dopamine receptor regulator PDE4C. Overall, methylation in CB also showed good correlation with methylation profiles seen in a published data set of late gestation foetal brain samples. Conclusion We show here clear alterations in DNA methylation at specific classes of neurodevelopmental genes in the same cohort of children, born to FA-supplemented mothers, who previously showed improved cognitive and psychosocial performance. Our results show measurable differences at neural genes which are important for transcriptional regulation and add to the supporting evidence for continued FA supplementation throughout later gestation. This trial was registered on 15 May 2013 at www.isrctn.com as ISRCTN19917787.Land and Food Systems, Faculty ofNon UBCReviewedFacultyResearche

Polyadenylation of <i>Dnmt1</i> in the mouse ovary.

<p>(<b>A</b>) Phylogenetic analysis of the <i>Dnmt1</i> 3′UTR in Clustal X shows that the CPE and MBE sites form part of a larger conserved element (CON) which is identical across diverse species. Grey shading denotes areas of 100% homology between species. The CPEB binding site (CPE: UUUUAU in mRNA) is boxed by a thick black line; the consensus RNA binding sequence for mammalian Musashi (MBE; G/AU_1–3AGU), is present as GTAGT (fine black line). The Hex sequence is also indicated. Species identity and RefSeq accession numbers are indicated at left; Hex- polyA hexanucleotide; CON- conserved region. (<b>B</b>) Rapid Amplification of cDNA End-Poly(A) Test (RACE-PAT) to assess polyadenylation levels. <i>Top panel:</i> Synthesis of cDNA was carried out with oligo dT primer, and subsequent PCR used a <i>Dnmt1</i> gene-specific primer and anchored oligo dT to prevent artificial shortening of the PCR products in subsequent cycles. <i>Bottom left panel:</i> Endogenous <i>Dnmt1</i> mRNA undergoes polyadenylation in ovaries where the first wave of oocytes are in the growth phase (6–24 days post partum-dpp). <i>Bottom right panel</i>: Southern blotting and hybridization with the DNMT1-specific probe indicated above. (<b>C</b>) RNA ligation-coupled RT-PCR was used to confirm polyadenylation. Following ligation of an oligo to the poly(A) tails of all mRNA species present in oocytes harvested from ovaries of the indicated age, RT-PCR was carried out using the gene-specific forward primers indicated at right and a primer anti-sense to the ligated oligo. For the <i>Gdf9</i> positive control, more of the signal is dispersed upwards on the gel (direction of arrow) as mRNA tails become longer in growing oocytes. <i>Dnmt1</i> mRNA can be seen to follow a similar pattern. Control PCR using gene-specific primers for β-actin demonstrates RNA integrity. (<b>D</b>) RT-PCR shows that transcripts of <i>Cpeb1</i> are present at high levels in ovaries throughout postnatal development, while <i>Msi1</i> is expressed in growing oocytes only (10–24 dpp). (<b>E</b>) Western blotting shows that only DNMT1^s is found in ES cells, whereas DNMT1^o begins to appear in growing oocytes and accumulates by 10 dpp. MSI1 shows strongest expression at 10 dpp. Signal for DNMT1^o is visible in 3 month old (3 m) adult ovaries on longer exposure. Knockout ES cells lacking DNMT1 (1KO) and GAPDH are shown as controls.</p

Msi plays a key role in polyadenylation in the <i>Xenopus</i> oocyte.

<p>(<b>A</b>) Polyadenylation occurs at GVBD in <i>Xenopus</i> oocytes. RNA ligation-coupled RT-PCR indicates that the endogenous <i>Xenopus Dnmt1</i> mRNA (<i>xDnmt1</i>) has a short poly(A) tail in immature oocytes, but becomes elongated during progesterone stimulated maturation (arrow indicates extent of polyadenylation). Oocytes were collected when 50% had undergone germinal vesicle breakdown (GVBD) and grouped into those which had (+) or had not (−) completed GVBD. The maximum size of poly(A) tail confirmed by sequencing is indicated in nucleotides (nt) beside the arrow. (<b>B</b>) Polyadenylation of mouse <i>Dnmt1</i> (<i>mDnmt1</i>) in <i>Xenopus</i> oocytes requires the MBE but not CPE. In vitro transcribed wildtype (WT), MBE mutant (MBE mut), or CPE mutant (CPE mut) <i>Dnmt1</i> 3′UTR constructs were injected into immature <i>Xenopus</i> oocytes, which were then stimulated with progesterone treatment and assayed as above. When the MBE is mutated, polyadenylation does not occur, while the pattern of polyadenylation of the CPE mutant is similar to wildtype. Max confirmed poly(A) tail size by cloning indicated as for (A).</p

Redundant control involving MSI and CPEin mouse embryonic stem cells.

<p>(A) Schematic to show the mutations used to test functional requirements in the <i>Dnmt1</i> 3′UTR after cloning it downstream of a luciferase reporter. As well as the CPE and MBE, the region contains a potential Pumilio binding element (PBE): all three were mutated (dark grey letters) using point mutation or insertions (MBE). The ΔCON construct contains a deletion of the entire conserved block indicated in Fig. 1. (<b>B</b>) Mutations in the CPE (ΔCPE) MBE (ΔMBE), or PBE significantly decrease translation of the luciferase reporter in mouse ES cells, but combined mutations (ΔCΔM; or triple mutant, TM) do not have an additive effect. Deletion of the conserved region reduces translation to below 40% of the wildtype <i>Dnmt1</i> 3′UTR. Readings in triplicate from more than three independent experiments are presented as mean +/−SEM; ***p<0.001 (by 2-tailed unpaired Students T-test) compared to expression of wildtype, which was arbitrarily set to 100%. (<b>C</b>) MSI1 interacts with endogenous <i>Dnmt1</i> mRNA. RNA-immunoprecipitation was performed in R63 mouse ES cells with anti-MSI1 antibody or normal rabbit IgG. Immunoprecipitated RNA was analysed by RT-PCR (top panel). The known MSI1 target <i>Numb</i> could be amplified, while <i>Actb</i> which is not a target, was absent, confirming the technique was working. <i>Dnmt1</i> could be amplified from MSI1 immunoprecipitates, but not negative controls, indicating MSI1 also binds to this mRNA. A no template control (NTC) was also included. Quantification by RT-qPCR (bottom panel) indicated significant enrichment of <i>Dnmt1,</i> to levels approx. half that of <i>Numb</i> (bottom right). Results are the average of three independent experiments carried out in triplicate. (<b>D</b>) Transient knockdown of RNA binding proteins in ESC. Levels of depletion of the respective target mRNAs following transfection by the indicated siRNA were assessed by RT-qPCR. A scrambled siRNA (mock) was used as a control. An example multiplex experiment (dark bars) is shown at right. All target mRNA were significantly reduced (p<0.05) in single or multiplex experiments. (<b>E</b>) Western blotting showing redundant control ensures DNMT1 translation in ESC. Transient depletion of any one of the RNA binding proteins MSI1, MSI2, CPEB or PUM2, or of combinations thereof such as MSI1 and MSI (MSi1&2) and MSI1, CPEB1 and PUM2 (TKD), had no detectable effect on DNMT1 protein levels in ESC. DNMT1 siRNA are shown as a positive control, mock and WT are negative controls.</p

Cells lacking MSI1 show greater CPEB dependence.

<p>(<b>A</b>) HeLa cells lack MSI1. Western blotting shows that while DNMT1 is expressed in HeLa, HCT116 and R63 ES cells, the expression of MSI1 is limited and detected only in HCT116 and R63 ES cells. Sizes are indicated in kilodaltons (kDa). Similar results were found for PUM2 (<b>B</b>) CPE is one important component of translational control in HeLa. Luciferase constructs as indicated in Fig. 3 were transfected into HeLa cells. Mutation of the CPE reduced expression of the reporter gene to a similar extent as observed in ESC cells. Significance **p<0.01; ***p<0.001 (<b>C</b>) CPEB1 was targeted for knockdown in HeLa cells by stable shRNA expression. Levels of <i>CPEB1</i> mRNA in two clonally-derived cell lines B11 and B12 carrying the shRNA are shown. (<b>D</b>) <i>Dnmt1</i> mRNA levels remain stable in CPEB1 knockdown cell lines. (<b>E</b>) Stable CPEB1 knockdown prevents efficient DNMT1 translation. Protein levels of CCNB1, which is known to be translationally repressed by CPEB1, show upregulation in both stable knockdown cell lines as expected. Reprobing the same membrane shows DNMT1 protein levels are reduced in these samples. Molecular weights are indicated to the left in kiloDaltons (KDa). (<b>F</b>) CPEB4 transient knockdown in HeLa. RT-qPCR shows mRNA levels relative to mock-transfected cells following transient knockdown of CPEB4 and DNMT1 in HeLa. (<b>G</b>) Transient depletion of CPEB4 reduces DNMT1 protein levels to an extent comparable to that achieved by <i>DNMT1</i> siRNA.</p