Expression of the SERPING1 gene is not regulated by promoter hypermethylation in peripheral blood mononuclear cells from patients with hereditary angioedema due to C1-inhibitor deficiency
© López-Lera et al.; licensee BioMed Central Ltd. 2014
Received: 24 February 2014
Accepted: 27 June 2014
Published: 22 July 2014
SERPING1 mutations causing Hereditary Angioedema type I (HAE-I) due to C1-Inhibitor (C1-INH) deficiency display a dominant-negative effect usually resulting in protein levels far below the expected 50%. To further investigate mechanisms for its reduced expression, we analyzed the promoter DNA methylation status of SERPING1 and its influence on C1-INH expression. Global epigenetic reactivation correlated with C1-INH mRNA synthesis and protein secretion in Huh7 hepatoma cells. However, PBMCs extracted from controls, HAE-I and HAE-II patients presented identical methylation status of the SERPING1 promoter when analyzed by bisulphite sequencing; the proximal CpG island (exon 2) is constitutively unmethylated, while the most distant one (5.7Kb upstream the transcriptional start site) is fully methylated. These results correlate with the methylation profile observed in Huh7 cells and indicate that there is not a direct epigenetic regulation of C1-INH expression in PBMCs specific for each HAE type. Other indirect modes of epigenetic regulation cannot be excluded.
C1-Inhibitor (C1-INH; OMIM#606860) is a SERPIN regulating the activation of the classical and lectin complement pathways, coagulation and fibrinolysis cascades []. It is mainly produced in the liver, although local synthesis by monocytes, fibroblasts and endothelial cells in inflammatory foci also exists. Deficiency of C1-INH due to mutations in the SERPING1 gene causes Hereditary Angioedema (HAE) types I and II. HAE type I (HAE-I) can be due to mutations located throughout the gene sequence and is characterized by functional and antigenic C1-INH deficiency. HAE type II (HAE-II) is caused by point mutations affecting the reactive centre of the protein or nearby residues and presents with reduced function but normal antigenic levels of the protein. HAE patients suffer from recurrent disabling episodes of swelling in the subcutaneous and submucosal layers which can affect any body location and, if not promptly and properly treated, can lead to suffocation when the larynx is affected [].
HAE is inherited in an autosomal dominant manner. Typically, HAE-I heterozygous patients exhibit C1-INH levels that are markedly below the expected 50% of controls, while type II patients have normal levels, but reduced function of C1-INH. Thus, regarding protein levels, HAE-I mutant alleles seem to exert dominant negative effects on their wild-type counterparts, rendering similarly reduced mRNA levels in both alleles to around 50% [-]. Additionally, a small percentage of HAE patients exhibiting reduced C1-INH function do not carry known SERPING1 mutations even after exhaustive sequence analyses and MLPA screening; therefore, alternative modes of regulating C1-INH remain to be identified.
DNA methylation is an epigenetic event that plays a role in development, aging, chromatin structure, genomic imprinting and gene and microRNA expression. Altered DNA methylation patterns have been described in many genes and microRNAs associated with disease [-], including some members of the SERPIN superfamily, whose epigenetic alterations have been described in human placental diseases []. To contribute to the understanding of C1-INH's regulation of expression in HAE, we have investigated the CpG methylation status of the C1-INH locus and its surrounding regions in the hepatoma cell line Huh7 and in stored DNA samples from PBMCs obtained from healthy donors, HAE-I and HAE-II patients.
Results and disussion
Identification of putative CpG islands in the SERPING1 locus
The screening for CpG islands was initially performed in the SERPING1’s genomic region containing its coding sequences (exons 1 to 8 and their adjacent introns). Next, we extended the search 6 kb in 5’ direction until the closest CpG cluster addressing the selection criteria:
More than 55% of GC dinucleotides (i), ObservedCpG vs. Expected CpG ratio: 0.65 (ii), Length: at least 200 bp (iii), Distance: 100 bp (iv).
Global demethylation induced by 5’Aza and TSA upregulates SERPING1 expression in Huh7 cells at the mRNA and protein levels
To our knowledge, no studies focused on quantifying C1-INH synthesis in Huh7 cells have been published, but the matter has been extensively addressed in other hepatocellular carcinoma cell lines, especially in the HepG2 line. HepG2 cells synthesize and secrete functional C1-INH and its expression is enhanced by IFN-γ administration in a dose-dependent manner [,].
We quantified C1-INH mRNA from 1 μg of total RNA extracted from 75% confluent Huh-7 control cell cultures and compared it to that of Huh7 cells treated with 5’Aza + TSA for 3 culture days as described []. Reverse-transcription with oligo-dT primers (Qiagen, Valencia, CA) and quantification using a SYBR Green system (Applied Biosystems, Foster City, CA) were performed as previously described [].
Similarly to what is seen in IFN-γ treated cells, global demethylation strongly correlated with enhanced SERPING1 mRNA synthesis. Furthermore, mRNA synthesis was higher in 5’Aza + TSA-treated cultures (38-fold) than in those exposed to 250UI/mL of IFN-γ for 3 days (20-fold). Combined addition of 5Aza, TSA and IFN-γ during 3 culture days produced a non-additive effect resulting in a 28-fold change upregulation of SERPING1 expression (Figure 1B). This probably reflects the consequences of global DNA/histones demethylation on the transcriptional/translational regulation of the IFNG gene (encoding human IFN-γ) or other loci involved in the transduction of IFN-γ signalling. In fact, DNA and histones’ epigenetic marks are known to play a role in transcriptional activation and silencing of IFN-γ signalling [].
C1-INH protein presents two different isoforms in serum and plasma by western blot analysis: a 105KDa protein representing its native, non-cleaved conformation and an additional 96 kDa form corresponding to its cleaved, latent, non-functional conformation. In our hands, Huh7 cultures exhibit a single 105 kDa native band, which suggests the absence of activity involving those serum proteolytic pathways regulated by C1-INH (Figure 1C). Western blot analysis of culture supernatants confirmed SERPING1 synthesis observed at the RNA level; no bands were observed in the supernatants of control Huh7 cells after three days of culture, while a single 105 kDa band corresponding to the native conformation of C1-INH was visible in treated Huh7 cells both by monoclonal anti-C1-INH antibody and biotinylated-C1s' detection systems (Figure 1C). Those results indicate a possible epigenetic regulation of SERPING1 gene expression in the Huh7 hepatoma cells.
CpG island 1 is unmethylated in HAE patients and controls while CpG island 2 is methylated
Bisulphite sequencing of stored genomic DNA samples obtained from HAE PBMCs (8 type I and 8 type II, Additional file 1), 10 healthy donor PBMCs and Huh7 cells was carried out as previously described [] These studies received approval from the ethical committee of Hospital La Paz.
We included genomic DNAs from a homozygous-deficient patient carrying the R378C missense mutation in exon 7 and a heterozygous relative (samples 3 and 3b, respectively). Genetic and biochemical characterization of these patients has been previously described []. The T157fsX78 frameshift mutation in HAE-I patients 4, 4b and 4c and the exon 4 deletion in patients 1 and 6 are expected to profoundly reduce SERPING1 mRNA levels due to the introduction of premature stop codons. In contrast, the remaining type I and type II mutations do not have obvious implications on translation or protein structure.
Considering the lack of evidence for differential epigenetic regulation at the DNA methylation level of promoter activity in the SERPING1 locus in HAE patients’ PBMCs, the fact that the demethylating agents 5'Aza-dC and TSA strongly increased mRNA and protein synthesis in Huh7 cells could reflect an upstream indirect epigenetic regulation of trans-acting factors like transcription factors or microRNAs in the hepatocyte. Those elements could be reactivated by demethylation as has been reported in previous studies using similar experimental epigenetic reactivation approaches [,]. On the other hand, we found no evidence for epigenetic mechanisms involving promoter CpG islands 1 and 2 playing a starring role in the negative dominance previously reported in HAE-I PBMCs.
The approach employed in this paper for the analysis of SERPING1 epigenetics is evidently hindered by the impossibility of obtaining hepatocyte samples from patients and controls in which to perform re-expression studies and bisulphite sequencing. Moreover, taking into consideration that monocytes do not undergo cell division, they are not suitable for 5’AZA + TSA re-expression studies, as a minimum of two cell divisions are required to completely remove epigenetic marks from DNA and histones. Therefore, these results are strictly limited to PBMCs and do not inform about epigenetic regulation in the hepatocyte, which is the main source of plasma C1-INH.
All considered, our data suggest that CpG island 1 is in a transcriptional-permissive chromatin conformation that would hypothetically allow the access of transcriptional machinery to the gene's promoter sites in PBMCs. The upregulating effect of demethylating agents on Huh7 cells would be due to epigenetic regulation of additional factors involved in SERPING1's control of transcription and suggests an analogous mechanism could account for the differential C1-INH plasma levels characteristic of each HAE type.
López-Trascasa Margarita and Ibanez deCaceres Inmaculada are Senior authors.
We are thankful to the patients, their families and clinicians for being part of these studies.
The work was funded by grants PS09/00122 by Instituto de Salud Carlos III (ISCIII) and Ministerio de Economía y Competitividad SAF2012-38636. OP and IIC were supported by FIS PS09/00472, PI12/00386 and Miguel Servet (CP 08/000689; PI-717). ALL was supported by CIBERER (Instituto de Salud Carlos III, ISCIII).
- Bowen T, Cicardi M, Farkas H, Bork K, Longhurst HJ, Zuraw B, Aygoeren-Pürsün E, Craig T, Binkley K, Hebert J, Ritchie B, Bouillet L, Betschel S, Cogar D, Dean J, Devaraj R, Hamed A, Kamra P, Keith PK, Lacuesta G, Leith E, Lyons H, Mace S, Mako B, Neurath D, Poon MC, Rivard GE, Schellenberg R, Rowan D, Rowe A, et al: 2010 International consensus algorithm for the diagnosis, therapy and management of hereditary angioedema. Allergy Asthma Clin Immunol. 2010, 6 (1): 24-10.1186/1710-1492-6-24. doi:10.1186/1710-1492-6-24View ArticlePubMedPubMed CentralGoogle Scholar
- Caballero T, Farkas H, Bouillet L, Bowen T, Gompel A, Fagerberg C, Björkander J, Bork K, Bygum A, Cicardi M, de Carolis C, Frank M, Gooi JH, Longhurst H, Martínez-Saguer I, Nielsen EW, Obtulowitz K, Perricone R, Prior N: International consensus and practical guidelines on the gynecologic and obstetric management of female patients with hereditary angioedema caused by C1 inhibitor deficiency. J Allergy Clin Immunol. 2012, 129 (2): 308-320. 10.1016/j.jaci.2011.11.025.View ArticlePubMedGoogle Scholar
- Kramer J, Rosen FS, Colten HR, Rajczy K, Strunk RC: Transinhibition of C1 inhibitor synthesis in type I hereditary angioneurotic edema. J Clin Invest. 1993, 91 (3): 1258-1262. 10.1172/JCI116290.View ArticlePubMedPubMed CentralGoogle Scholar
- Pappalardo E, Zingale LC, Cicardi M: C1 inhibitor gene expression in patients with hereditary angioedema: quantitative evaluation by means of real-time RT-PCR. J Allergy Clin Immunol. 2004, 114 (3): 638-644. 10.1016/j.jaci.2004.06.021.View ArticlePubMedGoogle Scholar
- Cugno M, Zanichelli A, Foieni F, Caccia S, Cicardi M: C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress. Trends Mol Med. 2009, 15 (2): 69-78. 10.1016/j.molmed.2008.12.001. doi:10.1016/j.molmed.2008.12.001View ArticlePubMedGoogle Scholar
- Feinberg AP: Phenotypic plasticity and the epigenetics of human disease. Nature. 2007, 447 (7143): 433-440. 10.1038/nature05919.View ArticlePubMedGoogle Scholar
- Toyota M, Suzuki H, Sasaki Y, Maruyama R, Imai K, Shinomura Y, Tokino T: Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer. Cancer Res. 2008, 68: 4123-4132. 10.1158/0008-5472.CAN-08-0325.View ArticlePubMedGoogle Scholar
- Grady WM, Parkin RK, Mitchell PS, Lee JH, Kim YH, Tsuchiya KD, Washington MK, Paraskeva C, Willson JK, Kaz AM, Kroh EM, Allen A, Fritz BR, Markowitz SD, Tewari M: Epigenetic silencing of the intronic microRNA hsa-miR-342 and its host gene EVL in colorectal cancer. Oncogene. 2008, 27: 3880-3888. 10.1038/onc.2008.10.View ArticlePubMedGoogle Scholar
- Chelbi ST, Mondon F, Jammes H, Buffat C, Mignot TM, Tost J, Busato F, Gut I, Rebourcet R, Laissue P, Tsatsaris V, Goffinet F, Rigourd V, Carbonne B, Ferré F, Vaiman D: Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia. Hypertension. 2007, 49 (1): 76-83. 10.1161/01.HYP.0000250831.52876.cb. Epub 2006 Nov 6View ArticlePubMedGoogle Scholar
- Takai D, Jones PA: The CpG island searcher: a new WWW resource. In Silico Biol. 2003, 3 (3): 235-240.PubMedGoogle Scholar
- Lappin DF, Guc D, Hill A, McShane T, Whaley K: Effect of interferon-gamma on complement gene expression in different cell types. Biochem J. 1992, 281 (Pt 2): 437-442.View ArticlePubMedPubMed CentralGoogle Scholar
- Morris KM, Aden DP, Knowles BB, Colten HR: Complement biosynthesis by the human hepatoma-derived cell line HepG2. J Clin Invest. 1982, 70 (4): 906-913. 10.1172/JCI110687.View ArticlePubMedPubMed CentralGoogle Scholar
- Ibanez de Caceres I, Dulaimi E, Hoffman AM, Al-Saleem T, Uzzo RG, Cairns P: Identification of novel target genes by an epigenetic reactivation screen of renal cancer. Cancer Res. 2006, 66 (10): 5021-5028. 10.1158/0008-5472.CAN-05-3365.View ArticlePubMedGoogle Scholar
- de la Cruz RM, López-Lera A, López-Trascasa M: Analysis of SERPING1 expression on hereditary angioedema patients: quantitative analysis of full-length and exon 3 splicing variants. Immunol Lett. 2012, 141 (2): 158-164. 10.1016/j.imlet.2011.07.011. doi:10.1016/j.imlet.2011.07.011View ArticlePubMedGoogle Scholar
- Aune TM, Collins PL, Collier SP, Henderson MA, Chang S: Epigenetic Activation and Silencing of the Gene that Encodes IFN-γ. Front Immunol. 2013, 4: 112-10.3389/fimmu.2013.00112. doi:10.3389/fimmu.2013.00112View ArticlePubMedPubMed CentralGoogle Scholar
- Ibanez de Caceres I, Cortes-Sempere M, Moratilla C, Machado-Pinilla R, Rodriguez-Fanjul V, Manguán-García C, Cejas P, López-Ríos F, Paz-Ares L, de CastroCarpeño J, Nistal M, Belda-Iniesta C, Perona R: IGFBP-3 hypermethylation-derived deficiency mediates cisplatin resistance in non-small-cell lung cancer. Oncogene. 2010, 29 (11): 1681-1690. 10.1038/onc.2009.454. doi:10.1038/onc.2009.454View ArticlePubMedGoogle Scholar
- López-Lera A, Favier B, de la Cruz RM, Garrido S, Drouet C, López-Trascasa M: A new case of homozygous C1-inhibitor deficiency suggests a role for Arg378 in the control of kinin pathway activation. J Allergy Clin Immunol. 2010, 126 (6): 1307-10.e3. 10.1016/j.jaci.2010.07.037. doi:10.1016/j.jaci.2010.07.037View ArticlePubMedGoogle Scholar
- Flicek P, Amode MR, Barrell D, Beal K, Billis K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fitzgerald S, Gil L, Girón CG, Gordon L, Hourlier T, Hunt S, Johnson N, Juettemann T, Kähäri AK, Keenan S, Kulesha E, Martin FJ, Maurel T, McLaren WM, Murphy DN, Nag R, Overduin B, Pignatelli M, Pritchard B, Pritchard E, Riat HS, et al: Ensembl 2014. Nucleic Acids Res. 2014, (Database issue): D749-D755. 10.1093/nar/gkt1196. doi:10.1093/nar/gkt1196View ArticleGoogle Scholar
- Agathanggelou A, Bièche I, Ahmed-Choudhury J, Nicke B, Dammann R, Baksh S, Gao B, Minna JD, Downward J, Maher ER, Latif F: Identification of novel gene expression targets for the Ras association domain family 1 (RASSF1A) tumor suppressor gene in non-small cell lung cancer and neuroblastoma. Cancer Res. 2003, 63 (17): 5344-5351.PubMedPubMed CentralGoogle Scholar
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