The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver.Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients

F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker

Association of COMT genotypes with S-COMT promoter methylation in growth-discordant monozygotic twins and healthy adults

<p>Abstract</p> <p>Background</p> <p>Catechol-O-Methyltransferase (COMT) plays a key role in dopamine and estrogen metabolism. Recently, COMT haplotypes rather than the single polymorphism Val158Met have been reported to underlie differences in protein expression by modulating mRNA secondary structure. So far, studies investigating the epigenetic variability of the S-COMT (soluble COMT) promoter region mainly focused on phenotypical aspects, and results have been controversial.</p> <p>Methods</p> <p>We assessed S-COMT promoter methylation in saliva and blood derived DNA with regard to early pre- and postnatal growth as well as to genotype for polymorphisms rs6269, rs4633, and rs4680 (Val158Met) in 20 monozygotic twin pairs (mean age 4 years), who were discordant for intrauterine development due to severe feto-fetal-transfusion syndrome. Methylation levels of two previously reported partially methylated cytosines were determined by the quantitative SIRPH (SNuPE- IP RP HPLC) assay.</p> <p>Results</p> <p>Overall, we observed a high variability of S-COMT promoter methylation, which did not correlate with individual differences in the pre- or postnatal growth pattern. Within the twin pairs however we noted a distinct similarity that could be linked to underlying COMT genotypes. This association was subsequently confirmed in a cohort of 93 unrelated adult controls. Interestingly, 158Val-alleles were found at both ends of the epigenotypical range, which is in accordance with a recently proposed model of COMT haplotypes corresponding to a continuum of phenotypical variability.</p> <p>Conclusion</p> <p>The strong heritable component of S-COMT promoter methylation found in our study needs to be considered in future approaches that focus on interactions between COMT epigenotype and phenotype.</p

NLRP7, Involved in Hydatidiform Molar Pregnancy (HYDM1), Interacts with the Transcriptional Repressor ZBTB16

<div><p>Mutations in the maternal effect gene <i>NLRP7</i> cause biparental hydatidiform mole (HYDM1). HYDM1 is characterized by abnormal growth of placenta and lack of proper embryonic development. The molar tissues are characterized by abnormal methylation patterns at differentially methylated regions (DMRs) of imprinted genes. It is not known whether this occurs before or after fertilization, but the high specificity of this defect to the maternal allele indicates a possible maternal germ line-specific effect. To better understand the unknown molecular mechanism leading to HYDM1, we performed a yeast two-hybrid screen against an ovarian library using NLRP7 as bait. We identified the transcriptional repressor ZBTB16 as an interacting protein of NLRP7 and verified this interaction in mammalian cells by immunoprecipitation and confocal microscopy. Native protein analysis detected NLRP7 and ZBTB16 in a 480kD protein complex and both proteins co-localize in the cytoplasm in juxtanuclear aggregates. HYDM1-causing mutations in NLRP7 did not show altered patterns of interaction with ZBTB16. Hence, the biological significance of the NLRP7-ZBTB16 interaction remains to be revealed. However, a clear effect of harvesting ZBTB16 to the cytoplasm when the NLRP7 protein is overexpressed may be linked to the pathology of the molar pregnancy disease.</p></div

Domain structure of NLRP7 and ZBTB16.

<p><b>A.</b> Domain structure of NLRP7. Location of analyzed HYDM1-causing mutations L398R, R693P, R693W and non-synonymous variant (NSV) K511R are marked with orange arrowheads. Apart from a full-length construct and the four individual NLRP7 domains, five additional deletion constructs were generated, either lacking one or two domains at a time. <b>B.</b> Domain structure of the transcription factor ZBTB16 identified as interaction partner by the yeast two-hybrid screening. Apart from the full-length construct, containing an N-terminal BTB/POZ, a RD2 and nine zinc-fingers, five additional ZBTB16 deletion constructs were generated (ZBTB16_del1-5). ZBTB16_del4 (aa 268–673) corresponds to “prey#1” identified by the yeast two-hybrid screen.</p

Yeast two-hybrid screen of full-length ZBTB16 against different NLRP7 deletion constructs and against full-length NLRP7 containing different HYDM1-causing mutations.

<p><b>A.</b> Yeast two-hybrid screen against the different NLRP7 constructs (fused to the GAL4 activation domain; pGADT7) using full-length ZBTB16 as bait (fused to the GAL4 binding domain; pGBKT7) performed with the Gal4-System (Clontech). ZBTB16 interacted with the highly reactive NAD domain and the LRR deleted constructs. An interaction of ZBTB16 with full-length NLRP7 failed. The self-interaction between full-length ZBTB16 served as positive control as it is already known that ZBTB16 forms a dimer by its N-terminal POZ/BTB domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130416#pone.0130416.ref030" target="_blank">30</a>]. <b>B.</b> Yeast two-hybrid mutation screen using ZBTB16 as bait (pGBKT7) against full-length NLRP7 containing one of the three HYDM1-causing mutations L398R (NACHT), R693P (LRR), R693W (LRR) or the NSV K511R (all pGADT7). The NACHT domain-associated mutation L398R resulted in the same strong interaction between full-length NLRP7 and full-length ZBTB16 as seen between NAD:ZBTB16 and ΔLRR:ZBTB16.</p

Blind docking of ZBTB16 and NLRP7 threaded protein models.

<p>The figure is split into three panels, each representing the ZBTB16/NLRP7 dock in differently colored formats. The models are depicted in stick form. <b>A.</b> The first panel shows the proteins ZBTB16 and NLRP7 in yellow and green. The interface regions are marked and specified. The red residues on the interface regions belong to ZBTB16, while the blue ones belong to NLRP7. <b>B.</b> and <b>C</b>. Both panels show the same docks but with the individual domains of ZBTB16 and NLRP7, respectively, colored according to the code described below. The apposing protein in each panel is uniformly colored in grey.</p

Co-Immunoprecipitation and Blue Native PAGE analysis between NLRP7 and ZBTB16.

<p><b>A.</b> Co-immunoprecipitation of transiently transfected ZBTB16 (Myc-tagged) and NLRP7 (Flag-tagged) in HEK293T cells. Immunoprecipitation was done using an anti-Flag specific antibody. Quantification of interaction between NLRP7 and ZBTB16 was determined relative to NLRP7 full-length. <b>B.</b> Co-immunoprecipitation of five ZBTB16 deletion constructs del1-5 (Myc-tagged) by immunoprecipitation of full-length NLRP7 (Lane 1) or one of four NLRP7 deletion constructs (lane 2–5; Flag-tagged) using an anti-Flag specific antibody. <b>C.</b> Blue Native gel electrophoresis of NLRP7 (Flag-tagged) and ZBTB16 (Myc-tagged) after transient transfection in HEK293T cells. Native protein extraction was performed using different concentrations of digitonin that is indicated above the picture. <b>Upper panels:</b> In the first dimension, the native Flag-NLRP7 appears as a smeary oligomer in a broad shift from 480–1000 kD (left side), while native ZBTB16-Myc was detected as a sharp band at 480 kD (right side). <b>Lower panels:</b> In the second dimension, the monomeric Flag-NLRP7 is visible as a thin line at 113kD, while ZBTB16-Myc occurs as individual spot at its expected size of 75 kD.</p

DataSheet1_The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression.docx

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.</p

Table1_The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression.xlsx

Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types.Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes.Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.</p