Metrics details
The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2
We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2
H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation
We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1
We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements
We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process
The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter
and the second is MIWI2 engagement with the nascent transcript
independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation
piRNA-directed transposon methylation requires precision
Failing to methylate every active transposon would be detrimental to the genomic integrity of the germline
but aberrant off-target methylation could result in germline-transmitted epimutations
piRNAs endow MIWI2 with the specificity to identify active transposon loci and
whether other mechanisms contribute to identifying active transposons and the exacting precision of the pathway remains unknown
AlphaFold2 co-folding prediction of the interaction between SPIN1 (Q61142) and SPOCD1 (B1ASB6; only amino acids 326–348 are shown)
Crosslinking mass spectrometry of mouse SPOCD1 fragment 1b (amino acids 203-409) with mouse SPIN1 (amino acids 49–262)
Phylogenetic tree from ray-finned fishes to mammals showing the presence of SPOCD1 and SPIN1 in the indicated animal clades
AlphaFold2 prediction of SPOCD1 from Anolis carolinensis (an anole lizard
XP_031752218.1) and Latimeria chalumnae (coelacanth
TFIIS-M domain and SPIN1-interacting β-hairpin are highlighted
Multiple sequence alignment of the SPOCD1 SPIN1-interacting β-hairpin region from different species
Numbering for mouse SPOCD1 is shown above the sequences and secondary-structure elements of mouse SPOCD1 are shown below
Sequences are coloured according to sequence identity
Representative Coomassie gel image of n = 3 co-precipitation experiments with the indicated recombinant SPOCD1 from different species with mouse SPIN1
SPIN1 (green) and DAPI (blue) staining of wild-type fetal testis sections from the indicated developmental time points
Images are representative of n = 3 biological replicates
Volcano plot showing enrichment (log2(mean label-free quantification ratio of anti-HA immunoprecipitates from Spocd1HA/HA/wild-type)) and statistical confidence (−log10(P-value of two-sided Student’s t-test)) of proteins co-purifying with HA-SPOCD1 from E14.5 fetal testes; n = 3
H3K9me3 and SPIN1 from E14.5 fetal germ cells
H3K9me3) and three (SPIN1) biological replicates
metaplot and heatmaps of signal over elements of different transposon families (e) are shown as well as young and old copies in the L1Md_T (f) and L1Md_A (g) families
Columns adjacent to the heatmaps show statistically significant peaks called for SPIN1 and the indicated histone modifications
the overlap of H3K4me3 and H3K9me3 peaks with SPIN1 peaks is significant for L1Md_A (P = 0.0099
Z-score = 2,007) by one-tailed permutation tests
enrichment of overlapping H3K4me3 and H3K9me3 peaks with SPIN1 peaks is significantly different between young and old L1Md_A (adjusted P < 2.2 × 10−16) and L1Md_T (adjusted P < 2.2 × 10−16) copies
as observed by two-tailed Fisher’s exact test
charts show overlap analysis of H3K4me3 and H3K9me3 peaks (h) and SPIN1 peaks (i) with the indicated genomic features
P-values and Z-scores from one-tailed permutation tests to assess the statistical significance of overlaps of CUT&TAG peaks with LINE1 elements are shown
a, Representative western-blot analyses of n = 3 immunoprecipitations of the mouse wild type and eight SPOCD1 alanine mutations (8 Ala mut) with SPIN1 in HEK 293 T cells. For whole-blot source data, see Supplementary Fig. 1
Representative Coomassie gel image of n = 3 co-precipitation experiments with the indicated recombinant proteins
Representative images of E16.5 gonocytes from n = 3 wild-type (WT) and Spocd1ΔSPIN1 mice stained for DNA (blue) and SPOCD1 (c)
Number of embryos per plug fathered by studs with the indicated genotype mated to wild-type females
from n = 6 wild-type (15 plugs in total) and n = 6 Spocd1ΔSPIN1 studs (12 plugs)
Testis weight of adult mice with the indicated genotype
from n = 8 wild-type and n = 8 Spocd1ΔSPIN1 mice
a representative image of testes from wild-type (left) and Spocd1ΔSPIN1 (right) mice
P-values in f and g were determined by unadjusted two-sided Student’s t-test
Representative images of PAS and haematoxylin-stained testes sections of wild-type and n = 5 Spocd1ΔSPIN1 adult mice
with different types of spermatogenic arrest observed in the tubules of the Spocd1ΔSPIN1 testes indicated
The percentage of each type of tubule is noted alongside
Adult testis sections stained for the DNA damage marker γH2AX (red) (i) and apoptotic cells (red) by TUNEL assay (j) from wild-type and Spocd1ΔSPIN1 mice (representative of n = 3 mice per genotype for γH2AX and n = 2 wild-type plus n = 3 Spocd1ΔSPIN1 mice for TUNEL)
Representative testis sections of n = 3 wild-type
Spocd1ΔSPIN and Spocd1−/− mice stained red for the LINE1 ORF1p (a) or IAP GAG protein (b)
RNA-seq heat maps showing fold changes in expression relative to wild type for the ten most upregulated LINE1 and ERVK transposable elements in Spocd1−/− P20 testes (n = 3 from each genotype)
***P < 0.001 of Bonferroni-corrected two-sided Wald’s test assuming n-binominal distribution
Only significant differences (P < 0.05) are shown
Genomic CpG methylation analysis of P14 undifferentiated spermatogonia from wild-type (n = 6)
Spocd1ΔSPIN (n = 4) and Spocd1−/− mice (n = 3)
Percentages of CpG methylation levels of the indicated genomic features (with genic
promoter and CpG island (CGI) regions defined as those not overlapping transposable elements
and intergenic regions as those not overlapping transposable elements or genes) or transposable elements (not overlapping genes) are shown as box plots
Boxes represent interquartile range from the 25th to the 75th percentile
and whiskers show the data range of the median ± twice the interquartile range
Significant differences (P < 0.05 of Bonferroni-corrected two-tailed Student’s t-tests) of Spocd1ΔSPIN (n = 4) and Spocd1−/− (n = 3) samples to wild-type (n = 6) are indicated
Metaplots of mean CpG methylation over the indicated transposable element
**P = 0.01–0.001 and ***P < 0.001 for Bonferroni-corrected two-tailed Student’s t-tests comparing the average CpG methylation of the promoter region to wild type for Spocd1ΔSPIN1 (red) and Spocd1−/− (blue)
Correlation analysis of mean CpG methylation loss relative to the wild type for individual transposable elements of the indicated LINE1 and ERVK families in relation to their divergence from the consensus sequence in Spocd1ΔSPIN spermatogonia
How SPOCD1 is recruited to IAPs remains unknown
but we speculate that another SPOCD1-associated protein could mediate this recruitment through the recognition of a distinct chromatin signature or sequence motif
The different mechanisms in LINE1 and IAPs reveals an unexpected complexity in the pathway
The prevailing notion is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of the piRNA–MIWI2 ribonucleoprotein complex with the nascent transcript
we demonstrate that multiple independent and developmentally choreographed events are required for LINE1 piRNA-directed DNA methylation
Our revised model posits that the recruitment of SPIN1–SPOCD1 through chromatin modification to young LINE1 elements constitutes a first licensing step
The engagement of MIWI2 with the nascent transcript is the second licensing event and triggers DNA methylation
we propose that a two-factor authentication system ensures the precision of LINE1 piRNA-directed methylation
Male fertility was assessed by mating studs to Hsd:ICR (CD1) wild-type females and counting the number of pups born for each plugged female
animal tissue samples were collected from one or more litters and allocated to groups according to genotype
No further randomization or blinding was applied during data acquisition and analysis
Animals were maintained at the University of Edinburgh
in accordance with the regulation of the UK Home Office
or at the Institute for Molecular Biology in Mainz
in accordance with local and European animal-welfare laws
Ethical approval for the UK mouse experimentation has been given by the University of Edinburgh’s Animal Welfare and Ethical Review Body and the work done under licence from the UK Home Office
Animal experiments done in Germany were approved by the ethical committees on animal care and use of the federal states of Rheinland-Pfalz
Duke University) 1:500; anti-γH2AX (Bethyl Laboratories) 1:500; anti-MIWI2 (a gift from R
Université de Genève) 1:500; anti-SPOCD1 rabbit serum rb175 1:500 (O’Carroll laboratory antibody); anti-SPIN1 (Cell Signaling Technologies) 1:500 (of a custom preparation of 1.1 μg μl−1 in PBS)
Images were taken on a Zeiss Observer or Zeiss LSM880 with an Airyscan module
Images acquired using the Airyscan module were deconvoluted with the Zeiss Zen software ‘Airyscan processing’ with settings 3D and a strength of 6
ImageJ and Zeiss Zen software were used to process and analyse the images
cells were washed twice with PBS and resuspended in 1 ml lysis buffer (IP buffer: 150 mM KCl
supplemented with 1× protease inhibitors (cOmplete ULTRA EDTA-free
Roche) with 37 units per ml benzonase (Millipore)) and lysed for 30 min
The lysate was cleared by centrifugation for 10 min at 21,000g
Cleared lysate (800 μl) was incubated with 20 μl of anti-HA beads (Pierce) that had been calibrated in lysis buffer and incubated for 1 h at 4 °C on a rotating wheel
The beads were washed four times with lysis buffer
Immunoprecipitates were eluted at 50 °C for 10 min in 20 μl 0.1% sodium dodecyl sulphate (SDS)
Lysates and eluates were run on a 4–12% bis–tris acrylamide gel (Invitrogen) and blotted onto a nitrocellulose membrane (Amersham Protran 0.45 NC) according to standard laboratory procedures
The membrane was blocked with blocking buffer (4% (w/v) skimmed milk powder (Sigma-Aldrich) in PBS-T (phosphate buffered saline
0.1% Tween-20)) and subsequently incubated for 1 h with primary antibodies (anti-HA (C29F4s
anti-SPOCD1 rabbit serum rb175 (O’Carroll laboratory antibody) 1:500 or anti-α-Tubulin (T9026
Sigma-Aldrich) 1:1,000) in blocking buffer
The anti-α-tubulin staining was used as loading control on the same blot as the experimental staining
the membrane was incubated with secondary antibodies (IRDye 680RD donkey anti-rabbit or IRDye 800CW donkey anti-mouse
It was washed three times for 10 min in PBS-T and imaged on a LI-COR Odyssey CLx system
Exposure of the entire images was optimized in Image Studio Lite (LI-COR)
and areas of interest were cropped for presentation
GST-tagged mouse SPOCD1 fragments (amino acids 203–409)
Latimeria SPOCD1 fragments (XP_014348336.1
amino acids 510–1009) and His-tagged SPIN1 (amino acids 49–262) were cloned in a pET-based backbone
Proteins were expressed in Escherichia coli BL21 (DE3)
Bacteria were grown in 2xTY media at 37 °C until an optical density of 0.8 was reached
the bacteria were induced with 1 mM IPTG and grown for another 14–16 h
Cells were collected and pellets were stored at −80 °C until purification
The pellets were resuspended in 50 ml lysis buffer (20 mM Tris-HCl
Roche cOmplete EDTA-free Protease Inhibitor Cocktail
0.01 mg ml−1 DNaseI (Sigma) and 2 mM AEBSF (Pefabloc) for SPIN1
0.01 mg ml−1 DNaseI (Sigma) and 2 mM AEBSF (Pefabloc) for SPOCD1) and cells were lysed with the Constant systems 1.1 kW TS cell disruptor at 25 kPSI
The cleared lysate was used to load on a cOmplete His-Tag Purification Column (Roche) for SPIN1 or incubated with 7 ml glutathione sepharose high-performance beads (Cytiva) for SPOCD1 calibrated in the respective buffer
Elution from column/beads with increasing (2.5–500 mM) imidazole gradient for SPIN1 or GST elution buffer containing 20 mM reduced glutathione for SPOCD1
The fractions of interest were pooled and dialysed overnight in 20 mM Tris-HCl
The SPIN1 construct was cleaved with GST–3C protease (made in our lab) overnight
The SPOCD1 constructs were concentrated and stored at −80 °C until used
SPIN1 was further purified by ion exchange with a gradient of 100–1,000 mM NaCl (Resource Q
Cytiva) and size-exclusion chromatography (HiLoad 16/600 Superdex 200 pg
the protein was concentrated and stored at −80 °C until used
starting from H3Δ1–31T32C C110A constructs that also contained the required H3X and H3Y mutations
H3X was used for H3K4me3 and H3Y for H3K9me3
A biotinylated 209-bp DNA fragment containing the 601 nucleosome positioning sequence was generated by PCR and purified by ion-exchange chromatography on a HiTrap Q column followed by ethanol precipitation
Mononucleosomes were then assembled from histone octamers and 601 DNA by gradient dialysis
Nucleosome assembly was verified by native gel electrophoresis on 6% acrylamide gels in 0.5× TGE buffer (12.5 mM Tris
After incubation with recombinant proteins
beads were washed three times with high-salt pull-down buffer (as above but with 350 mM NaCl) for 5 min
Nucleosomes and bound proteins were eluted by boiling in 1.5× SDS sample buffer (95 mM Tris HCl
Binding was analysed by western blotting with antibodies against His tag (Sigma H1029
Antibodies against histone H3 (Abcam ab176842
H3K4me3 (Cell Signaling) 1:2,000 and H3K9me3 (Abcam ab176916) 1:1,000 were used to verify nucleosome loading and modification state
For analytical size-exclusion chromatography
125 μg SPIN1 and/or 500 μg mouse GST–SPOCD1-F1b were used for each run
Proteins were diluted in 250 μl size-exclusion chromatography buffer (20 mM HEPES
1 mM DTT) and injected on a Superdex 200 10/300 GL column
loaded on an SDS–PAGE gel and visualized by Coomassie staining
IP-MS of SPOCD1–HA from Spocd1HA/+ E14.5 fetal testis using 50 μl of anti-HA beads (Pierce, 88837) was done as previously described4
with a reduced number of 25 testes per replicate
Wild-type fetal testes were used as controls
To purify foetal germ cells for CUT&Tag analysis, E14.5 testes were dissected from embryos carrying the Oct4eGFP allele34
A single cell suspension was obtained by sequential treatment with 100 µl collagenase solution at 37 °C for 8 min (10 units of collagenase A (Sigma-Aldrich 10103578001); 2× NEAAs (Gibco); 2× Na-pyruvate (Gibco); 25 mM HEPES–KOH
pH 7.5) and 200 µl TryPLE Express (Gibco) at 37 °C for 5 min with gentle flicking and pipetting of the solution to aid dissociation
Digestion was neutralized by 70 µl prewarmed FBS and cells were collected by spinning at 600g for 4 min at room temperature followed by two washes in FACS buffer (1× PBS; 2 mM EDTA
10% FBS; 2 µg ml−1 DAPI) and filtering (Corning
Cell sorting was done on an Invitrogen Bigfoot using a 100 μm nozzle and gating for DAPI-negative (live)
OCT4–eGFP-positive (germ cells) populations into collection tubes containing 100 µl 1× PBS
For EM-seq, CD9+ spermatogonia were sorted from P14 testes as described previously52 using Fc block (eBioscience
lot 2297433) 1:50; biotin-conjugated anti-CD45 (eBioscience
and biotin-conjugated anti-CD51 (Biolegend
lot B308465) 1:100 anti-CD9APC (eBioscience
Cells were sorted into DMEM media on a BD Aria II sorter
pelleted for 5 min at 500g and snap frozen in liquid nitrogen
For gating strategies, see Supplemental Fig. 2
using pA–Tn5 at a 1:400 dilution (Diagenode
C01070001) and 15 PCR cycles of library amplification
Libraries were cleaned up by magnetic bead-based solid-phase separation and assessed on a Tapestation (Agilent)
Antibodies and dilutions used for CUT&Tag were rabbit IgG control (Abcam
and guinea pig anti-rabbit IgG (Antibodies Online
Pooled libraries were sequenced using paired-end 150 bp on a NextSeq 2000 instrument (Illumina)
using the following parameters: -bs 1 --normalizeUsing CPM —exactScaling --ignoreForNormalization MT
Log2 enrichment profiles of CUT&Tag samples over IgG controls were generated with deepTools bamCompare using the following parameters: -bs 1 --normalizeUsing CPM --exactScaling --ignoreForNormalization MT --scaleFactorsMethod None
Monomers associated with inert promoters (subtypes 6 and 2) were removed from the analysis
The central regions of repetitive elements were length-normalized to 5 kb with flanking regions ±2 kb from the start and end positions
Heatmaps and profile plots show data in consecutive 10b bins with regions subdivided by elements and arranged in descending order of total enrichment across all samples
Histology experiments on mouse samples were done as previously described4
TUNEL assay experiments were done as previously described4
two-tailed Student’s t-tests were used to compare the differences between groups and adjusted for multiple testing using Bonferroni correction where indicated
Averaged data are presented as mean ± s.e.m.
No statistical methods were used to predetermine the sample size
The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article
The scripts used for the EM-seq and RNA-seq analysis are available from github at https://github.com/rberrens/SPOCD1-piRNA_directed_DNA_met, and the scripts used for ChIP and CUT&Tag analysis are available from github at https://github.com/swebb1/heep-et-al_2024
PIWI-interacting RNAs: small RNAs with big functions
The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements
TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing
SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation
Zoch, A. et al. C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line. Mol Cell https://doi.org/10.1016/j.molcel.2024.01.014 (2024)
Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation
Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells
Jr A novel active L1 retrotransposon subfamily in the mouse
Developmentally regulated piRNA clusters implicate MILI in transposon control
Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L
MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline
Transcription of IAP endogenous retroviruses is constrained by cytosine methylation
The diverse roles of DNA methylation in mammalian development and disease
A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice
Two waves of de novo methylation during mouse germ cell development
Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes
Distinct mode of methylated lysine-4 of histone H3 recognition by tandem tudor-like domains of Spindlin1
Active genes are tri-methylated at K4 of histone H3
Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain
Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins
Highly accurate protein structure prediction with AlphaFold
Highly accurate protein structure prediction for the human proteome
Broad heterochromatic domains open in gonocyte development prior to de novo DNA methylation
Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse
Efficient low-cost chromatin profiling with CUT&Tag
A tudor domain protein SPINDLIN1 interacts with the mRNA-binding protein SERBP1 and is involved in mouse oocyte meiotic resumption
The DNA methyltransferase DNMT3C protects male germ cells from transposon activity
encoding a DNA methyltransferase homolog required for meiosis and transposon repression in the mouse male germline
DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes
A transit-amplifying population underpins the efficient regenerative capacity of the testis
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering
One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering
Oct4 expression is not required for mouse somatic stem cell self-renewal
A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming
G9a co-suppresses LINE1 elements in spermatogonia
ColabFold: making protein folding accessible to all
PyMOL Molecular Graphics System v.1.8 (Schrodinger
scalable generation of high-quality protein multiple sequence alignments using Clustal Omega
Jalview Version 2−a multiple sequence alignment editor and analysis workbench
ChromID identifies the protein interactome at chromatin marks
A synthetic biology approach to probing nucleosome symmetry
The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication
Trimmomatic: a flexible trimmer for Illumina sequence data
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013)
The Sequence Alignment/Map format and SAMtools
deepTools2: a next generation web server for deep-sequencing data analysis
Subtype classification and functional annotation of L1Md retrotransposon promoters
SeqPlots – interactive software for exploratory data analyses
pattern discovery and visualization in genomics
Wickham, H. Welcome to the Tidyverse. J. Open Source Softw. https://doi.org/10.21105/joss.01686 (2019)
Defective germline reprogramming rewires the spermatogonial transcriptome
The nf-core framework for community-curated bioinformatics pipelines
regioneR: an R/Bioconductor package for the association analysis of genomic regions based on permutation tests
Gaspar, J. M. Improved peak-calling with MACS2. Preprint at bioRxiv https://doi.org/10.1101/496521 (2018)
Software for computing and annotating genomic ranges
pyGenomeTracks: reproducible plots for multivariate genomic datasets
The Perseus computational platform for comprehensive analysis of (prote)omics data
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
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This research was supported by funding from Wellcome to D.O’C
(095021 and 200885); the Wellcome Centre for Cell Biology (203149 and multi-user equipment grants 108504 and 092076); funding for the Wellcome Discovery Research Platform for Hidden Cell Biology (226791); and support from the microscopy
proteomics and bioinformatics cores of the Wellcome Discovery Research Platform for Hidden Cell Biology
was funded by a German Research Foundation fellowship (DFG award ZO 376/1-1); J.B
were funded by a German Research Foundation collaborative research centre grant (DFG grant SFB 1361
Work in P.V.’s lab was supported by Wellcome (104175/Z/14/Z)
and the UK Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0421)
are funded by the Darwin Trust of Edinburgh
This work used the University of Edinburgh Protein Production Facility (EPPF)
the Wellcome Centre for Cell Biology’s Centre Optical Instrumentation Laboratory (COIL)
proteomics and bioinformatics core platforms
and the Centre for Regenerative Medicine’s FACS facility
We also thank staff at the EMBL GeneCore facility in Heidelberg
for preparing the methyl-seq libraries and sequencing all libraries; S
Nick at IMB flow-cytometry core facility for assistance with operating the Bigfoot cell sorter instrument (project number 511658729); and M
Heinen at the IMB protein production core facility for the recombinant Tn5 protein fusions
These authors contributed equally: Ansgar Zoch
Zhejiang University-University of Edinburgh Institute (ZJU-UoE Institute)
execution and analysis of most of the experiments
did the immunofluorescence and IP-MS experiments
did the CUT&RUN and CUT&Tag experiments under the guidance of J.B
did the bioinformatic analysis of the EM-seq
generated site-specifically modified histones
and designed and performed the nucleosome pull-down experiments
did the molecular-biology and histology experiments
analysed the crosslinking-mass spectrometry data
contributed to analyses and experimental design
The authors declare no competing interests
reviewer(s) for their contribution to the peer review of this work
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
HA (red) and DAPI (blue) staining of E16.5 foetal testis sections from Spocd1HA/+ mice treated with PBS or RNase A prior to fixation
HA (red) and DAPI (blue) staining of E16.5 foetal testis sections from E16.5 Miwi2−/−;Spocd1HA/+ and Miwi2+/−;Spocd1HA/+ mice
SPIN1 (green) and DAPI (blue) staining of E16.5 Miwi2+/− and Miwi2−/− E16.5 foetal testis sections
(c) shows a zoom-in of the cell highlighted with a dashed rectangle in (d)
Images of (a-d) are representative of n = 3 biological replicates
sequences are coloured according to sequence identity
Numbering above according to mouse sequence
Panels show H3K4me3 (a) and H3K9me3 (b) ChIP-seq signal in reads per million (RPM) over young and old elements within the indicated LINE1 family
Metaplot and heat maps of indicated CUT&Tag signal of H3K4me3
H3K9me3 and SPIN1 over young and old L1MD_F elements
Columns adjacent to the heatmaps show peaks called for SPIN1 and the indicated histone modifications
Data depicts element plus adjacent 2 kb for each of the transposon families indicated
Genome snapshots showing datatracks of CUT&Tag signal of H3K4me3
H3K9me3 and SPIN1 over selected genome regions containing a young L1Md_A
Enrichment of overlapping H3K4me3 and H3K9me3 peaks with SPIN1 peaks is not significantly different between young and old L1Md_F copies
as observed by a two-tailed Fisher’s exact test
Representative images of sections from n = 3 wild-type foetal testis stained for SPIN1 (green) and DAPI (blue) from indicated timepoints
Cell shown in (a) is highlighted with a white box in (b)
a, Representative western blot analyses of n = 3 anti-HA immunoprecipitations of the HA epitope-tagged mouse wild-type, SPOCD1 8 alanine mutated proteins or GFP control with FLAG-tagged DNMT3L in HEK 293 T cells. For whole blot source data, see Supplementary Fig. 1
Schematic representations of the mouse Spocd1 locus and encoded 1015 amino acid protein are shown
sgRNA used for generation of the Spocd1ΔSPIN1 allele and adjacent PAM site are indicated
Schematic of CRISPR targeting strategy showing the location of single-stranded oligo DNA donor (ssODN) and homology arms (HA) used
and sequencing trace of the part of Spocd1ΔSPIN1 exon 4 harbouring the mutation sites
a 30 bp sequence creating the 8 alanine mutation is highlighted in red
Representative image of genotyping result for n = 3 Spocd1+/+
Representative images of E16.5 gonocytes from n = 3 Spocd1ΔSPIN1 and wild-type control mice stained for SPOCD1 (e)
Supplementary Figures containing uncropped scans of the western-blot experiments shown in Figs
and the FACS gating strategy for sorting fetal germ cells and undifferentiated spermatogonia (Supplementary Fig
Further data relating to crosslinking mass spectrometry data
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The Alheira is a beautiful thing. A type of Portuguese sausage known for garlic and breadcrumbs, the meat link is so beloved in Portugal that it was voted one of the nation’s seven gastronomic wonders in a 2011 vote. Oh, and it was invented by Jews to save their lives
Portugal’s Jews went to great lengths to hide their religion
and in a mountain town in northern Portugal called Mirandela
the Portuguese would hang preserved pork sausages from their rafters to keep them fed through winter
But Mirandela’s secret Jewish community didn’t eat pork
and thus: an easy target for anti-Semites looking to turn in their Jewish neighbors
So the Jews did what they always do: they turned to food. Mirandela’s Jews invented a new sausage, the Alheira de Mirandela
which they made with kosher chicken and bread and promptly hung from their rafters
maybe the kosher version of this heroic Jewish sausage will make a comeback
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a high proportion of filler is not generally considered a positive
In a time when Jews were being persecuted in Rossio Square
just metres from where Manteigaria Silva’s cream awning extends today
Yet Portugal’s cuisine is more narrative-heavy than most
a complex tapestry of invasions and colonisations that slips and slides between continents and religions
• The ugly story behind a breakfast meat
• The dessert that’s blocked at borders
• Why you can’t get ‘Jewish food’ in Israel
the most popular and time-honoured ones have stayed with us over many centuries from the period of Moorish rule – also known as a golden era for Jews in Western Europe,” Paolo Scheffer
the sophisticated Muslim culture from North Africa that outsiders called the Moors ruled much of Iberia
including the hilly city known as Al-Ushbuna
A Jewish community had long lived and flourished here
Jews and Muslims both left their gastronomic mark on modern-day LisbonFrom marzipan and rosewater pastries to soups
citizens of both religions left their gastronomic mark on what is today the city of Lisbon
Moorish fish dishes and even Moorish broth
which is now a seafood dish called cataplana,” Scheffer observed
“But those dishes would have adhered to Judaic and Islamic dietary laws without the popular ingredients added today like shellfish
when Christian crusaders first roared through Lisbon
the city already had its own culinary culture: Christian elements
merged with this established set of flavours
as Portuguese navigators spread across the globe
chilli and black pepper would leave their mark in turn
it’s hard to separate what’s now identified as Christian Portuguese food from the established Arab and Jewish cuisines
Ferdinand of Aragon and his warrior queen Isabella of Castile defeated the last Moorish emirate – Granada – and took the Alhambra Palace as their own
Ferdinand and Isabella believed that practising Jews might encourage those who had converted to Christianity to go back to their old religion
They appointed interrogators to persecute the Jews in their kingdom: their rule of terror would be known as the Spanish Inquisition
tens of thousands of Jews who had flourished in Moorish Al-Andalus were thrown out of Spain
After overcrowding caused a plague outbreak
Christian citizens forced all Jews to live outside the city walls
Portugal’s Jews were also forced to convert to Christianity
rampaging citizens and sailors killed thousands of converted Jews in a citywide pogrom
the Inquisition formally arrived in Portugal
and soon both practising Jews and Jews who had converted to Christianity were among the unfortunates parading in penance or burnt at the pyre in Rossio Square
Jews went to huge lengths to conceal their faithDisguising themselves as Christian converts
Portugal’s secret Jews went to huge lengths to conceal their religion – from writing Hebrew prayers in Catholic prayer books to combining Jewish words with Catholic rituals
(One community in Belmonte kept its faith alive in secret for more than 400 years.) In the rugged mountains of northern Portugal’s Trás-os-Montes
one of these hidden communities created Portugal’s best-known alheira sausage: Alheira de Mirandela
In Trás-os-Montes every home preserved pork sausages to see the family through the winter
hanging them from the rafters in meaty coils
Jews – who did not eat pork – were conspicuous for their missing sausages
“They were seeking refuge from the Inquisition,” Scheffer explained
“So the town of Mirandela developed a bread sausage that could fool informers and local zealots who denounced them to the Inquisition for not eating pork.”
the Alheira de Mirandela seems very like kishke
meal and flavourings that’s often served in the slow-cooked Jewish Sabbath bean stew known as cholent
The Jews of Trás-os-Montes traditionally made theirs with bread and chicken
although a present-day Alheira de Mirandela is no longer kosher and can include everything from pork to game
the alheira has travelled far beyond the mountains
It’s ubiquitous in supermarkets and appears alongside steak and eggs in workers’ cafes or neighbourhood diners
At Zé dos Cornos
a white-tiled little place below Lisbon’s Castelo de São Jorge
I watched local workers chow down from huge rectangular plates
chunks of juicy game mingled with chunks of sour
the hillside district where they lived is known as Mouraria (Moorsville)
But it was not until the early 19th Century that Jews began to return and
there were no more than 1,000 Jews in Lisbon
the neutral city again became a refuge for Europe’s Jews
Portuguese diplomat Aristides de Sousa Mendes issued travel documents to thousands of Jews: more than 10,000 Jews would set sail from Lisbon to safety across the Atlantic
although towns and cities across Portugal are beginning to rediscover their Jewish history
the alheira is more a part of mainstream Portuguese cooking than a symbol of the people who created it
Like the Portuguese word for ‘Saturday’ – 'Sábado'
for the Jewish Sabbath – and the brilliant Arab-influenced tiles that illuminate Lisbon's teetering streets
the sausage is an indicator of a past as cosmopolitan as it is complex
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GM Carlos Daniel Albornoz Cabrera from Cuba emerged as the winner of the III Open Xadrez Terras Trás-os-Montes 2022
one of the strongest open tournaments held in Portugal in recent years
took place in Mirandela from August 17-23 and brought together 131 participants from 26 federations
confirmed his status as the main favourite and took the first prize scoring an impressive 8/9
the winner was awarded a chess set signed by Judit Polgar
IM Arthur Pijpers (Netherlands) and IM Jakub Kosakowski (Poland) finished a full point behind the champion and tied for second place
The Buchholz tiebreak favoured Leniart and Pijpers taking second and third positions on the podium
Several young players came to the final round seeking different norms
but only FM Guy Levin from Israel succeeded and completed an IM norm
Check out the complete results here
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Portugal’s CTM Mirandela triumphed over Czech club HB Ostrov in the opening leg of the Stage three of the Europe Cup Women
Annamaria ERDELYI delivered two wins against Veronika POLAKOVA and Zdena BLASKOVA
Mariana SANTA COMBA added the third point with a hard-fought five-game victory over Jana VASENDOVA
BLASKOVA secured the sole point for HB Ostrov by defeating Ines MATOS
We seized the opportunity to secure an advantage before the return leg in Czechia,” said ERDELYI
It’s great that Mariana managed to beat VASENDOVA
The return leg is scheduled for February 16th
jovem jogador com raízes familiares em Mirandela
assinou contrato com o Vitória de Guimarães para as próximas quatro épocas
confirmou o clube no seu canal de comunicação
O jogador de 21 anos vai ser orientado pelo também mirandelense
passou pelo Padroense e posteriormente pelo Desportivo de Chaves
O jovem mirandelense estreou-se oficialmente pela seleção nacional de Sub/17
ao marcar presença nos três jogos da 43.ª edição do Torneio Internacional do Algarve
Mirandela went the full distance but succeeded in France
In the opening leg of the Europe Cup quarterfinal Clube Ténis de Mesa de Mirandela secured an away win against JOUE LES TOURS TT
After a full five matches they overcame French club
Li HE handled the pressure exceptionally well defeating both Jingyi ZHOU and Annamaria ERDELYI
ZHOU and ERDELYI on the other hand showed no weaknesses in their duel against Audrey ZARIF
Equally important Ines MATOS won a very close duel against Nolwenn FORT
JOUE LES TOURS TT – Clube Ténis de Mesa de Mirandela 2-3
Li HE – Jingyi ZHOU 3-2 (11-6 9-11 11-5 9-11 6-1)
Audrey ZARIF – Annamaria ERDELYI 0-3 (7-11 9-11 4-11)
Nolwenn FORT – Ines MATOS 2-3 (12-14 11-9 11-7 7-11 1-6)
Li HE – Annamaria ERDELYI 3-0 (12-10 11-7 11-9)
Audrey ZARIF – Jingyi ZHOU 1-3 (7-11 11-8 7-11 9-11)
The European Champions League Women (ECLW) organised by the European Table Tennis Union (ETTU)
is the most important international club competition which replaced European Club Cup of Champions (ECCC)
Hungarian club Statisztika Budapest still holds remarkable record
They were on the top 11 consecutive seasons (1976-86)
Romanian Vointa Arad was the winner of the inaugural 1963/64 season
The competition came into a new era in 2005/06
when the European Champions League Women started
In an all-French Champions League Women’s semifinal
Metz TT overcame Saint-Quentin TT in the second..
Reigning champions KTS ENEA SIARKOPOL Tarnobrzeg were forced to surrender their title even before reaching..
Reigning champions KTS ENEA SIARKOPOL Tarnobrzeg and Saint-Quentin TT emerged victorious in the opening leg..
The Champions League Women semifinal lineup is now complete
In their debut appearance in the Europe Cup CSM Constanta reached the semifinals of the Women’s Cup
Joining the Romanian club Portugal’s Clube Ténis de Mesa de Mirandela also secured a place in the penultimate stage
CSM Constanta defeated GRAND-QUEVILLY ALCL 3-1 in the away match in France after prevailing 3-2 at home
Reaching the semifinal in our first year of the ETTU Cup season is a significant achievement
I am thrilled to have won two matches for my team; last time my sister contributed with two victories as well
Additionally Bianca did her best showing her high level and securing one match win
Given the high stakes we felt the pressure but we overcame it together
Supporting each other we remained focused from the beginning until the end
Despite facing really strong opponents we were fully aware of their capabilities
In the end the collective power of our team prevailed ” said Elena ZAHARIA
Clube Ténis de Mesa de Mirandela also prevailed in both legs against JOUE LES TOURS TT
In the opening leg it was a long-distance duel while tonight Annamaria ERDELYI Jingyi ZHOU and Ines MATOS showed no mercy
“After we won the opening leg we knew that we had a chance
That opportunity became even better when we saw the lineup of the opposing team without Audrey ZARIF
Of course it gave us a certain dose of confidence but winning 3-0 was never on the table
I opened the match very well and the crucial moment was the victory of our player from Singapore Jingyi ZHOU over the Chinese player Li HE
After that the very good performance of Ines MATOS sealed the match ” said Annamaria ERDELYI
In the semis Clube Ténis de Mesa de Mirandela awaits the winner of the match HB Ostrov z.s
GRAND-QUEVILLY ALCL – CSM Constanta 1-3
Samson LI – Alina MUSAT (ZAHARIA) 3-0 (11-6 11-4 11-9)
Jiaduo WU – Elena ZAHARIA 1-3 (7-11 7-11 11-6 11-13)
Anais SALPIN – Bianca MEI ROSU 2-3 (8-11 8-11 11-7 11-2 5-6)
Samson LI – Elena ZAHARIA 1-3 (5-11 11-8 6-11 7-11)
Clube Ténis de Mesa de Mirandela – JOUE LES TOURS TT 3-0
Annamaria ERDELYI – Clemence CHEVALLIER 3-0 (11-6 11-8 11-2)
Jingyi ZHOU – Li HE 3-1 (6-11 11-7 11-6 11-9)
Ines MATOS – Nolwenn FORT 3-1 (13-11 17-19 12-10 11-6)
After an intense final series in the Portugal League play-off Sporting CP emerged victorious over CTM Mirandela clinching their 15th title in the club’s history
The defending champions handed over the trophy to the new winners
In the opening match Sporting CP defeated Mirandela by a close score of 3-2
However Mirandela quickly leveled the score setting the stage for a decisive clash
Once again Sporting CP found themselves pushed to the limit but ultimately came out on top
The pivotal moment in the quest for the title came during the opening duel
In the fifth and final game of the fifth match Galia DVORAK of Sporting CP triumphed over Annamaria ERDELYI of Mirandela sealing the victory with a score of 15-13
“I believe that the moment that truly made the difference was the first match when we won 3-2 and sealed it with a 15-13 victory in the deciding game
The level of both teams was incredibly even and winning two matches in a row seemed unlikely for either side
Despite our loss in the second encounter we remained positive and knew that if we capitalized on our chances we could secure the title… and we did ” expressed Galia DVORAK
She further added “I am incredibly satisfied because I wasn’t sure if I could still perform at this level but I proved that I could
We have faced each other many times in the past so we both understand the intensity of this battle.”
Sporting CP’s triumph in securing their 15th trophy highlights their dominance in Portuguese table tennis
The team’s resilience and determination throughout the final series ultimately paid off solidifying their place in the club’s rich history
Annamaria ERDELYI Matilde PINTO – Anna HURSEY Galia DVORAK 3-2 (9-11 12-10 14-16 11-2 11-6)
Inês MATOS – Anna HURSEY 0-3 (9-11m 6-11 10-12)
Annamaria ERDELYI – Galia DVORAK 0-3 (9-11 7-11 8-11)
Matilde PINTO – Patrícia SANTOS 3-1 (11-9 11-5 8-11 11-7)
Inês MATOS – Galia DVORAK 0-3 (10-12 10-12 5-11)
Annamaria ERDELYI Matilde Pinto – Anna HURSEY Galia DVORAK 3-1 (11-5 11-9 7-11 11-9)
Inês MATOS – Galia DVORAK 1-3 (11-4 5-11 12-14 10-12)
Annamaria ERDELYI – Patrícia SANTOS 3-0 (11-3 11-3 11-5)
Matilde PINTO – Anna HURSEY 0-3 (7-11 8-11 8-11)
Inês MATOS – Patrícia SANTOS 3-2 (5-11 11-7 11-8 8-11 11-8)
Patrícia SANTOS Anna HURSEY – Inês MATOS Matilde PINTO 3-0 (11-5 12-10 11-6)
Galia DVORAK – Inês MATOS 1-3 (8-11 8-11 12-10 11-13)
Patrícia SANTOS – Annamaria ERDELYI 1-3 (18-16 9-11 12-14 9-11)
Anna HURSEY – Matilde PINTO 3-0 (11-6 11-8 11-5)
Galia DVORAK – Annamaria ERDELYI 3-2 (11-5 14-12 6-11 9-11 15-13)
The Europe Trophy Grand Final in Mirandela concluded with the won of the French TT Joué les Tours
On the final hurdle they succeeded against Greek club ASEA Sarises Florinas
In the ultimate stage Audrey ZARIF LI He and Nolwenn FORT overcame Bernadett BALINT LIU Judith and Despoina AMPA in straight matches
“It is first time for the club that we won the European Trophy and we are very happy
I hope we will be equally successful next season in t he Europe Cup ” said coach Hugo BERGER
Audrey ZARIF added: “It was a long week-end with a lot of matches
It was really important for the club to win this title so we gave our best
Some matches was more difficult than other we won all the matches 3/0 so that nice.”
In the semi final Entente Saint Pierraise TT faced first defeat at the tournament
Greece’s ASEA Sarises Florinas caused the big upset and won the difficult five matches duel to secure the ultimate stage
They were 2:0 up before they withstood spirited comeback from Entente Saint Pierraise TT to win the place in the final in the decisive match
“We had very tough match to reach the final but we know how difficult will be against next adversary
We already lost to Joue Les Tours in the group phase
However we will stay focused and we will fight hard ” said coach Mark PINK before the final
Joue Les Tours was the first team proceeding to the final
They overcame Croatia’s STK Aquaestil Duga Resa
“We are really happy that we won this match
We will give our best to win this Trophy ” said Audrey ZARIF
On the opening day Joue Les Tours overcame Portugal’s Uniao Sebastianense F.C Norway’s B-72 and Greek ASEA Sarises Florinas
ASEA Sarises Florinas – TT Joué les Tours 0:3
Bernadett BALINT – Audrey ZARIF 2:3 (11:7 8:11 4:11 11:9 1:6)
LIU Judith – LI He 1:3 (6:11 9:11 13:11 4:11)
Despoina AMPA – Nolwenn FORT 0:3 (9:11 7:11 4:11)
Entente Saint Pierraise TT – ASEA Sarises Florinas 2:3
Stephanie LOEUILLETTE – LIU Judith 2:3 (11:7 11:4 4:11 7:11 4:6)
Lucie GAUTHIER – Bernadett BALINT 0:3 (9:11 8:11 5:11)
Jeanne ROBBES – Despoina AMPA 3:0 (11:8 11:5 11:6)
Stephanie LOEUILLETTE – Bernadett BALINT 3:0 (11:6 11:9 11:6)
Lucie GAUTHIER – LIU Judith 1:3 (11:9 7:11 8:11 6:11)
TT Joué les Tours – STK Aquaestil Duga Resa 3:0
Audrey ZARIF – Leeloo HAN VUKELJA 3:0 (12:10 11:5 11:5)
LI He – Xue HAN VUKELJA 3:2 (11:5 11:2 9:11 5:11 6:4)
Nolwenn FORT – Sofija SHEREDEHA 3:0 (11:3 11:6 13:11)
CTM MIRANDELA – Uniao Sebastianense F.C
Ines MATOS – Cecilia AKPAN 3:2 (11:9 11:5 3:11 5:11 6:2)
Matilde PINTO – Raquel MARTINS 3:0 (11:7 11:2 11:4)
Mariana SANTA COMBA – Leila OLIVEIRA 0:3 (7:11 9:11 12:14)
Ines MATOS – Raquel MARTINS 0:3 (9:11 8:11 9:11)
Matilde PINTO – Cecilia AKPAN 2:3 (11:9 9:11 11:7 3:11 0:6)
Tereza PYTLIKOVA – Maria HORGEN 3:0 (11:2 11:5 11:6)
Aneta SIRUCKOVA – Vivian HUYNH 3:0 (11:4 11:2 11:5)
(ANS - Mogofores) - The Extraordinary Visitation that the General Councilor for Social Communication
continues to the Salesian Province "Saint Anthony" of Portugal
The last stops were at the Salesians of Mogofores and Mirandela
Fr Mendes visited Mogofores in the last days from 22 to 24 April and
punctually at 8 am on the first of the 3 days
he was received by the Director and by the other Salesians of the community
Then he offered the thought of the Salesian “Good morning” to the school's students and explained all the meaning of his visit: to be with his brothers
to praise God for what is being done there
to offer useful suggestions and indications
He also brought the greeting of the Rector Major
and his paternal "concern" to help the Congregation grow in the beauty of the Salesian charism
in the apostolic holiness of every religious
and in pastoral service to the poorest and most marginalized young people
he spent a lot of time listening: to fully understand the characteristics of the work of Mogofores and the environment in which it is inserted; before offering
the best ways to follow to achieve goals and projects
He conversed with the entire Educational Community of the School
with the members of the Salesian Family and the Parish Pastoral Council
and often interacted with the students of the institute
the General Councilor for Social Communication reached Mirandela
where he carried out the previously planned program
involving the people who animate the main sectors of the Salesian presence - the Salesian community
the “St John Bosco” parish with its pastoral project
He also met the diocesan bishop and dedicated himself to getting to know the surrounding situation
Fr Mendes had various meetings with the Salesian community and met the community council
the members of the technical team and had a meeting with all the collaborators who make up the educational and support teams
He then gathered with the leaders of the parish animation teams
whom he congratulated on their ability to engage in the process of education and evangelization typical of a Salesian parish
He expressed his appreciation of the geographical position of the Salesian house from the point of view of landscape and ecology and noted the good relationship that exists with the various entities and institutions of the city
And during the visit to the diocesan bishop
the work carried out by various generations of Salesians who have passed through that city
the Salesian imprint of the parish of St John Bosco and the need to strengthen the educational-pastoral intervention in the Polytechnic Institute of Bragança
members of the community of the Salesian parish of "St
John Bosco" stood in solidarity with the people of Ukraine by raising their prayers for peace in that country
would inspire those with decision-making abilities
The prayer was led by the parish priest Fr João de Brito and enriched by various interventions
a video was projected with some images of the events
the choirs of young people and adults sang some songs of peace to Our Lady
representatives of each catechetical group
the prayer for peace in Ukraine was recited
which ended with a song entitled "Miraculous Queen of Heaven"
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pastoral teams and Salesian Social Services join together at this time of year to promote traditional Christmas Campaigns in the various communities
the solidarity campaign will help two associations this Christmas
The value of admission tickets to the three schools' Christmas parties will be donated to the "Ajuda de Berço" Association
will be given to "Turma Solidária," a social solidarity association in Alcabideche
"SolSal - Family Support Service" is promoting the "Hands that Multiply" food collection campaign among the educational community
invited everyone to live the true spirit of Christmas and to convert the usual exchange of gifts into help for Christmas offerings to be delivered to more than 200 families accompanied by the "SolSal" service and the parish "Solidarity Bazaar."
A solidarity campaign has also been underway at the Salesian School in Funchal since the beginning of December to provide a little more comfort to the neediest families in and around the school
Cycle 1 students have been asked to contribute basic necessities for personal hygiene
Cycle 2 and 3 students brought food items from a list requested by the school
and staff of the Lisbon Salesians were asked to collaborate with donations for the Christmas Campaign to benefit needy families
The main needs were studied and a list was compiled and distributed to the various classes and groups; until December 16
students were able to bring hygiene products
Particular and well thought out was the campaign initiated in Manique
where the 57 classes of the Salesian institute were invited to make a Christmas basket
based on the characteristics of each family (number of items
which lasted from November 21 to December 16
the Christmas collection campaign will benefit the Children and Youth Home and the needy in the eight parishes entrusted to the Salesians
the Salesians in Mirandela also support students in some of the city's institutions through the distribution of clothing
The National Shrine of Our Lady Help of Christians in Mogofores is collecting food
and gifts with the help of the parish's social store and youth groups that will participate in WYD Lisbon 2023
The distribution of gifts during the Christmas celebration is a Shrine tradition
And parishioners can contribute by delivering their offerings during the Eucharists on the four Sundays of Advent or weekdays directly to the Shrine's Pastoral Office
the Association of Parents and Guardians of Pupils of the Porto Salesians has also launched a food collection drive
The baskets will benefit needy families in the various work areas of the school
(ANS - Dili) - Fr João Pires de Deus
the oldest Salesian of the East Timor Vice Province
after 61 years of missionary life spent in East Timor and 53 as a Salesian
Fr João de Deus was born in Mirandela
in 1963 he founded the "Fatumaka Technical School of Agriculture" and supported the development of Catholic missions in Baucau
the Portuguese government ordered the immediate evacuation of all Catholic missionaries from East Timor
but Fr João de Deus decided to stay in the country
despite the risks and dangers he was running
In that year he was captured by the Indonesians and was released only a few weeks later
After the dismantling of the bases in 1978
the Catholic Church was the only institution that remained with the population during the Indonesian occupation
the Portuguese missionaries built orphanages in Baguia
Fr João remained there and continued to support the local population
In 2012 he was awarded the Medal of the Order "Martinho Lopes"
an order created by the State to recognize the contribution of the Catholic Church in the national liberation of East Timor and to offer thanks for the support and closeness to the local population
President of the Democratic Republic of East Timor
personally offered his last farewell to Fr João de Deus
going to the Chapel of Don Bosco in Comoro
the President expressed the greatest gratitude for the contribution that Fr João had made for national independence
President Guterres then signed a presidential decree
where he once again expresses his thanks for what Fr João de Deus did in the most difficult years for the country
The East Timorese Head of State then brought his condolences to the Salesian Family for this great loss
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