Fig 1: Lamin A/C-chromatin associations are rearranged in LAP2alpha-deficient versus wild-type cells. (A) Screenshots of IGV tracks of Chromosome 11 (mm9) representing loge (ChIP/Input) [−0.4;0.4], peak regions identified by EDD, MEF LADs, the RefSeq gene track, and histone mark tracks. Shown are data for LAP2alpha and lamin A/C (precipitated with 3A6 or N18 antibodies in Tmpo WT and KO cells) after 12 cycles of sonication and data of histone marks after 12 cycles of sonication (scale for active marks H3K4me3 and H3K9ac is loge (ChIP/Input) [0;0.3] and for repressive marks H3K27me3 and H3K9me3 loge (ChIP/Input) [−0.6;0.6]). Black arrowheads point to regions of loss of lamin A/C or histone marks and white arrowheads to regions of gain of lamin A/C or histone marks in Tmpo KO cells. (B) Venn diagrams of EDD peak overlaps [Mb] between lamin A/C in Tmpo WT and KO samples after 12 cycles of sonication. Numbers below Venn diagram show overlap (in Mb and %) of lamin A/C EDD peak fractions (occurring only in WT, in WT and KO, or KO only) with LAP2alpha EDD peaks. (C) Redistribution of lamin A/C within LAP2alpha-associated sites. Pie chart depicting percentage of lamin A/C-associated regions (identified by antibodies N18 or 3A6) within the LAP2alpha-associated regions that retained, lost, or gained lamin A/C binding in Tmpo KO versus WT samples. (D) SICER peak correlations (peaks/Mb) corresponding to data in B.
Fig 2: Consequences of lamin A/C redistribution in LAP2alpha-deficient cells on chromatin organization and gene expression. (A) MEF LAD overlap. Degree of overlap (% of bp) of EDD peaks obtained after 12 cycles of sonication for lamin A/C-N18 and -3A6 in Tmpo WT and KO mouse fibroblasts with MEF LADs. Error bars indicate the interval that contains 95% of all mean overlaps obtained through random permutation tests. All overlaps are significantly smaller or larger than expected under the null model (P < 10−4). (B) Change in repressive and active histone marks. EDD peak regions of lamin A/C (N18 and 3A6) in Tmpo WT and KO cells were divided into regions occurring in both samples (“WT^KO”), losing association in Tmpo KO (“loss”), and gaining associations Tmpo KO (“gain”). Percent difference (Tmpo KO vs. WT) in the abundance of repressive (H3K27me3, H3K9me3) and active (H3K4me3, H3K9ac) histone marks present in all lamin A/C-N18 and -3A6 “WT^KO,” “loss,” and “gain” regions, all regions of LAP2alpha loss (ALL REGIONS) and promoter regions of up- (UP, gene promoter) and down-regulated (DOWN, gene promoter) in lamin A/C-N18 and -3A6 “WT^KO,” “loss,” and “gain” regions, after 12 sonication cycles. Asterisk (*) denotes significant change in histone mark abundance (P < 0.05) compared to random permutation testing. (C) Gene expression change. Average log2-fold change of differentially regulated genes in lamin A/C-N18 and -3A6 “WT^KO,” “loss,” and “gain” regions and regions of LAP2alpha loss after 12 and 30 sonication cycles. Asterisk (*) denotes significant gene expression change (P < 0.05) compared to random permutation testing. (D) Redistribution of lamin A/C upon loss of LAP2alpha alters gene expression and histone marks on promoters of affected genes. IGV track compilation of up- and down-regulated genes in regions of loss and gain of lamin A/C in euchromatin-enriched (12 cycle) and heterochromatin-enriched (30 cycle) regions. Shown are EDD peak tracks for lamin A/C-N18 and -3A6 in Tmpo WT and KO cells obtained after 12 and 30 cycles of sonication, followed by RefSeq tracks of the respective genes (blue arrow denotes direction of transcription) and histone mark data tracks obtained after 12 cycles of sonication; scale for active marks H3K4me3 and H3K9ac is loge (ChIP/Input) [−5;5] and for repressive marks H3K27me3 and H3K9me3 loge (ChIP/Input) [−3;3]. Black and white arrowheads point to regions of loss and gain of histone marks in Tmpo KO, respectively.
Fig 3: LAP2 expression in human CE (a) RT-PCR (Sybr Green) showing up and down-regulation of LAP2α and β in HCEnCs isolated from the tissues (T) and at subsequent passages (P0-P3) in S and C HCEnCs culture in vitro. (b) Representative images of LAP2 (red) immunofluorescence staining in cryosections of the human cornea, showing CE and stroma. DAPI (blue) counterstained nuclei. (c) Representative images of LAP2 (green) and ki67 (red) immunofluorescence staining on HCEnCs in vitro, at P1. DAPI (blue) counterstained nuclei. (d) Quantification of the immunofluorescence data shown in (c).
Fig 4: LAP2 is upregulated in sub-confluent rCEnCs (a). RT-PCR (Sybr Green) showing up and down-regulation of LAP2α and β across the passages in rCEnCs sub-confluent (S) and confluent (C) cultures. The LAP2 ability of being up and down-regulated decreases with passages (b). RT-PCR, divided between S and C populations highlighted how LAP2 expression decreases with passages till P6 when it is drastically reduced (c). Delta of LAP2 expression between S and C populations, studied across passages (P1 to P6) (d). RT-PCR of LAP2α and β expression in rCEnCs isolated from the Descemet tissues (T), compared with rCEnCs in culture at P0. Data are presented as mean ± SE. The statistical significance was determined by Students’ t-test. * p-value < 0.05. ** p < 0.01.
Fig 5: Localization of NE proteins in nuclei from differentiated myotubes of LMNA-mutant iPSCs. (A) Lamin B1, emerin, NUP153, and LAP2 were double-stained with lamin A/C and a DAPI nuclear counterstain. Yellow arrows indicate the representative localizations of nuclear envelope proteins in abnormal regions of myonuclei. (B) Accumulation of emerin expression in abnormal regions of myonuclei from LMNA hiPSCs cultured on a glass-bottom dish, a plastic-bottom dish, and a silicone chamber. Scale bar = 50 μm.
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