A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha

Lamins are components of the peripheral nuclear lamina and interact with heterochromatic genomic regions, termed lamina-associated domains (LADs). In contrast to lamin B1 being primarily present at the nuclear periphery, lamin A/C also localizes throughout the nucleus, where it associates with the chromatin-binding protein lamina-associated polypeptide (LAP) 2 alpha. Here, we show that lamin A/C also interacts with euchromatin, as determined by chromatin immunoprecipitation of euchromatin- and heterochromatin-enriched samples. By way of contrast, lamin B1 was only found associated with heterochromatin. Euchromatic regions occupied by lamin A/C overlap with those bound by LAP2alpha, and lack of LAP2alpha in LAP2alpha-deficient cells shifts binding of lamin A/C toward more heterochromatic regions. These alterations in lamin A/C-chromatin interactions correlate with changes in epigenetic histone marks in euchromatin but do not significantly affect gene expression. Loss of lamin A/C in heterochromatic regions in LAP2alpha-deficient cells, however, correlated with increased gene expression. Our data show a novel role of nucleoplasmic lamin A/C and LAP2alpha in regulating euchromatin.

For ChIP-qPCR cells were harvested, obtained chromatin was processed and ChIPs were performed as described. ReChIP-qPCR was performed according to (Truax and  results were calculated as % of Input.

ChIP-seq data analysis
To quantify the degree of overlap between EDD peaks of two ChIP samples (A and B) we counted the number of base pairs covered by peaks in only the first (A only), only the second (B only) or both samples (A and B) using BEDTools (Quinlan and Hall 2010). The resulting numbers were visualized in Venn diagrams. To compare the lamin A/C, LAP2alpha and lamin B1 regions to published LAD annotations we computed for each set of EDD peaks the percentage of base pairs that overlap with ciLADs and MEF LADs obtained from the GEO database (see Data viewing). In contrast to lamin and LAP2alpha data presented here, ciLADs and MEF LADs are based on peaks called with a two-state HMM from DamID tiling arrays. However, the large overlap between MEF LADs and our lamin B1 data shows, that overall both protocols produce comparable results. Similar to (Gel et al. 2015), the statistical significance of an overlap was assessed by comparing to a null distribution of overlaps obtained from 10,000 random permutations. For all random permutation tests, we excluded all "gap" regions on the genome (retrieved from UCSC Genome Browser). Genes per megabase were computed individually for each peak. Only genes that overlap by at least 50% with a peak were considered. The final number of overlapping genes for each peak was then normalized to its length. P-values were computed using the Wilcoxon Rank Sum Test.
Peaks detected by SICER are much shorter than typical lamin and LAP2alpha binding domains (Lund et al. 2014). Thus, regions of enrichment are covered by a high number of short peaks instead of one long peak. Since the exact position of these short peaks within an enriched region can vary between samples, simple overlaps are not suitable to quantify similarity in SICER peak coverage between lamin and LAP2alpha samples. Therefore, we divided the genome into non-overlapping 1 Mb bins and used the number of SICER peaks per bin to measure the similarity between samples. To this end, each bin b is represented by two peak counts (x b , y b ), where x b and y b represent the number of SICER peaks in sample A and B, respectively. For all bins the peak counts were illustrated in a scatter-plot. Finally, we computed the Pearson Correlation coefficient between the peak counts per bin in sample A and sample B, and used this as a measure of similarity of SICER peak coverage.

Histone mark abundance
To determine histone mark abundance on promoters, we defined promoters as regions in the genome that extend from 2,200 bp upstream to 500 bp downstream of transcription start sites (TSS), similar to Lund et al. (Lund et al. 2013). For each gene we counted the number of histone ChIP and the number of input reads that mapped to its promoter region. After normalizing the read counts by the total library size, we computed the log ratio of the normalized ChIP count and the normalized input count.
The same approach was used to measure histone mark abundance in lamin A/C and LAP2alpha binding regions. Instead of individual promoter regions, we used all regions covered by EDD peaks in a sample to compute log ratios. In addition, we computed the log ratio separately for regions that gained, lost or maintained lamin A/C or LAP2alpha binding in Lap2alpha KO cells. For data in Figure 1E in main manuscript, the significance of differences in histone mark enrichment between 12 and 30 cycle samples was assessed using the Wilcoxon Rank Sum Test. For data in Figure 4B and Supplemental Figure 6 in main manuscript, the significance of differences in histone mark enrichment changes was assessed by random permutation testing (n = 10,000).  (Anders et al. 2013). Briefly, after adapter 6 removal using cutadapt (Martin 2011), reads were mapped to the mouse genome (NCBI37/mm9 annotation from July 2007) using TopHat2 (Kim et al. 2013) version 2.0.9 and RefSeq gene annotations retrieved from the UCSC Genome Browser.

RNA sequencing and analysis
Htseq-count (Anders et al. 2015) was used to create a count table taking into account only reads with a mapping quality larger than 20. All annotated transcripts that show at least one mapped read per one million sequenced reads (counts per million) in both replicates were tested for differential expression using edgeR (Robinson et al. 2010) and a false discovery rate (FDR) cutoff of 0.05. All significantly up-or downregulated genes are listed in Supplemental Table 1. Significant changes in expression in Lamin A/C gain, loss and WT^KO regions were detected by comparing the actual log2 fold change to fold changes obtained from 10,000 random permutations of differentially expressed genes. (p < 0.05).