Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes

Abstract

The nuclear lamina is implicated in the organization of the eukaryotic nucleus. Association of nuclear lamins with the genome occurs through large chromatin domains including mostly, but not exclusively, repressed genes. How lamin interactions with regulatory elements modulate gene expression in different cellular contexts is unknown. We show here that in human adipose tissue stem cells, lamin A/C interacts with distinct spatially restricted subpromoter regions, both within and outside peripheral and intra-nuclear lamin-rich domains. These localized interactions are associated with distinct transcriptional outcomes in a manner dependent on local chromatin modifications. Down-regulation of lamin A/C leads to dissociation of lamin A/C from promoters and remodels repressive and permissive histone modifications by enhancing transcriptional permissiveness, but is not sufficient to elicit gene activation. Adipogenic differentiation resets a large number of lamin-genome associations globally and at subpromoter levels and redefines associated transcription outputs. We propose that lamin A/C acts as a modulator of local gene expression outcome through interaction with adjustable sites on promoters, and that these position-dependent transcriptional readouts may be reset upon differentiation.

Figure 1.

LMNA interacts with promoters in adipose stem cells. (A) Immunofluorescence detection of lamin A/C in ASCs. (Inset) Phase contrast. Bar, 10 μm. (B) Western blot analysis of lamin A/C in ASCs, ASC chromatin (input), and antibody-unbound and -bound fractions after LMNA ChIP. (C) Browser view of LMNA occupancy on two regions of chromosome 1; (left) nucleotide 1,230,000–1,260,000 (TNFRSF18, TNFRSF4, SDF4, B3GALT6); (right) nucleotide 12,810,000–12,888,000 (PRAMEF11, HNRNPCL1, PRAMEF2, PRAMEF4, PRAMEF12) in ASCs before and after LMNA KD. (D) Number of genes with a LMNA-associated promoter in ASCs and in LMNA-KD ASCs. (E) GAGA and A/T-rich motifs enriched in promoter regions underlying LMNA peaks.

Figure 2.

3D immuno-FISH unveils peripheral and intra-nuclear LMNA-associated loci. (A) LMNA enrichment in sliding 31-gene windows (x, x+31) across chromosomes. Scale shows the numbers of LMNA-enriched genes in the window. (B) Distribution of FISH signals from LMNA-enriched (DEFA3, SCN10A, DNAL4) and nonenriched (HOXB9) loci relative to peripheral LMNA (n ≥ 30 loci per gene). (C) 3D immuno-FISH (green; arrows) of loci shown in B relative to peripheral LMNA labeling (red). DNA is stained with DAPI. Scale bars, 5μm.

Figure 3.

LMNA associates with a repressive chromatin environment and with distinct promoter subregions. (A) Expression level of genes enriched in LMNA and indicated histone modification. (*) P < 10⁻⁶ relative to RefSeq genes; Wilcoxon rank sum test. (B) Percentage of LMNA-associated and RefSeq genes enriched in H3K4me3, H3K9me3, or H3K27me3. (*) P < 10⁻⁴ relative to RefSeq; χ² with Yates' correction. (C) Heat map of LMNA peak position on promoters (scale, no. of genes with a LMNA peak at a given position). (D) Corresponding expression frequency heat map (scale, ratio of expressed genes/all genes for a given offset from TSS). (E) 2D heat maps of LMNA and H3K4me3 peak position on co-enriched promoters, and corresponding expression heat map; scales are as in C and D.

Figure 4.

LMNA down-regulation results in chromatin rearrangement. (A) Expression level of genes that lose or retain LMNA after LMNA KD. (*) P < 10⁻⁶ relative to RefSeq genes; Wilcoxon ran sum test. (B) Proportion of H3K4me3-, H3K9me3-, or H3K27me3-marked genes that lose or retain LMNA after LMNA KD. (*) P < 10⁻⁴ compared to the percentage of genes bound by LMNA and enriched in the indicated histone modifications in ASCs; Fisher's exact test. (C) Proportion of RefSeq genes enriched in LMNA and H3K4me3, H3K9me3, or H3K27me3 in ASCs before (Native) and after LMNA KD. (*) P < 10⁻⁴ relative to native ASCs; Fisher's exact test. (D) LMNA peak density map on promoters retaining LMNA after LMNA KD. (E) H3K4me3 peak density map on all H3K4me3-enriched promoters before and after LMNA KD.

Figure 5.

Adipogenic differentiation resets LMNA-promoter interactions. (A) Differentiation of ASCs into adipocytes (21 d; lipids are stained with Oil Red-O). Arrows point to nuclei showing nuclear compaction in adipocytes. Bars, 50 μm. (B) Number of genes interacting with LMNA in ASCs and adipocytes. (C) LMNA peak density map on promoters in adipocytes. (D) Percent of genes that maintain or change expression in adipocytes relative to ASCs, as a function of retention, gain, or loss of LMNA. (Right) Proportions of up- and down-regulated genes that retain, lose, or gain LMNA. (E) Expression heat map for genes with a LMNA-bound promoter in adipocytes (scale, ratio of expressed genes/all genes for a given offset from TSS). (F) Adipogenic promoters lose LMNA association after adipogenic differentiation. Profiles show loss of LMNA from the PPARG P2 promoter and the FABP locus (green arrows), and significant gain of LMNA on the RUNX2 P1 promoter (red arrow). (G) Retention of LMNA on nonadipogenic, lineage-specific promoters (F,G) (log₂ ChIP/input ratios).

Figure 6.

Developmental regulation of LMNA association of promoters important for lineage commitment. In undifferentiated adipocyte progenitors (ASCs), master regulator genes of differentiation into adipogenic and nonadipogenic lineages are tethered to LMNA and not expressed. Adipogenic differentiation results in disengagement of adipogenic loci from LMNA and their transcriptional activation. However, nonadipogenic loci remain associated with LMNA in adipocytes. These include genes important for differentiation into, e.g., osteogenic or myogenic pathways, endodermal and ectodermal lineages, as well as pluripotency-associated genes.