Depletion of lamins B1 and B2 alters chromatin mobility and induces differential gene expression by a mesoscale-motion dependent mechanism

Abstract

B-type lamins are critical nuclear envelope proteins that interact with the 3D genomic architecture. However, identifying the direct roles of B-lamins on dynamic genome organization has been challenging as their joint depletion severely impacts cell viability. To overcome this, we engineered mammalian cells to rapidly and completely degrade endogenous B-type lamins using Auxin-inducible degron (AID) technology. Paired with a suite of novel technologies, live-cell Dual Partial Wave Spectroscopic (Dual-PWS) microscopy, in situ Hi-C, and CRISPR-Sirius, we demonstrate that lamin B1 and lamin B2 depletion transforms chromatin mobility, heterochromatin positioning, gene expression, and loci-positioning with minimal disruption to mesoscale chromatin folding. Using the AID system, we show that the disruption of B-lamins alters gene expression both within and outside lamin associated domains, with distinct mechanistic patterns depending on their localization. Critically, we demonstrate that chromatin dynamics, positioning of constitutive and facultative heterochromatic markers, and chromosome positioning near the nuclear periphery are significantly altered, indicating that the mechanism of action of B-type lamins is derived from their role in maintaining chromatin dynamics and spatial positioning. Our findings suggest that the mechanistic role of B-type lamins is stabilization of heterochromatin and chromosomal positioning along the nuclear periphery. We conclude that degrading lamin B1 and lamin B2 has several functional consequences related to both structural disease and cancer.

Nuclear blebs are associated with destabilized chromatin packing domains

“…4C, E), it is visually apparent that nanoscopic heterochromatin domains are observed. Within the nuclear body, as previously demonstrated [32,33], auxin-induced depletion of B-type lamins resulted in reduced peripheral heterochromatic cores at the nuclear periphery, however, domains formed within the nuclear interior were typically larger in size (Fig. S2).…”

“…S2 ). With respect to heterochromatin nanodomains in GSK343 treated HCT116 cells[32, 33] and TSA treated U2OS cells, these were smaller than those in control cells and in lamin B-depletion as expected due to the inhibition of heterochromatin enzymes within the nuclear body ( Fig. 4D-F; Fig.…”

“…[2, 34] To assess the impact of lamin degradation on nuclear morphology, we applied the AID system to HCT116 colorectal carcinoma epithelial cells to induce simultaneous degradation of lamin B1 and lamin B2 as previously described. [32, 33, 35] Using immunofluorescence imaging, we quantified the percentages of nuclear blebbing in HCT116 LMN(B1&B2)-AID cells before and after depletion of B-type lamins by auxin treatment for 24 hours. We found that in comparison to untreated controls, auxin treatment promoted a significant increase in the percentage of cells containing nuclear blebs (2.07% vs 6.23%; 4.163 ± 1.033 (Mean difference ± SEM), p-value = 0.016, Student’s t-test) ( Fig.…”

“…In addition to quantifying packing domain structure in live cells, PWS microscopy allows measurement of the effective diffusion coefficient, D e , and the fractional moving mass (FMM) which quantifies the fraction of chromatin demonstrating coherent motion within a diffraction limited volume. Utilizing this technique, we have previously demonstrated that B-type lamin depletion is associated with increased levels of chromatin fractional moving mass and repositioning of heterochromatin cores [32, 33].…”

Formamide denaturation of double-stranded DNA for fluorescencein situhybridization (FISH) distorts nanoscale chromatin structure

“…A pericentromeric region (PR1) on the p-arm of chromosome 19 was detected using the target sequence CCnGTTCACTGTCAC (start 21049341, end 21099156, 160 copies)[31] with the forward primer 5' ACC GGT GAC AGT GAA C 3' and the reverse primer 5' AAA CGT TCA CTG TCA C 3'. Repeats on an intronic region of CR1 encoding for complement receptor 1 (CR1) were detected with the forward primer 5' ACC GGA GAG GCT GGG 3' and the reverse primer 5' AAA CCC CAG CCT CTC C 3'.Xyloside xylosyltransferase 1 (XXYLT1) repeats on an intronic region of the XXYLT1 gene located on chromosome 3 were detected with the target sequence ATGATATCACAGTGG (start 195199025, end 195233876, 333 copies)[61] with the forward primer 5' ACC GTG ATA TCA CAG 3' and the reverse primer 5' AAA CCT GTG ATA TCA C 3'. Repeats of fibrillin 3 (FBN3) on an intron of the gene FBN3 located on chromosome 19 were detected with the target sequence ATCCCTCCAACCnGG (start 8201581, end 8202360, 22 copies)[31] with the forward primer 5' ACC GAT CCC TCC AAC C 3' and the reverse primer 5' AAA CGG TTG GAG GGA TC 3'.Lentiviral packagingLentiviral particles were produced in HEK293T cells using Fugene HD (Promega, #E2311) transfection reagent following the manufacturer's protocol.…”