Distal Gene Expression Governed by Lamins and Nesprins via Chromatin Conformation Change
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eLife Assessment
This study provides useful information on the impact of Lamin A/C knockdown on gene expression using RNA-Seq analysis. In addition, the impact of Lamin A/C knockdown on telomere dynamics is explored using live cell imaging. The conclusions, however, are inadequately supported by the data presented. Weaknesses include excessive reliance on gene ontology analysis without further validation of direct versus indirect effects, use of only one shRNA, which may have off target effects, validation of knockdown only from gene expression rather than protein levels, lack of discussion on previous studies showing the presence of Lamin A/C in the nuclear interior among others.
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Abstract
Abstract
The nuclear lamina is a vital structural component of eukaryotic cells, playing a pivotal role in both physiological processes, such as cell differentiation, and pathological conditions, including laminopathies and cancer metastasis. Lamina associated proteins, particularly lamins and nesprins, are integral to mechanosensing, chromatin organization, and gene regulation. However, their precise contributions to gene regulation remain incompletely understood. This study explores the functions of lamin A, LMNA, and SYNE2 in gene expression, with a particular focus on their influence on distal chromatin interactions and conformational changes. Using inducible shRNA knockdown, RNA-seq analysis, and dCas9-mediated live imaging of chromosomes, we demonstrate that lamin A affects RNA synthesis, LMNA governs chromatin spatial organization, and SYNE2 regulates chromatin modifications. Furthermore, both lamins and nesprins enhance telomere dynamics. These findings elucidate nuclear envelope-associated mechanisms in gene regulation, offering valuable insights into chromatin dynamics under both physiological and pathological contexts.
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eLife Assessment
This study provides useful information on the impact of Lamin A/C knockdown on gene expression using RNA-Seq analysis. In addition, the impact of Lamin A/C knockdown on telomere dynamics is explored using live cell imaging. The conclusions, however, are inadequately supported by the data presented. Weaknesses include excessive reliance on gene ontology analysis without further validation of direct versus indirect effects, use of only one shRNA, which may have off target effects, validation of knockdown only from gene expression rather than protein levels, lack of discussion on previous studies showing the presence of Lamin A/C in the nuclear interior among others.
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Reviewer #1 (Public review):
This manuscript reports a descriptive study of changes in gene expression after knockdown of the nuclear envelope proteins lamin A/C and Nesprin2/SYNE2 in human U2OS cells. The readout is RNA-seq, which is analyzed at the level of gene ontology and focused investigation of isoform variants and non-coding RNAs. In addition, the mobility of telomeres is studied after these knockdowns, although the rationale in relation to the RNA-seq analyses is rather unclear.
RNA-seq after knockdown of lamin proteins has been reported many times, and the current study does not provide significant new insights that help us to understand how lamins control gene expression. This is particularly because the vast majority of the observed effects on gene expression appear to occur in regions that are not bound by lamin A. It seems …
Reviewer #1 (Public review):
This manuscript reports a descriptive study of changes in gene expression after knockdown of the nuclear envelope proteins lamin A/C and Nesprin2/SYNE2 in human U2OS cells. The readout is RNA-seq, which is analyzed at the level of gene ontology and focused investigation of isoform variants and non-coding RNAs. In addition, the mobility of telomeres is studied after these knockdowns, although the rationale in relation to the RNA-seq analyses is rather unclear.
RNA-seq after knockdown of lamin proteins has been reported many times, and the current study does not provide significant new insights that help us to understand how lamins control gene expression. This is particularly because the vast majority of the observed effects on gene expression appear to occur in regions that are not bound by lamin A. It seems likely that these effects are indirect. There is also virtually no overlap between genes affected by laminA/C and by SYNE2, which remains unexplained; for example, it would be good to know whether laminA/C and SYNE2 bind to different genomic regions. The claim in the Title and Abstract that LMNA governs gene expression / acts through chromatin organization appears to be based only on an enrichment of gene ontology terms "DNA conformation change" and "covalent chromatin conformation" in the RNA-seq data. This is a gross over-interpretation, as no experimental data on chromatin conformation are shown in this study. The analyses of transcript isoform switching and ncRNA expression are potentially interesting but lack a mechanistic rationale: why and how would these nuclear envelope proteins regulate these aspects of RNA expression? The effects of lamin A on telomere movements have been reported before; the effects of SYNE2 on telomere mobility are novel (to my knowledge), but should be discussed in the light of previously documented effects of SUN1/2 on the dynamics of dysfunctional telomeres (Lottersberger et al, Cell 2015).
As indicated below, I have substantial concerns about the experimental design of the knockdown experiments.
Altogether, the results presented here are primarily descriptive and do not offer a significant advance in our understanding of the roles of LaminA and SYNE2 in gene regulation or chromatin biology, because the results remain unexplained mechanistically and functionally. Furthermore, the RNAseq datasets should be interpreted with caution until off-target effects of the shRNAs can be ruled out.
Specific comments:
(1) Knockdowns were only monitored by qPCR. Efficiency at the protein level (e.g., Western blots) needs to be determined.
(2) For each knockdown, only a single shRNA was used. shRNAs are infamous for off-target effects; therefore, multiple shRNAs for each protein, or an alternative method such as CRISPR deletion or degron technology, must be tested to rule out such off-target effects.
(3) It is not clear whether the replicate experiments are true biological replicates (i.e., done on different days) or simply parallel dishes of cells done in a single experiment (= technical replicates). The extremely small standard deviations in the RT-qPCR data suggest the latter, which would not be adequate.
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Reviewer #2 (Public review):
Summary:
This study focused on the roles of the nuclear envelope proteins lamin A and C, as well as nesprin-2, encoded by the LMNA and SYNE2 genes, respectively, on gene expression and chromatin mobility. It is motivated by the established role of lamins in tethering heterochromatin to the nuclear periphery in lamina-associated domains (LADs) and modulating chromatin organization. The authors show that depletion of lamin A, lamin A and C, or nesprin-2 results in differential effects of mRNA and lncRNA expression, primarily affecting genes outside established LADs. In addition, the authors used fluorescent dCas9 labeling of telomeric genomic regions combined with live-cell imaging to demonstrate that depletion of either lamin A, lamin A/C, or nesprin-2 increased the mobility of chromatin, suggesting an …
Reviewer #2 (Public review):
Summary:
This study focused on the roles of the nuclear envelope proteins lamin A and C, as well as nesprin-2, encoded by the LMNA and SYNE2 genes, respectively, on gene expression and chromatin mobility. It is motivated by the established role of lamins in tethering heterochromatin to the nuclear periphery in lamina-associated domains (LADs) and modulating chromatin organization. The authors show that depletion of lamin A, lamin A and C, or nesprin-2 results in differential effects of mRNA and lncRNA expression, primarily affecting genes outside established LADs. In addition, the authors used fluorescent dCas9 labeling of telomeric genomic regions combined with live-cell imaging to demonstrate that depletion of either lamin A, lamin A/C, or nesprin-2 increased the mobility of chromatin, suggesting an important role of lamins and nesprin-2 in chromatin dynamics.
Strengths:
The major strength of this study is the detailed characterization of changes in transcript levels and isoforms resulting from depletion of either lamin A, lamin A/C, or nesprin-2 in human osteosarcoma (U2OS) cells. The authors use a variety of advanced tools to demonstrate the effect of protein depletion on specific gene isoforms and to compare the effects on mRNA and lncRNA levels.
The TIRF imaging of dCas9-labeled telomeres allows for high-resolution tracking of multiple telomeres per cell, thus enabling the authors to obtain detailed measurements of the mobility of telomeres within living cells and the effect of lamin A/C or nesprin-2 depletion.
Weaknesses:
Although the findings presented by the authors overall confirm existing knowledge about the ability of lamins A/C and nesprin to broadly affect gene expression, chromatin organization, and chromatin dynamics, the specific interpretation and the conclusions drawn from the data presented in this manuscript are limited by several technical and conceptual challenges.
One major limitation is that the authors only assess the knockdown of their target genes on the mRNA level, where they observe reductions of around 70%. Given that lamins A and C have long half-lives, the effect at the protein level might be even lower. This incomplete and poorly characterized depletion on the protein level makes interpretation of the results difficult. The description for the shRNA targeting the LMNA gene encoding lamins A and C given by the authors is at times difficult to follow and might confuse some readers, as the authors do not clearly indicate which regions of the gene are targeted by the shRNA, and they do not make it obvious that lamin A and C result from alternative splicing of the same LMNA gene. Based on the shRNA sequences provided in the manuscript, one can conclude that the shLaminA shRNA targets the 3' UTR region of the LMNA gene specific to prelamin A (which undergoes posttranslational processing in the cell to yield lamin A). In contrast, the shRNA described by the authors as 'shLMNA' targets a region within the coding sequence of the LMNA gene that is common to both lamin A and C, i.e., the region corresponding to amino acids 122-129 (KKEGDLIA) of lamin A and C. The authors confirm the isoform-specific effect of the shLaminA isoform, although they seem somewhat surprised by it, but do not confirm the effect of the shLMNA construct. Assessing the effect of the knockdown on the protein level would provide more detailed information both on the extent of the actual protein depletion and the effect on specific lamin isoforms. Similarly, given that nesprin-2 has numerous isoforms resulting from alternative splicing and transcription initiation. In the current form of the manuscript, it remains unclear which specific nesprin-2 isoforms were depleted, and to what extent (on the protein level).
Another substantial limitation of the manuscript is that the current analysis, with the exception of the chromatin mobility measurements, is exclusively based on transcriptomic measurements by RNA-seq and qRT-PCR, without any experimental validation of the predicted protein levels or proposed functional consequences. As such, conclusions about the importance of lamin A/C on RNA synthesis and other functions are derived entirely from gene ontology terms and are not sufficiently supported by experimental data. Thus, the true functional consequences of lamin A/C or nesprin depletion remain unclear. Statements included in the manuscript such as "our findings reveal that lamin A is essential for RNA synthesis, ..." (Lines 79-80) are thus either inaccurate or misleading, as the current data do not show that lamin A is ESSENTIAL for RNA synthesis, and lamin A/C and lamin A deficient cells and mice are viable, suggesting that they are capable of RNA synthesis.
Another substantial weakness is that the data and analysis presented in the manuscript raise some concerns about the robustness of the findings. Given that the 'shLMNA' construct is expected to deplete both lamin A and C, i.e., its effect encompasses the depletion of lamin A, which is achieved by the 'shLaminA' construct, one would expect a substantial overlap between the DEGs in the shLMNA and shLaminA conditions, with the shLMNA depletion producing a broader effect as it targets both lamin A and C. However, the Venn Diagram in Figure 4a, the genomic loci distribution in Figure 4b, and the correlation analysis in Supplementary Figure S2 show little overlap between the shLMNA and shLaminA conditions, which is quite surprising. In the mapping of the DEGs shown in Figure 4b, it is also surprising not to see the gene targeted by the shRNA, LMNA, found on chromosome 1, in the results for the shLMNA and shLamin A depletion.
The correlation analysis in Supplementary Figure S2 raises further questions. The authors use doc-inducible shRNA constructs to target lamin A (shLaminA), lamin A/C (shLMNA), or nesprin-2 (shSYNE2). Thus, the no-dox control (Ctr) for each of these constructs would be expected to be very similar to the non-target scrambled controls (Ctrl.shScramble and Dox.shScramble). However, in the correlation matrix, each of the no-dox controls clusters more closely with the corresponding dox-induced shRNA condition than with the Ctrl.shScramble or Dox.shScramble conditions, suggesting either a very leaky dox-inducible system, strong effects from clonal selection, or substantial batch effects in the processing. Either of these scenarios could substantially affect the interpretation of the findings. For example, differences between different clonal cell lines used for the studies, independent of the targeted gene, could explain the limited overlap between the different shRNA constructs and result in apparent differences when comparing these clones to the scrambled controls, which were derived from different clones.
The manuscript also contains several factually inaccurate or incorrect statements or depictions. For example, the depiction of the nuclear envelope in Figure 1 shows a single bilipid layer, instead of the actual double bi-lipid layer of the inner and outer nuclear membranes that span the nuclear lumen. The depiction further lacks SUN domain proteins, which, together with nesprins, form the LINC complex essential to transmit forces across the nuclear envelope. The statement in line 214 that "Linker of nucleoskeleton and cytoskeleton (LINC) complex component nesprin-2 locates in the nuclear envelope to link the actin cytoskeleton and the nuclear lamina" is not quite accurate, as nesprin-2 also links to microtubules via dynein and kinesin.
The statement that "Our data show that Lamin A knockdown specifically reduced the usage of its primary isoform, suggesting a potential role in chromatin architecture regulation, while other LMNA isoforms remained unaffected, highlighting a selective effect" (lines 407-409) is confusing, as the 'shLaminA' shRNA specifically targets the 3' UTR of lamin A that is not present in the other isoforms. Thus, the observed effect is entirely consistent with the shRNA-mediated depletion, independent of any effects on chromatin architecture.
The premise of the authors that lamins would only affect peripheral chromatin and genes at LADs neglects the fact that lamins A and C are also found in the nuclear interior, where they form stable structure and influence chromatin organization, and the fact that lamins A and C and nesprins additionally interact with numerous transcriptional regulators such as Rb, c-Fos, and beta-catenins, which could further modulate gene expression when lamins or nesprins are depleted.
The comparison of the identified DEGs to genes contained in LADs might be confounded by the fact that the authors relied on the identification of LADs from a previous study (ref #28), which used a different human cell type (human skin fibroblasts) instead of the U2OS osteosarcoma cells used in the present study. As LADs are often highly cell-type specific, the use of the fibroblast data set could lead to substantial differences in LADs.
Another limitation of the current manuscript is that, in the current form, some of the figures and results depicted in the figures are difficult to interpret for a reader not deeply familiar with the techniques, based in part on the insufficient labeling and figure legends. This applies, for example, to the isoform use analysis shown in Figure 3d or the GenometriCorr analysis quantifying spatial distance between LADs and DEGs shown in Figure 4c.
Overall appraisal and context:
Despite its limitations, the present study further illustrates the important roles the nuclear envelope proteins lamin A, lamin C, and nesprin-2 have in chromatin organization, dynamics, and gene expression. It thus confirms results from previous studies (not always fully acknowledged in the current manuscript) previously reported for lamin A/C depletion. For example, the effect of lamin A/C depletion on increasing mobility of chromatin had already been demonstrated by several other groups, such as Bronshtein et al. Nature Comm 2015 (PMID: 26299252) and Ranade et al. BMC Mol Cel Biol 2019 (PMID: 31117946). Additionally, the effect of lamin A/C depletion on gene and protein expression has already been extensively studied in a variety of other cell lines and model systems, including detailed proteomic studies (PMIDs 23990565 and 35896617).
The finding that that lamin A/C or nesprin depletion not only affects genes at the nuclear periphery but also the nuclear interior is not particularly surprising giving the previous studies and the fact that lamins A and C are also founding within the nuclear interior, where they affect chromatin organization and dynamics, and that lamins A/C and nesprins directly interact with numerous transcriptional regulators that could further affect gene expression independent from their role in chromatin organization.
The authors provide a detailed analysis of isoform switching in response to lamin A/C or nesprin depletion, but the underlying mechanism remains unclear. Similarly, their analysis of the genomic location of the observed DEGs shows the wide-ranging effects of lamin A/C or nesprin depletion, but lets the reader wonder how these effects are mediated. A more in-depth analysis of predicted regulator factors and their potential interaction with lamins A/C or nesprin would be beneficial in gaining more mechanistic insights.
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Reviewer #3 (Public review):
Summary:
This manuscript describes DOX inducible RNAi KD of Lamin A, LMNA coded isoforms as a group, and the LINC component SYNE2. The authors report on differentially expressed genes, on differentially expressed isoforms, on the large numbers of differentially expressed genes that are in iLADs rather than LADs, and on telomere mobility changes induced by 2 of the 3 knockdowns.
Strengths:
Overall, the manuscript might be useful as a description for reference data sets that could be of value to the community.
Weaknesses:
The results are presented as a type of data description without formulation of models or explanations of the questions being asked and without follow-up. Thus, conceptually, the manuscript doesn't appear to break new ground.
Not discussed is the previous extensive work by others on the …
Reviewer #3 (Public review):
Summary:
This manuscript describes DOX inducible RNAi KD of Lamin A, LMNA coded isoforms as a group, and the LINC component SYNE2. The authors report on differentially expressed genes, on differentially expressed isoforms, on the large numbers of differentially expressed genes that are in iLADs rather than LADs, and on telomere mobility changes induced by 2 of the 3 knockdowns.
Strengths:
Overall, the manuscript might be useful as a description for reference data sets that could be of value to the community.
Weaknesses:
The results are presented as a type of data description without formulation of models or explanations of the questions being asked and without follow-up. Thus, conceptually, the manuscript doesn't appear to break new ground.
Not discussed is the previous extensive work by others on the nucleoplasmic forms of LMNA isoforms. Also not discussed are similar experiments- for instance, gene expression changes others have seen after lamin A knockdowns or knockouts, or the effect of lamina on chromatin mobility, including telomere mobility - see, for example, a review by Roland Foisner (doi.org/10.1242/jcs.203430) on nucleoplasmic lamina. The authors need to do a thorough search of the literature and compare their results as much as possible with previous work.
The authors don't seem to make any attempt to explore the correlation of their findings with any of the previous data or correlate their observed differential gene expression with other epigenetic and chromatin features. There is no attempt to explore the direction of changes in gene expression with changes in nuclear positioning or to ask whether the genes affected are those that interact with nucleoplasmic pools of LMNA isoforms. The authors speculate that the DEG might be related to changing mechanical properties of the cells, but do not develop that further.
The technical concerns include: 1) Use of only one shRNA per target. Use of additional shRNAs would have reduced concern about possible off-target knockdown of other genes; 2) Use of only one cell clone per inducible shRNA construct. Here, the concern is that some of the observed changes with shRNA KDs might show clonal effects, particularly given that the cell line used is aneuploid. 3) Use of a single, "scrambled" control shRNA rather than a true scrambled shRNA for each target shRNA.
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