The nuclear membrane protein Samp1 links peripheral genome organization to the myogenic transcriptional program
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eLife Assessment
This useful study shows that loss of Samp1, a protein at the boundary of the cell nucleus, disrupts muscle cell development and is accompanied by changes in gene activity and genome organization. Some of the evidence is solid, but data to support the overall claims is judged to be incomplete. Controlled experiments and mutants to establish whether the changes in organization contribute to impaired development or follow from it would elevate the work. This work will be of interest to those in the genome organization and developmental fields.
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Abstract
Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.
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eLife Assessment
This useful study shows that loss of Samp1, a protein at the boundary of the cell nucleus, disrupts muscle cell development and is accompanied by changes in gene activity and genome organization. Some of the evidence is solid, but data to support the overall claims is judged to be incomplete. Controlled experiments and mutants to establish whether the changes in organization contribute to impaired development or follow from it would elevate the work. This work will be of interest to those in the genome organization and developmental fields.
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Reviewer #1 (Public review):
Summary:
The authors ask whether Samp1 contributes to the reorganization of the genome and the regulation of gene expression during muscle-cell differentiation. Using C2C12 cells, they find that Samp1 depletion impairs differentiation and is accompanied by altered transcriptional responses and changes in peripheral chromatin and chromosome 8 distribution. These findings support Samp1's involvement in the differentiation program, although the causal relationship between the spatial and transcriptional phenotypes remains unresolved.
Strengths:
The study addresses a biologically significant question: how proteins at the nuclear envelope contribute to the coordinated changes in genome organization and gene expression that accompany cell differentiation. Its main contribution is to extend the established …
Reviewer #1 (Public review):
Summary:
The authors ask whether Samp1 contributes to the reorganization of the genome and the regulation of gene expression during muscle-cell differentiation. Using C2C12 cells, they find that Samp1 depletion impairs differentiation and is accompanied by altered transcriptional responses and changes in peripheral chromatin and chromosome 8 distribution. These findings support Samp1's involvement in the differentiation program, although the causal relationship between the spatial and transcriptional phenotypes remains unresolved.
Strengths:
The study addresses a biologically significant question: how proteins at the nuclear envelope contribute to the coordinated changes in genome organization and gene expression that accompany cell differentiation. Its main contribution is to extend the established requirement for Samp1 in myogenesis to measurable spatial and transcriptional phenotypes, providing a more specific basis for investigating its function.
The complementary imaging approaches examine chromatin organization at different scales. Fluorescence ratiometric imaging assesses the relative distribution of chromatin reporters across the nucleus, whereas chromosome 8 imaging provides a chromosome-specific measurement. Agreement between these approaches strengthens the observation that Samp1-depleted cells undergo an altered spatial response to differentiation conditions, while leaving the underlying mechanism open.
The four-time-point transcriptional analysis adds temporal information that an endpoint comparison would miss. It allows readers to examine baseline differences and subsequent responses to differentiation conditions, and provides a potentially useful resource for identifying genes and pathways associated with the impaired differentiation program.
The work also builds on earlier validation with an independent depletion construct and rescue of differentiation by RNAi-resistant Samp1. This prior evidence strengthens the biological foundation of the present study. Together, the results identify Samp1 as a useful target for further investigation of the relationship between nuclear organization and muscle development.
Weaknesses:
The study leaves open how the differentiation, spatial, and transcriptional changes are connected. By day three, control cells have formed myotubes, whereas Samp1-depleted cells largely remain myoblasts. This is a consequence of the depletion and does not invalidate the comparison. However, it means that altered chromosome positioning could contribute to the differentiation defect or arise because differentiation has been disrupted. The authors acknowledge that the mechanism remains unresolved; the findings should therefore be interpreted as evidence that these processes depend on Samp1, without establishing that changes in genome positioning drive the transcriptional response.
The earlier rescue of differentiation strengthens the evidence for Samp1's role in myogenesis. The new spatial and transcriptional findings, however, have not been tested by rescue. This leaves some uncertainty about whether these particular effects are specific to Samp1 depletion in the stable cell line used here.
The connection between impaired gene repression and association with the nuclear lamina remains indirect. The published Lamin B1 DamID data describe normal differentiation and do not measure what happens to lamina association after Samp1 depletion. They support the proposed connection but cannot establish it.
The imaging measurements also need a cautious interpretation. The peripheral chromatin index describes relative fluorescence enrichment, rather than directly measuring heterochromatin abundance. The biological meaning of changes such as an index of approximately 1.1 versus 1.3 is therefore not immediately clear. Similarly, the chromosome 8 analysis measures the distribution of chromosome-paint signal within five optical sections rather than the entire chromosome territory. The results support spatial differences, but their magnitude may depend on nuclear geometry, the sampled sections, and image-processing choices whose influence is not fully assessed.
Some conclusions about the transcriptional response go beyond what the statistical comparisons directly test. A significant change during differentiation in control cells and a nonsignificant change in depleted cells do not, by themselves, establish that the responses differ. The comparison between groups at day three provides evidence of expression differences, but does not determine whether the changes from their respective starting points differ.
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Reviewer #2 (Public review):
Summary:
Kašnik et al. set out test whether the previously reported requirement for the inner nuclear protein Samp1in myogenic differentiation and its also previously reported role in chromatin organization at the nuclear periphery are functionally linked. The authors confirm using a previously characterized shRNA system in cultured C2C12 cells that Samp1 loss leads to myogenic differentiation defects. Loss of Samp1 results in gene expression changes, including of genes associated with myogenesis and also interfered with the formation of peripheral heterochromatin and peripheralization of chromosome 8 which has previously been reported to be associated with myogenic differentiation. Finally, many of the downregulated genes by Samp1 loss were found in chromatin domains bound by lamin B.
Strengths:
The …
Reviewer #2 (Public review):
Summary:
Kašnik et al. set out test whether the previously reported requirement for the inner nuclear protein Samp1in myogenic differentiation and its also previously reported role in chromatin organization at the nuclear periphery are functionally linked. The authors confirm using a previously characterized shRNA system in cultured C2C12 cells that Samp1 loss leads to myogenic differentiation defects. Loss of Samp1 results in gene expression changes, including of genes associated with myogenesis and also interfered with the formation of peripheral heterochromatin and peripheralization of chromosome 8 which has previously been reported to be associated with myogenic differentiation. Finally, many of the downregulated genes by Samp1 loss were found in chromatin domains bound by lamin B.
Strengths:
The strength of this study is that is represents a rather straightforward analysis of the loss of Samp1 in myogenic differentiation in vitro. The RNAseq data is informative and is in line with the observed differentiation defects. In addition, the experiments are clearly designed and cleanly performed.
Weaknesses:
The major weakness of the study is that major conclusions are strongly overinterpreted and the degree of advance compared to prior studies is limited.
The study does not - as claimed by the authors - functionally link Samp1 to myogenic differentiation via its role in genome organization. As outlined by the authors there is already considerable evidence for a role of Samp1 in myogenic differentiation. There is also prior data to show that Samp1 loss prevents peripheralization of chromosome 8. The data reported in this study add further data to a possible functional link between these two events, but similar to the currently available data, the evidence remain circumstantial and they fall short of the authors' claim - and stated goal - of directly demonstrating a functional role of Samp1 in myogenesis via genome organization. The presented data does not rule out that the two effects represent unrelated functions of Samp1. The authors have not shown as claimed that Samp1 exerts its effect on myogenesis via genome reorganization.
The most relevant piece of data to point to such a link is the DamID data in Figure 3 showing that many downregulated Samp1-sensitive genes reside in lamin B associated chromatin. However, in this experiment, critical controls are missing to demonstrate that this effect represents an enrichment of Samp1-sensitive genes (see Recommendations).
While the RNAseq data offers the opportunity to speculate on pathways involved in myogenesis, none of the proposed pathways are experimentally tested to validate their functional roles. They remain hypothetical but are presented as demonstrated facts.
Finally, many of the findings are confirmatory in nature, such as the finding that Samp1 inhibits myogenic differentiation, that differentiation is accompanied by chromatin peripheralization or affects MAPK signaling.
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Reviewer #3 (Public review):
The study by Kašnik et al investigates the role of Samp1, an inner nuclear membrane protein, in muscle cell differentiation. Samp1 was already known to be required for myogenic differentiation and to influence chromatin organization at the nuclear periphery, but whether these two functions were connected was unclear. The authors set out to address this by asking whether the differentiation defect caused by Samp1 depletion is accompanied by failures in genome reorganization at the nuclear periphery.
Using C2C12 mouse myoblasts with stable Samp1 knockdown, they combine RNA sequencing, immunofluorescence, FRIC analysis, and chromosome 8 DNA-FISH to track both the transcriptional and spatial consequences of Samp1 loss during differentiation. The goal is to show that Samp1 is needed not just for muscle gene …
Reviewer #3 (Public review):
The study by Kašnik et al investigates the role of Samp1, an inner nuclear membrane protein, in muscle cell differentiation. Samp1 was already known to be required for myogenic differentiation and to influence chromatin organization at the nuclear periphery, but whether these two functions were connected was unclear. The authors set out to address this by asking whether the differentiation defect caused by Samp1 depletion is accompanied by failures in genome reorganization at the nuclear periphery.
Using C2C12 mouse myoblasts with stable Samp1 knockdown, they combine RNA sequencing, immunofluorescence, FRIC analysis, and chromosome 8 DNA-FISH to track both the transcriptional and spatial consequences of Samp1 loss during differentiation. The goal is to show that Samp1 is needed not just for muscle gene activation but also for the peripheral repositioning of chromatin that normally accompanies differentiation, and that these two processes are linked.
The biggest weakness of this study is that it does not explain how Samp1 actually controls chromatin organization at the nuclear periphery. Is it acting through emerin, the LINC complex, or lamin A/C? This is never addressed. At minimum, the authors should test whether a Samp1 mutant that cannot bind emerin or SUN2 can still rescue the chromatin and differentiation defects.
Related to this, only chromosome 8 is examined for repositioning defects. This chromosome was deliberately chosen because it is known to move during myogenesis, so it is not surprising that it fails to reposition upon Samp1 depletion. A chromosome that does not normally reposition should be included to show the effect is specific, and genes that are expected to remain away from the periphery should also be examined to test whether Samp1 depletion causes indiscriminate rather than selective chromatin reorganization defects.
More broadly, the study is largely descriptive. The main finding is that knocking down Samp1 disrupts gene expression and chromatin repositioning, and that these two things correlate. But which comes first? Does the chromatin move and then genes get silenced, or the other way around? This question is central to the paper's argument and is never tested. Without some attempt to address this, the study reads more as an observation than a mechanistic advance.
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Author response:
We thank the reviewers for their careful evaluation of our manuscript and their constructive feedback. We appreciate the points raised and will take them into consideration when revising the manuscript.
We plan to clarify and refine certain aspects of the interpretation and discussion, particularly regarding the relationship between Samp1, chromatin organization, and myogenic differentiation. We will also aim to strengthen the study with additional experiments and analyses where feasible, addressing the reviewers’ suggestions to the extent possible within the scope of the study.
We believe these revisions will help improve the clarity and strength of the manuscript.
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