NF- κ B is a Central Regulator of Hypoxia-Induced Gene Expression

  1. Dilem Shakir
  2. Michael Batie
  3. Chun-Sui Kwok
  4. Simon J. Cook
  5. Niall S. Kenneth
  6. Sonia Rocha

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Abstract

Hypoxia is both a physiological and pathological signal in cells. Changes in gene expression play a critical role in the cellular response to hypoxia, enabling cells to adapt to reduced oxygen availability. These changes are primarily mediated by the HIF family of transcription factors, however other transcription factors such as NF-κB, are also activated by hypoxia. Although NF-κB is known to be activated by hypoxia, the extent to which NF-κB contributes to the hypoxic response remains poorly understood. Here, we analysed hypoxia-induced, NF-κB-dependent gene expression, to define the NF-κB-dependent hypoxic signature. Our analysis reveals that most genes downregulated by hypoxia require NF-κB for their repression. We show that although the NF-κB-mediated hypoxic response may vary between cell types, a core subset of hypoxia-inducible genes requires NF-κB across multiple cell backgrounds. We demonstrate that NF-κB is critical for reactive oxygen species (ROS) generation and regulation of genes involved in oxidative phosphorylation under hypoxia. This work highlights NF-κB’s central role in the hypoxia response and offering new insights into gene expression regulation by hypoxia and NF-κB.

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    Reply to the reviewers

    1. General Statements [optional]

    *We would like to thank all the reviewers for their positive comments and valuable feedback. In addition, we would like to address reviewer 1 query on novelty, which was not questioned by the other 2 reviewers. Our study uncovered two main aspects of hypoxia biology: first we addressed the role of NF-kappaB contribution towards the transcriptome changes in hypoxia, and second, this revealed a previously unknown aspect, that NF-kappaB is required for gene repression in hypoxia. While we know a lot about gene induction in hypoxia, much less is known about repression of genes. In times of energy preservation, gene repression is as important as gene induction. *

    .

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    The work from Shakir et al uses different cell line models to investigate the role of NF-kB in the transcriptional adaptation of cells to hypoxia, which is relevant. In addition, the manuscript contains a large amount of data that could be of interest and even useful for researchers in the field of hypoxia and NF-kB. However, in my opinion, there are several concerns that should be revised and additional experiments that could be included to strengthen the relevance of the work.

    We thank this reviewer for their positive comments.

    Specific issues: In Figure 1A, the authors examine which of the genes induced by hypoxia require NF-kB by RNA sequencing analysis of cells knocked down for specific NF-kB subunits and exposed to hypoxia for 24 hours. The knockdown is about 40-60% at the RNA level, but it would be helpful to show the effect of knockdown at the protein level.

    *We agree with this and have added Western blot data (Sup. Figure S1F), which shows the effects of the siRNA are much more pronounced at the protein level. *

    All the data regarding genes induced by hypoxia in control or NF-kB siRNA-treated cells are somewhat confusing. If I understand correctly, when the data from the three different siRNAs are crossed, only 1070 genes are upregulated and 295 are downregulated in an NF-kB-independent manner. If this is the case, I think it would be easier to use this information in Figure 2 to define the hypoxia-induced genes that are NF-kB-dependent by simply considering those induced in the control that are not in the NF-kB-independent subset (rather than repeating the integration of the data without additional explanation). If the authors do this simple analysis, are the resulting genes the same or similar? In any case, the way these numbers are obtained should be shown more clearly (i.e., a new Venn diagram showing genes up- or down-regulated in the siRNA control that are not up- or down-regulated in any of the siRNA-NF-kB treatments).

    Figure 1 shows the effects on gene expression of hypoxia in control and NF-____k____B ____subunit____-depleted cells compared to normoxia control cells. Figures 1F/1G compares genes up/downregulated in hypoxia when RelA, RelB, and cRel are depleted, compared to normoxia control. Figure 1 does not display N____F-____k____B____-dependent/independent hypoxia-responsive genes____, but rather the overall effect of siRNA control and siNF-____k____B treatments in hypoxia, compared to siRNA control in normoxia. Figure 2 then defines NF-____k____B-dependent ____and independent hypoxia-responsive genes. We actually define these exactly as the reviewer suggested and agree that we should show the way these numbers are obtained more clearly. We have added the suggested Venn diagrams (Sup. Figure S2) and added extra information to the methods section (page 5 of revised manuscript). We felt it was important to show all the data upfront in Figure 1 and then integrate and focus on NF-____k____B-dependent ____hypoxia-induced genes in Figure 2.

    * *

    Figure 2H shows that approximately 80% of the NF-kB-dependent genes up- or down-regulated in hypoxia were identified as RelA targets, which is statistically significant compared to RelB or cRel targets. However, what is the proportion of genes identified as RelA targets in the subset of NF-kB-independent hypoxia-induced genes? And in a randomly selected set of 500-600 genes? In my opinion, this statistical analysis should be included to demonstrate a relationship between NF-kB recruitment and hypoxia-induced upregulation (expected) and downregulation (unexpected). In this context, it is surprising that HIF consensus sites are preferentially detected in the genes that are supposed to be NF-kB dependent instead of RelA consensus.

    We thank the reviewer for this question, which is really helpful. The way we have displayed the stars on the graph for Figure 2H was slightly misleading we realize now. As such, we have amended the graph. RelA, RelB, and cRel bound genes (from the ChIP atlas) are all significantly enriched within our N____F-____k____B-dependent hypoxia-responsive genes, there is no statistical testing between RelA bound vs RelB bound or cRel bound. We have also performed this analysis on the NF-____k____B____-independent hypoxia-responsive genes ____and see the same trend (Sup. Figure S5B). This indicates that the enrichment of Rel binding sites from the ChIP atlas is not specific to NF-____k____B____-dependent hypoxia-responsive genes____. We have moved Figure 2H to (Sup. Figure S5A) and amended our description of the finding. This showcases how DNA binding does not necessarily mean functionality. We have amended our description of this result and limitation of the study.

    * *

    Figure 3 is just a confirmation by qPCR of the data obtained in the RNA-seq analysis, which in my opinion should be included as supplementary information. Moreover, both the effects of hypoxia and reversion by RelB siRNA are modest in several of the genes tested. The same is true for Figures 4 and 5 with very modest and variable results across cell types and genes.

    *We appreciate this comment; we would like to keep this as a main figure for full transparency and show validation of our RNA-sequencing results. *

    * *

    Figure 6 shows the effect of NF-kB knockdown on the induction of ROS in response to hypoxia. In the images provided, the effect of hypoxia is minimal in control cells, with the only clear differences shown in RelA-depleted cells.

    The quantification of the IF data (Figure 6B) shows ROS induction in hypoxia which is reduced in Rel-depleted cells, with RelA depletion having the strongest effect. ROS generation in hypoxia, although counterintuitive, is well documented and used for important signalling events. We believe our data supports the previously reported levels of ROS induction (reviewed in {Alva, 2024}) in hypoxia and importantly, that NF-____k____B depletion can at least partially____* reverse this. *

    In 6B it is not clear what the three asterisks in the normoxia control represent (compared to the hypoxia siRNA control?). This should be clarified in the figure legend or text.

    *We apologize for the lack of clarity we have now added this information to the figure legend. *

    In the Western blot of 6C, there are no differences in the levels of SOD1 after RelA depletion. Again, there is no reason not to include the NF-kB subunits in the Western blot analysis.

    We have added the Western blot analysis to this figure. We were trying to simplify it. Although depletion of RelA does not rescue the hypoxia-induced repression of SOD1, depletion of RelB does. Furthermore, cRel although not statistically significant, has a trend for the rescue of this effect, see Figure 6C-D.

    Finally, regarding Figure 7, the authors mention that "we confirmed that hypoxia led to a reduction in several proteins represented in this panel (of proteins involved in oxidative phosphorylation), such as UQCRC2 and IDH1 (Figure 7A-B)". The authors cannot say this because it is not seen in the Western blot in 7A or in the quantification shown in 7B. In my personal opinion, stating something that is not even suggested in the experiments is very negative for the credibility of the whole message.

    We really do not agree with this comment. We do see reductions in the levels of the proteins we mentioned. We have made the figure less complex given that some proteins are very abundant while others are not. We hope the changes are now clear and apparent. We have changed the quantification normalisation to reflect this as well and modified our description of the results, see Figure 7 and Sup. Figure S18.

    In conclusion, this paper contains a large amount of relevant information, but i) non-essential data should be moved to Supplementary, ii) protein levels of relevant players need to be shown in addition to RNA, iii) minimal or undetectable differences need to be considered as no-differences, and iv) a model showing what is the interpretation of the data provided is needed to better understand the message of the paper. I mean, is it p65 or RelB binding to some of these genes leading to their activation or repression, or is it RelA or RelB inducing HIF1beta leading to NF-kB-dependent gene activation by hypoxia? If this were the case, experimental evidence that NF-kB regulates a subset of hypoxia genes through HIF1beta would make the story more understandable.

    We apologise but we do not know why the reviewer mentions HIF1beta. For gene induction, there is cooperation with the HIF system in some genes but not all. The most interesting and unexpected finding is that NF-kappaB is required for gene repression in hypoxia. We have added a new figure, investigating how HDAC inhibition could reverse the repression. A mechanism known to be employed by NF-kappaB when repressing genes. We have added all the blots for NF-kB, clarified the quantification and included other approaches including a CRISPR KO cell lines for both IKKs. We hope this is now clear.

    Reviewer #1 (Significance (Required)):

    The work presented here is interesting but does not provide a major advance over previous publications, the main message being that a subset of hypoxia-regulated genes are NF-kB dependent. However, there is no mechanistic explanation of how this regulation is achieved and several data that are not clearly connected. A more comprehensive analysis of the data and additional experimental validation would greatly enhance the significance of the work.

    We politely disagree with the reviewer. Our main finding is that NF-____k____B____* does play an important role in gene regulation in hypoxia but unexpectedly, this occurs mostly via gene repression. While there is vast knowledge on gene induction in hypoxia, gene repression, which typically does not occur directly via HIF, is virtually unknown. A previous study had identified Rest as a transcriptional repressor {PMID: 27531581} but this could only account for 20% of gene repression. Our findings reveal up to 60% of genes repressed in hypoxia require NF-____k____B____, hence this is a significant finding and a major advance over previous knowledge. Furthermore, we feel this paper is an excellent data resource for the field, as it is, to our knowledge, the first study characterising the extent to which NF-____k____B is required for hypoxia-induced gene changes, on a transcriptome-wide scale. Furthermore, we have validated this across multiple cell types and also used different approaches to investigate the role of NF-kB in the hypoxia transcriptional response. We are happy that the other reviewers agree with our novel findings.*

    * *

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this study, the authors have interrogated the role of NF-kappaB in the cellular transcriptional response to hypoxia. While HIF is considered the master regulator of the cellular response to hypoxia, it has long been known that mutliple transcription factors also play a role both independently of HIF and through the regulation of HIF-1alpha levels. Chief amongst these is NF-kappaB, a regulator of cell death and inflammation amongst other things. While NF-kappB has been known to be activated in hypoxia through altered PhD activity, the impact of this on global gene expression has remained unclear and this study addresses this important question. Of particular interest, genes downregulated in hypoxia appear to be repressed in a NF-kappaB-dependent manner. Overall, this nice study reveals an important role for NF-kappaB in the control of the global cellular transcriptional response to hypoxia.

    We thank this reviewer for their positive comments.

    Reviewer #2 (Significance (Required)):

    Some questions for the authors to consider with experiments or discussion: -One caveat of the current study which should be discussed is that while interesting and extensive, the analysis is restricted to cancer cell lines which have dysfunctional gene expression systems which may differ from "normal" cells. This should be discussed.

    We thank the reviewer for these comments. This is indeed an important aspect, which we now expand on in the discussion section. We also took advantage of RNA-seq datasets for HUVECs (a non-transformed cell lines) in response to hypoxia (Sup. Figure S15), TNF-alpha with and without RelA depletion (Sup. Figure S16). These data support our findings that in hypoxia NF-kB is important for transcriptional repression, with some contributions to gene induction, even in a non-transformed cell system.

    In the publicly available data sets analyzed, were the same hypoxic conditions used as in this study. This information should be included.

    *We apologize if this was not clear, the hypoxia RNA-seq studies are the same oxygen level and time (1%, 24 hours), this is in the legend of Figure 4A and Sup. Figure S9 and in Sup. Table S2. We have added this information to the main text also. *

    • What is known about NF-kappaB as a transcriptional repressor in other systems such as the control of cytokine or infection driven inflammation? This is briefly discussed but should be expanded. This is important as a key question in the study of hypoxia is what regulates gene repression.

    We have included this in the discussion and also analysed available data in HUVECs in response to cytokine stimulation with and without RelA depletion (Sup. Figure S16). This analysis revealed equal importance of RelA for activation and repression of genes upon TNF-alpha stimulation. Around 40% of genes require RelA for their induction or repression in response to TNF-a. In the discussion we have also included other references where NF-kappaB has been found to repress genes.

    NF-kappaB has previously been shown to regulate HIF-1alpha transcription. What are the effects of NF-kappaB subunit siRNAs on basal HIF-1alpha transcription? In figure 7, it appears that NF-kappaB subunit siRNA is without effect on hypoxia-induced HIF protein expression. Could this account for some of the effects of NF-kappaB depletion on the hypoxic gene signature? This point needs to be clarified in light of the data presented.

    We have included data for HIF-1α RNA levels in HeLa cells with/without NF-____k____B____* depletion followed by 24 hours of hypoxia (Sup. Figure S20) and we see a small reduction (~10-20%). The reviewer is correct, there was not much effect of NF-____k____B____ depletion on HIF-1α protein levels following 24 hours hypoxia in HeLa cells. Effects of NF-kappaB depletion can be found usually with lower times of hypoxia exposure or when more than one subunit is depleted at the same time. We have added this as a discussion point in the revised manuscript.*

    NRF-2 is a key cellular sensor of oxidative stress in a similar way to HIF being a hypoxia sensor. The authors demonstrate using a dye that ROS are paradoxically increased in hypoxia (a more controversial finding than the authors present). It would be of interest to know if NFR-2 is induced in hypoxia as a marker of cellular oxidative stress. Similarly, it would be interesting to determine by metabolic analysis whether oxidative phosphorylation (O2 consumption) is decreased as the transcriptional signature would suggest (although the difficulty of performing metabolic analysis in hypoxia is acknowledged).

    To investigate if NRF2 is induced, we performed a western blot at 0, 1, and 24 hours 1% oxygen, but didn’t see any induction of NRF2 protein levels (____Sup. Figure S17A). We also overlapped our hypoxia upregulated genes with NRF2 target genes from {PMID:24647116 and PMID: 38643749} (Sup. Figure S17B) and found limited evidence of NRF2 target genes being induced. Based on these findings, it seems that NRF2 is not being induced in hypoxia, at least not at the *hypoxia level/time point we have analysed. We also agree it would be ideal to measure oxygen consumption in hypoxia, but unfortunately, we do not have the technical ability to do this at present. *

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Strengths This manuscript attempts to integrate multiple strands of data to determine the role of NFkB in hypoxia -induced gene expression. This analysis looks at multiple NFkB subunits in multiple cell lines to convincingly demonstrate that NFkB does indeed play a central role in the regulation of hypoxia-induced gene expression. This broad approach integrates new experimental data with findings from the published literature.

    A significant amount of work has been performed both experimentally and bioinformatically to test experimental hypotheses.

    We thank this reviewer for their positive comments.

    Limitations

    The main analysis in the paper involves comparing the impact of knocking down different NFkB family members in hypoxia and comparing transcriptional responses. I am surprised that the authors did not include the impact of knockdown of the NFkB family members in normoxia too. The absence of these control experiments allows us to understand the role of NFkB in hypoxia, but does not give us information as to how many of those impacts are specific/ induced in hypoxic conditions. i.e. many of the observed effects of NFkB knockdown could be due to basal suppression of NFkB target genes that happen to be hypoxia sensitive. This finding is obviously important, but it would be nice to know how many of those genes are only / preferentially regulated by NFkB in hypoxia. This would give a much deeper insight into the role of NFkB in hypoxia induced gene expression.

    We agree this would have been ideal. For financial reasons we limited our analysis to hypoxia samples. We have performed qPCR analysis depleting RelA, RelB and cRel under normal oxygen conditions in HeLa (Sup. Figure S8). We find that the majority of the validated genes in HeLa cells which require____* NF-____k____B* for gene changes in hypoxia, are not regulated by N____F-____k____B under normal oxygen conditions____*. We have also added this limitation into our discussion section. *

    * *

    The broad experimental approach while a strength of the paper in many ways also has its limitations e.g. Motif analysis revealing e.g. HIF-1a binding site enrichment in RelA and RelB-dependent DEGs is correlative observation and does not prove HIF involvement in NFkB-dependent hypoxia induced gene activation. Comparing responses with responses seen in one cell type with responses that have been described in a database comprised of many studies in a variety of different cells also has some limitations. These points can be described more fully in the discussion

    *We agree these are mere correlations and hence a limitation and we have not formerly tested the involvement of HIF. We have included this in the discussion as suggested. For HIF binding site correlation, we do also compare to HIF ChIP-seq in HeLa cells exposed to 1% oxygen, albeit at 8 hours and not 24 hours (Sup. Figure S4). *

    For siRNA transfections, single oligonucleotide sequences were used for RelA, RelB and cRel. This increases the potential likelihood of 'off targets' compared to pooled oligos delivered at lower concentrations. This limitation should at least be mentioned.

    We agree and have now included this as a limitation in the discussion section. We have now also included analysis using wild type and 2 different IKK____⍺____/β____* double KO CRISPR cell lines generated in the following publication {PMID: 35029639}. Out of the 9 genes we identified as NF-____k____B-dependent hypoxia upregulated genes from HeLa cell RNA-seq and validated by qPCR, which are also hypoxia-responsive in HCT116 cells (Sup. Figure S11D), 6 displayed ____NF-____k____B dependence in HCT116 cells (Sup. Figure S14). We also provide new protein data in this cell system for oxidative phosphorylation markers, which show as with the siRNA depletion, rescue of repression of these proteins when NF-____k____B is inactivated.*

    * *

    RNA-seq experiments are performed on n=2 data which means relatively low statistical power. How has the statistical analysis been performed on normalised counts (corresponding to 2 n- numbers) to yield statistical significance? I am not familiar with hypergeometric tests - please justify their use here.

    __*We use DESeq2 for differential expression analysis and filter for effect size (> -/+ 0.58 log2 fold change) and statistical significance (FDR I am not familiar with hypergeometric tests - please justify their use here.

    The hypergeometric test (equivalent to a one-sided Fisher's exact test) is routinely used to determine whether the observed overlap between two gene lists is statistically significant compared to what would be expected by chance. It is also the statistical test of choice for popular bioinformatics tools which perform over representation analysis (ORA) to see which gene sets/groups/pathways/ontologies are over-represented in a gene list, examples include Metascape, clusterProfiler, WebGestalt (used in this study), and *gProfiler. *

    P14 RelB is described as having the most widespread impact of hypoxia dependent gene changes across all cell systems tested. Could this be due to a more potent silencing of RelB and / or due to particularly high/ low expression of RelB in these cells in general?

    This is an excellent point, at the RNA level the RelB depletion is slightly more efficient (Sup. Figure S1), at the protein level, silencing is highly potent with all 3 siRNAs (Sup. Figure S1). We looked at the RNA levels of RelA, RelB and cRel in HeLa cells at basal conditions, and RelA shows the highest abundance compared to RelB and cRel, while RelB and cRel have similar expression levels (see below). However, RelB is very dynamic in response to hypoxia, something we have observed but have not published yet.

    * *

    * *

    P18 For western blot analysis best practise is to have 2 MW markers per blot presented

    * *

    *We have and have added the second MW markers suggested. *

    For quantification, I suggest avoiding performing statistical analysis on semi-quantitative data unless a dynamic range of detection (with standards) has been fully established.

    We agree this has many limitations, we will keep the quantification but moved into supplementary information.

    P19 There is clearly an effect of reciprocal silencing with the NFkB knockdown experiments ie. siRelA affects RelB levels in hypoxia and vice versa. The implications of this for data interpretation should be discussed.

    Indeed, it is well known that RelB and cRel are RelA targets. Less is known about RelA as it is not a known NF-____k____B____* target. We have added a discussion in the revised manuscript.*

    P20 The literature can be better cited in relation RelB and hypoxia A brief search reveals a few papers that should be mentioned/ discussed. Oliver et al. 2009 Patel et al. 2017 Riedl et al. 2021

    We have looked into these suggestions. Oliver et al, refer to hypercapnia, not hypoxia and the other two only briefly mentioned RelB with no effects toward the goals of their studies. We have tried to incorporate what is currently known as much as possible.

    I suggest leaving out mention of IkBa sumoylation and supplementary figure 10. I'm not sure the data in the paper as a whole merits focus on this very specific point.

    We thank the reviewer for this suggestion and we have removed this aspect from the manuscript.

    * *

    There is a very strong reliance on mRNA and TPM data. Some additional protein data in support of key findings will enhance

    * *

    We have added additional protein level analysis where we could obtain antibodies, see Figures 6, 7 and Sup. Figures S17, S18, and S19 for our protein level analysis.

    A graphical abstract summarising key findings with exemplar genes highlighted will enhance.

    * *

    We have added a model to summarise our findings as suggested.

    Both HIF and NFKB are ancient evolutionarily conserved pathways. Can lessons be learned from evolutionary biology as to how NFkB regulation of hypoxia induced genes occured. Does the HIF pathway pre-date the NFkB pathway or vice versa. This approach could be valuable in supporting the findings from this study.

    We have investigated this. Unfortunately, there are very little available data on hypoxia gene expression in lower organisms. However, we have added a few sentences on the evolution of NF-____k____B____* and HIF.*

    Minor comments P2 please briefly explain how 5 genes give rise to 7 proteins

    * *

    *We have added this to the introduction as requested. *

    P2 there seems to be some recency bias in the studies cited as being associated with NFkB activation in response to hypoxia. Mention of Koong et al (1994) and Taylor et al (1999) and other early papers in the field will enhance

    We have added these as suggested.

    P3 The role of PHD enzymes in the regulation of NFkB in hypoxia can be introduced and / or discussed

    We have added a reference to this aspect as suggested.

    P8 I suggest use of proportional Venn diagrams to demonstrate the patterns more clearly

    *We have added these as suggested. *

    P11 To what extent might NFkB and Rest co-operate/ co-regulate gene repression in hypoxia?

    *This is a good question. We have overlapped our datasets with Rest-dependent hypoxia-regulated genes identified by Cavadas et al., (Figure below), and find that these appear to act independently of each other for the most part, with very few genes co-regulated by both. *

    Reviewer #3 (Significance (Required)):

    Shakir et al. present a manuscript titled 'NFkB is a central regulator of hypoxia-induced gene expression'.

    The research group are experts in both NFkB and hypoxia signaling and are the ideal group to perform these studies.

    Hypoxia and inflammation are co-incident in many physiological and pathophysiological conditions, where the microenvironment affects disease severity and patient outcome. The cross talk between inflammatory and hypoxia signaling pathways is not fully described. Thus, this manuscript takes a novel approach to an established question and concludes clearly that NFkB is a central regulator of hypoxia-induced gene expression.

    We thank the reviewer for these positive comments.

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    Referee #3

    Evidence, reproducibility and clarity

    Strengths

    This manuscript attempts to integrate multiple strands of data to determine the role of NFkB in hypoxia -induced gene expression. This analysis looks at multiple NFkB subunits in multiple cell lines to convincingly demonstrate that NFkB does indeed play a central role in the regulation of hypoxia-induced gene expression. This broad approach integrates new experimental data with findings from the published literature.

    A significant amount of work has been performed both experimentally and bioinformatically to test experimental hypotheses.

    Limitations

    The main analysis in the paper involves comparing the impact of knocking down different NFkB family members in hypoxia and comparing transcriptional responses. I am surprised that the authors did not include the impact of knockdown of the NFkB family members in normoxia too. The absence of these control experiments allows us to understand the role of NFkB in hypoxia, but does not give us information as to how many of those impacts are specific/ induced in hypoxic conditions. i.e. many of the observed effects of NFkB knockdown could be due to basal suppression of NFkB target genes that happen to be hypoxia sensitive. This finding is obviously important, but it would be nice to know how many of those genes are only / preferentially regulated by NFkB in hypoxia. This would give a much deeper insight into the role of NFkB in hypoxia induced gene expression.

    The broad experimental approach while a strength of the paper in many ways also has its limitations e.g. Motif analysis revealing e.g. HIF-1a binding site enrichment in RelA and RelB-dependent DEGs is correlative observation and does not prove HIF involvement in NFkB-dependent hypoxia induced gene activation. Comparing responses with responses seen in one cell type with responses that have been described in a database comprised of many studies in a variety of different cells also has some limitations. These points can be described more fully in the discussion

    For siRNA transfections, single oligonucleotide sequences were used for RelA, RelB and cRel. This increases the potential likelihood of 'off targets' compared to pooled oligos delivered at lower concentrations. This limitation should at least be mentioned.

    RNA-seq experiments are performed on n=2 data which means relatively low statistical power. How has the statistical analysis been performed on normalised counts (corresponding to 2 n- numbers) to yield statistical significance? I am not familiar with hypergeometric tests - please justify their use here.

    P14 RelB is described as having the most widespread impact of hypoxia dependent gene changes across all cell systems tested. Could this be due to a more potent silencing of RelB and / or due to particularly high/ low expression of RelB in these cells in general?

    P18 For western blot analysis best practise is to have 2 MW markers per blot presented

    For quantification, I suggest avoiding performing statistical analysis on semi-quantitative data unless a dynamic range of detection (with standards) has been fully established.

    P19 There is clearly an effect of reciprocal silencing with the NFkB knockdown experiments ie. siRelA affects RelB levels in hypoxia and vice versa. The implications of this for data interpretation should be discussed.

    P20 The literature can be better cited in relation RelB and hypoxia A brief search reveals a few papers that should be mentioned/ discussed. Oliver et al. 2009 Patel et al. 2017
    Riedl et al. 2021

    I suggest leaving out mention of IkBa sumoylation and supplementary figure 10. I'm not sure the data in the paper as a whole merits focus on this very specific point.

    There is a very strong reliance on mRNA and TPM data. Some additional protein data in support of key findings will enhance

    A graphical abstract summarising key findings with exemplar genes highlighted will enhance.

    Both HIF and NFKB are ancient evolutionarily conserved pathways. Can lessons be learned from evolutionary biology as to how NFkB regulation of hypoxia induced genes occured. Does the HIF pathway pre-date the NFkB pathway or vice versa. This approach could be valuable in supporting the findings from this study.

    Minor comments

    P2 please briefly explain how 5 genes give rise to 7 proteins

    P2 there seems to be some recency bias in the studies cited as being associated with NFkB activation in response to hypoxia. Mention of Koong et al (1994) and Taylor et al (1999) and other early papers in the field will enhance

    P3 The role of PHD enzymes in the regulation of NFkB in hypoxia can be introduced and / or discussed

    P8 I suggest use of proportional Venn diagrams to demonstrate the patterns more clearly

    P11 To what extent might NFkB and Rest co-operate/ co-regulate gene repression in hypoxia?

    Significance

    Shakir et al. present a manuscript titled 'NFkB is a central regulator of hypoxia-induced gene expression'.

    The research group are experts in both NFkB and hypoxia signaling and are the ideal group to perform these studies.

    Hypoxia and inflammation are co-incident in many physiological and pathophysiological conditions, where the microenvironment affects disease severity and patient outcome. The cross talk between inflammatory and hypoxia signaling pathways is not fully described. Thus, this manuscript takes a novel approach to an established question and concludes clearly that NFkB is a central regulator of hypoxia-induced gene expression.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    In this study, the authors have interrogated the role of NF-kappaB in the cellular transcriptional response to hypoxia. While HIF is considered the master regulator of the cellular response to hypoxia, it has long been known that mutliple transcription factors also play a role both independently of HIF and through the regulation of HIF-1alpha levels. Chief amongst these is NF-kappaB, a regulator of cell death and inflammation amongst other things. While NF-kappB has been known to be activated in hypoxia through altered PhD activity, the impact of this on global gene expression has remained unclear and this study addresses this important question. Of particular interest, genes downregulated in hypoxia appear to be repressed in a NF-kappaB-dependent manner. Overall, this nice study is reveals an important role for NF-kappaB in the control of the global cellular transcriptional response to hypoxia.

    Significance

    Some questions for the authors to consider with experiments or discussion:

    • One caveat of the current study which should be discussed is that while interesting and extensive, the analysis is restricted to cancer cell lines which have dysfunctional gene expression systems which may differ from "normal" cells. This should be discussed.
    • In the publicly available data sets analysed, were the same hypoxic conditions used as in this study. This information should be included.
    • What is known about NF-kappaB as a transcriptional repressor in other systems such as the control of cytokine or infection driven inflammation? This is briefly discussed but should be expanded. This is important as a key question in the study of hypoxia is what regulates gene repression.
    • NF-kappaB has previously been shown to regulate HIF-1alpha transcription. What are the effects of NF-kappaB subunit siRNAs on basal HIF-1alpha transcription? In figure 7, it appears that NF-kappaB subunit siRNA is without effect on hypoxia-induced HIF protein expression. Could this account for some of the effects of NF-kappaB depletion on the hypoxic gene signature? This point needs to be clarified in light of the data presented.
    • NRF-2 is a key cellular sensor of oxidative stress in a similar way to HIF being a hypoxia sensor. The authors demonstrate using a dye that ROS are paradoxically increased in hypoxia (a more controversial finding than the authors present). It would be of interest to know if NFR-2 is induced in hypoxia as a marker of cellular oxidative stress. Similarly it would be interesting to determine by metabolic analysis whether oxidative phosphorylation (O2 consumption) is decreased as the transcriptional signature would suggest (although the difficulty of performing metabolic analysis in hypoxia is acknowleged).
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    Referee #1

    Evidence, reproducibility and clarity

    The work from Shakir et al uses different cell line models to investigate the role of NF-kB in the transcriptional adaptation of cells to hypoxia, which is relevant. In addition, the manuscript contains a large amount of data that could be of interest and even useful for researchers in the field of hypoxia and NF-kB. However, in my opinion, there are several concerns that should be revised and additional experiments that could be included to strengthen the relevance of the work.

    Specific issues:

    In Figure 1A, the authors examine which of the genes induced by hypoxia require NF-kB by RNA sequencing analysis of cells knocked down for specific NF-kB subunits and exposed to hypoxia for 24 hours. The knockdown is about 40-60% at the RNA level, but it would be helpful to show the effect of knockdown at the protein level. All the data regarding genes induced by hypoxia in control or NF-kB siRNA-treated cells are somewhat confusing. If I understand correctly, when the data from the three different siRNAs are crossed, only 1070 genes are upregulated and 295 are downregulated in an NF-kB-independent manner. If this is the case, I think it would be easier to use this information in Figure 2 to define the hypoxia-induced genes that are NF-kB-dependent by simply considering those induced in the control that are not in the NF-kB-independent subset (rather than repeating the integration of the data without additional explanation). If the authors do this simple analysis, are the resulting genes the same or similar? In any case, the way these numbers are obtained should be shown more clearly (i.e., a new Venn diagram showing genes up- or down-regulated in the siRNA control that are not up- or down-regulated in any of the siRNA-NF-kB treatments). Figure 2H shows that approximately 80% of the NF-kB-dependent genes up- or down-regulated in hypoxia were identified as RelA targets, which is statistically significant compared to RelB or cRel targets. However, what is the proportion of genes identified as RelA targets in the subset of NF-kB-independent hypoxia-induced genes? And in a randomly selected set of 500-600 genes? In my opinion, this statistical analysis should be included to demonstrate a relationship between NF-kB recruitment and hypoxia-induced upregulation (expected) and downregulation (unexpected). In this context, it is surprising that HIF consensus sites are preferentially detected in the genes that are supposed to be NF-kB dependent instead of RelA consensus. Figure 3 is just a confirmation by qPCR of the data obtained in the RNA-seq analysis, which in my opinion should be included as supplementary information. Moreover, both the effects of hypoxia and reversion by RelB siRNA are modest in several of the genes tested. The same is true for Figures 4 and 5 with very modest and variable results across cell types and genes. Figure 6 shows the effect of NF-kB knockdown on the induction of ROS in response to hypoxia. In the images provided, the effect of hypoxia is minimal in control cells, with the only clear differences shown in RelA-depleted cells. In 6B it is not clear what the three asterisks in the normoxia control represent (compared to the hypoxia siRNA control?). This should be clarified in the figure legend or text. In the Western blot of 6C, there are no differences in the levels of SOD1 after RelA depletion. Again, there is no reason not to include the NF-kB subunits in the Western blot analysis. Finally, regarding Figure 7, the authors mention that "we confirmed that hypoxia led to a reduction in several proteins represented in this panel (of proteins involved in oxidative phosphorylation), such as UQCRC2 and IDH1 (Figure 7A-B)". The authors cannot say this because it is not seen in the Western blot in 7A or in the quantification shown in 7B. In my personal opinion, stating something that is not even suggested in the experiments is very negative for the credibility of the whole message. In conclusion, this paper contains a large amount of relevant information, but i) non-essential data should be moved to Supplementary, ii) protein levels of relevant players need to be shown in addition to RNA, iii) minimal or undetectable differences need to be considered as no-differences, and iv) a model showing what is the interpretation of the data provided is needed to better understand the message of the paper. I mean, is it p65 or RelB binding to some of these genes leading to their activation or repression, or is it RelA or RelB inducing HIF1beta leading to NF-kB-dependent gene activation by hypoxia? If this were the case, experimental evidence that NF-kB regulates a subset of hypoxia genes through HIF1beta would make the story more understandable.

    Significance

    The work presented here is interesting but does not provide a major advance over previous publications, the main message being that a subset of hypoxia-regulated genes are NF-kB dependent. However, there is no mechanistic explanation of how this regulation is achieved and several data that are not clearly connected. A more comprehensive analysis of the data and additional experimental validation would greatly enhance the significance of the work.

  5. Version published to 10.1101/2025.01.08.631917 on bioRxiv