KMT2A modulates the epigenetic landscape of rDNA by facilitating the recruitment of histone lysine acetyltransferase PCAF to the rDNA locus
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eLife Assessment
This useful manuscript presents evidence that the major contribution of the lysine methyltransferase KMT2A to ribosomal gene transcription regulation is through promoting histone acetylation rather than H3K4 methylation, in part through interactions of KMT2A with PCAF. Some of the claims in the paper are unsupported by the evidence and therefore viewed as incomplete. Additional experiments demonstrating a more direct and causal link between PCAF and KMT2A would elevate the key conceptual advance that this new axis influences epigenetic pathways through an unconventional histone acetylation pathway. This work will be of interest to those studying transcriptional regulation and chromatin remodeling.
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Abstract
Histone acetylation is often associated with transcriptional activation across a wide range of genes, playing a key role in RNA Polymerase II dynamics. However, its specific role in transcriptional activation of RNA Polymerase I (RNA Pol I) remains unclear. In this study, we demonstrate that KMT2A associates with ribosomal DNA (rDNA) loci. Notably, the loss of KMT2A in our inducible KO cell line does not affect the levels of H3K4me3 on rDNA. We support these observations with analyses of ChIP-seq data from mouse embryonic stem cells. While H3K4 methylation remains unchanged, the absence of KMT2A causes a significant decrease in H3 acetylation levels on rDNA, especially in H3K9 acetylation levels. To identify the histone lysine acetyl transferases (KATs) that cooperate with KMT2A in promoting rDNA transcription, we examined the occupancy of multiple KATs and their associated histone acetylation marks on the rDNA locus. Our analyses identify PCAF (p300/CBP associated factor) as the KAT that contributes to KMT2A-mediated transcriptional activation of rDNA. Depletion of KMT2A reduces the levels of PCAF on rDNA, suggesting that KMT2A plays the role of a co-activator of RNA Pol I in the recruitment of KATs on the rDNA loci. Finally, the depletion of KMT2A leads to the disruption of the pre-initiation complex from the rDNA 47S promoter, resulting in a stalled RNA Pol I complex at the spacer promoter. Our findings elucidate the non-redundant, distinct function of KMT2A in the regulation of RNA Pol I transcription.
Article activity feed
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eLife Assessment
This useful manuscript presents evidence that the major contribution of the lysine methyltransferase KMT2A to ribosomal gene transcription regulation is through promoting histone acetylation rather than H3K4 methylation, in part through interactions of KMT2A with PCAF. Some of the claims in the paper are unsupported by the evidence and therefore viewed as incomplete. Additional experiments demonstrating a more direct and causal link between PCAF and KMT2A would elevate the key conceptual advance that this new axis influences epigenetic pathways through an unconventional histone acetylation pathway. This work will be of interest to those studying transcriptional regulation and chromatin remodeling.
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Reviewer #1 (Public review):
Summary:
In this manuscript the authors study the distribution of the KMT2A methytransferase across the human ribosomal gene loci (rDNA) and the effects of its depletion on rDNA chromatin modification using a KMT2A conditional cell line. They used ChIP mapping data and claim to show that KMT2A depletion does not affect H3K4me3 levels but does reduce acetylation of H3K9 and reduces PCAF (KAT2B) binding. They further claim that KMT2A depletion causes stalling of RNA Polymerase I at the upstream "Spacer" promoter duplication on the rDNA.
Weaknesses:
I found the manuscript very confusing. In particular, I found it short on short on experimental detail and I found the data in general to be unconvincing. It is my opinion that the authors overinterpret their data, leading to unrealistic and unsupported claims. The …
Reviewer #1 (Public review):
Summary:
In this manuscript the authors study the distribution of the KMT2A methytransferase across the human ribosomal gene loci (rDNA) and the effects of its depletion on rDNA chromatin modification using a KMT2A conditional cell line. They used ChIP mapping data and claim to show that KMT2A depletion does not affect H3K4me3 levels but does reduce acetylation of H3K9 and reduces PCAF (KAT2B) binding. They further claim that KMT2A depletion causes stalling of RNA Polymerase I at the upstream "Spacer" promoter duplication on the rDNA.
Weaknesses:
I found the manuscript very confusing. In particular, I found it short on short on experimental detail and I found the data in general to be unconvincing. It is my opinion that the authors overinterpret their data, leading to unrealistic and unsupported claims. The manuscript also avoided discussing relevant recent studies on the regulation of the rDNA and of rDNA chromatin, most citations being to data 15 to 20 or more years old. This is most apparent when discussing the factors such as UBTF and SL1 that determine preinitiation complex assembly and the status of active versus inactive rDNA chromatin. Both have seen significant advances in recent years, and these should be thoroughly discussed.
Specific comments:
The authors do not describe the generation of the KMT2A conditional HEK293T cells or their treatment. Since these cells form the basis for subsequent experiments, they must be explained fully. I found that the conditional cell line had already been used in a previous unrelated publication "Chinchole et al. 2022". This manuscript explained that the KMT2A knockout is induced via Dox controlled Cas9 in a HEK cell clone constitutively expressing guide RNAs to KMT2A. The KMT2A knockout requires a 7 day treatment with Dox, leaving wide open the question of direct versus indirect effects of KMT2A loss, and perhaps explaining some of the discrepancies with published data.
In mapping the interactions of KMT2A across the rDNA by ChIP they use 18 PCR amplicons, some of which the authors inaccurately state as targeting specific rDNA elements. For example, they state Primers (that is, Amplicons) #1-3 target the spacer and core 47S promoters. In fact, amplicon #2 overlaps neither of these promoters and lies centrally within the 1kbp of enhancer repeats between them. Exactly why emphasis is put on KMT2A chipping at these two promoters is anyhow questionable since KMT2A levels (Figure 1) appear equally high at multiple IGS amplicons.
Discrepancies with published data are particularly apparent in their mapping of H3K4me2, H3K4me3 and H3K9ac, all directly relevant to their conclusions. These modifications have been shown in several publications to map to a single major site a few hundred bp upstream of the spacer promoter in multiple human and mouse cell types. Since the authors fail to monitor H3K4 methylation and H3K9 acetylation at this dominant site they also fail to provide significant evidence that KMT2A in fact affects H3 methylation or H3 acetylation on the rDNA.
The KMT2A deletion drastically reduces PolI and UBF (UBTF) engagement across the rDNA, and consistent with this, SL1 and RRN3 engagement is reduced as are 47S levels. Thus, whatever the 7-day KMT2A deletion is doing to the cells it is certainly inducing a very significant shut-down of active rDNA. How such an extreme experimental model can tell anything of KMT2A functions is unclear.
In the last results section, the authors further claim "Loss of KMT2A results in a stalled RNA Pol I complex at the spacer promoter" (Figure 4 and S4). To support this the authors try to map spacer promoter transcripts but mistakenly claim that the amplicon #1 can detect these transcripts. In fact, this amplicon overlaps the Spacer Promoter initiation site and so could not detect transcripts coming from this promoter. Published data, not cited, have demonstrated that PolI stalling at the spacer promoter occurs as a normal phenomenon on the active rDNA. Its loss is associated with suppression, not the opposite as inferred in this manuscript.
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Reviewer #2 (Public review):
Summary:
This important manuscript presents solid evidence that the major contribution of KMT2A to ribosomal gene transcription is through promoting histone acetylation rather than H3K4 methylation. It is suggested this occurs in part through interactions of KMT2A with PCAF.
Strengths:
This manuscript advances an important concept that chromatin modifying enzymes can also function to promote transcription through interactions with other transcriptional regulators independently of their catalytic functions. The data are overall supportive of this conclusion for KDM2A.
Weaknesses:
(1) Care needs to be taken regarding interpreting the lack of effect of depleting KMTs or HATs on histone acetylation or methylation levels. There are several overlapping HATS or KMTs that generate these modifications. The most that …
Reviewer #2 (Public review):
Summary:
This important manuscript presents solid evidence that the major contribution of KMT2A to ribosomal gene transcription is through promoting histone acetylation rather than H3K4 methylation. It is suggested this occurs in part through interactions of KMT2A with PCAF.
Strengths:
This manuscript advances an important concept that chromatin modifying enzymes can also function to promote transcription through interactions with other transcriptional regulators independently of their catalytic functions. The data are overall supportive of this conclusion for KDM2A.
Weaknesses:
(1) Care needs to be taken regarding interpreting the lack of effect of depleting KMTs or HATs on histone acetylation or methylation levels. There are several overlapping HATS or KMTs that generate these modifications. The most that can be said from these negative results is that the depleted HAT or KMT is not the sole enzyme generating these modifications at these loci.
(2) The biochemistry supporting an interaction between KDM2A and PCAF is under-developed and not strong enough to support this conclusion at this point.
(3) It is concluded that acetylation causes increased rDNA transcription. However, acetylation may be a consequence of transcription instead, especially over transcribed regions.
(4) Some conclusions, for example implicating PCAF, are based on overexpression experiments that are very artificial and difficult to draw conclusions from.
(5) Several figures are generated using publicly available data sets that were reanalyze using updated reference genomes, sometimes from different cell lines/types. This seems to be a useful exercise but the methods should include information regarding the robustness of these data sets.
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Reviewer #3 (Public review):
Summary:
This manuscript examines a key mechanism by which KMT2A regulates RNA polymerase I transcription at ribosomal DNA. The authors suggest that KMT2A facilitates the recruitment of PCAF to rDNA. This process enhances histone acetylation and supports assembly of the RNA polymerase I transcription machinery. The study addresses an intriguing question by proposing a catalytic-independent role for KMT2A in rDNA transcription, potentially linking the KMT2A-dependent pathway to histone acetylation. However, several aspects of the proposed mechanism require further experimental validation. Notably, while the current evidence demonstrates associations among KMT2A, PCAF occupancy, histone acetylation, and rRNA transcription, it does not fully support the proposed objectives.
Strengths:
This could be the first …
Reviewer #3 (Public review):
Summary:
This manuscript examines a key mechanism by which KMT2A regulates RNA polymerase I transcription at ribosomal DNA. The authors suggest that KMT2A facilitates the recruitment of PCAF to rDNA. This process enhances histone acetylation and supports assembly of the RNA polymerase I transcription machinery. The study addresses an intriguing question by proposing a catalytic-independent role for KMT2A in rDNA transcription, potentially linking the KMT2A-dependent pathway to histone acetylation. However, several aspects of the proposed mechanism require further experimental validation. Notably, while the current evidence demonstrates associations among KMT2A, PCAF occupancy, histone acetylation, and rRNA transcription, it does not fully support the proposed objectives.
Strengths:
This could be the first report of a lysine methyltransferase regulating an acetyltransferase in Pol I-mediated gene expression.
Weaknesses:
(1) The causal relationship between KMT2A and the recruitment of PCAF needs to be demonstrated. Simply showing reduced PCAF occupancy after depleting KMT2A does not, on its own, prove direct recruitment. The authors should investigate whether KMT2A and PCAF physically interact, and whether this interaction depends on DNA or RNA.
(2) The main prediction of the model is that restoring PCAF activity should reverse the molecular effects caused by the depletion of KMT2A. The authors should test whether PCAF restoration can mitigate the consequences of KMT2A loss. They should also assess PCAF occupancy, H3K9 acetylation levels, Pol I occupancy, and nascent rRNA synthesis.
(3) The catalytic-independent function of KMT2A should be directly investigated. The absence of a significant change in H3K4me3 levels following KMT2A depletion does not confirm that KMT2A's catalytic activity is not essential. Rescue experiments comparing wild-type KMT2A with a catalytically inactive variant would provide a much stronger assessment of the proposed non-catalytic mechanism.
(4) The manuscript highlights H3K9ac; however, various acetyltransferases and histone acetylation marks are linked to active rDNA. The authors should investigate whether KMT2A specifically regulates H3K9ac or leads to broader changes in histone acetylation at rDNA.
(5) Due to the highly repetitive nature of rDNA, the manuscript must include detailed information about the reference assembly, mapping strategy, management of multimapping reads, normalisation processes, and biological replicates. The authors should demonstrate that any observed changes in occupancy are not due to technical differences in rDNA mapping or variations in copy number.
(6) KMT2A regulates a wide range of transcriptional programs, and its depletion can affect proliferation, cell-cycle distribution, or nucleolar function. The authors should include appropriate controls to demonstrate that the rDNA phenotype is not a result of changes in cellular proliferation or a generalised response to transcriptional or nucleolar stress.
(7) The CSB and B-WICH complex have previously been linked to the recruitment of PCAF and the acetylation of H3K9 at active rDNA. The authors should investigate whether KMT2A operates independently of these factors or if KMT2A functions within an established pathway for remodeling rDNA chromatin.
(8) The distinction between active and silent rDNA should be taken into account. The authors need to determine whether KMT2A/PCAF occupancy is preferentially associated with active rDNA copies. Sequential ChIP analysis or the use of established markers for active, poised, and silent rDNA would help clarify the chromatin context in which the proposed mechanism operates.
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Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript the authors study the distribution of the KMT2A methyltransferase across the human ribosomal gene loci (rDNA) and the effects of its depletion on rDNA chromatin modification using a KMT2A conditional cell line. They used ChIP mapping data and claim to show that KMT2A depletion does not affect H3K4me3 levels but does reduce acetylation of H3K9 and reduces PCAF (KAT2B) binding. They further claim that KMT2A depletion causes stalling of RNA Polymerase I at the upstream "Spacer" promoter duplication on the rDNA.
Weaknesses:
I found the manuscript very confusing. In particular, I found it short on short on experimental detail and I found the data in general to be unconvincing. It is my opinion that the authors overinterpret their data, …
Author response:
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript the authors study the distribution of the KMT2A methyltransferase across the human ribosomal gene loci (rDNA) and the effects of its depletion on rDNA chromatin modification using a KMT2A conditional cell line. They used ChIP mapping data and claim to show that KMT2A depletion does not affect H3K4me3 levels but does reduce acetylation of H3K9 and reduces PCAF (KAT2B) binding. They further claim that KMT2A depletion causes stalling of RNA Polymerase I at the upstream "Spacer" promoter duplication on the rDNA.
Weaknesses:
I found the manuscript very confusing. In particular, I found it short on short on experimental detail and I found the data in general to be unconvincing. It is my opinion that the authors overinterpret their data, leading to unrealistic and unsupported claims. The manuscript also avoided discussing relevant recent studies on the regulation of the rDNA and of rDNA chromatin, most citations being to data 15 to 20 or more years old. This is most apparent when discussing the factors such as UBTF and SL1 that determine preinitiation complex assembly and the status of active versus inactive rDNA chromatin. Both have seen significant advances in recent years, and these should be thoroughly discussed.
Specific comments:
The authors do not describe the generation of the KMT2A conditional HEK293T cells or their treatment. Since these cells form the basis for subsequent experiments, they must be explained fully. I found that the conditional cell line had already been used in a previous unrelated publication "Chinchole et al. 2022". This manuscript explained that the KMT2A knockout is induced via Dox-controlled Cas9 in a HEK cell clone constitutively expressing guide RNAs to KMT2A. The KMT2A knockout requires a 7-day treatment with Dox, leaving wide open the question of direct versus indirect effects of KMT2A loss, and perhaps explaining some of the discrepancies with published data.
We thank the Reviewer for their comment. As the Reviewer correctly states, these KMT2A knockouts have been used in previous studies: Chinchole et al., 2022; PMID: 36111497 and Malik et al., 2023; PMID: 37379335. In both cases, the generation of KMT2A inducible knockout was described in detail in the Methods section; hence, in this manuscript we referred to these publications. However, if the Reviewer feels that details should still be provided, we will do so again here.
As the Reviewer is aware, KMT2A is an essential chromatin-bound protein, which is quite stable in its heterodimer form. As we described in our publication, Malik et al., 2023, the inducible knockout clones were obtained after extensive screening and thoroughly characterized. While the protein diminishes in the WB after 3 days, we thought it necessary to confirm that it does not persist on the chromatin in ChIP assays.
We are pasting the excerpt from the manuscript describing the relevant section below.
“Our initial attempts to generate MLL knock-outs in several different cell lines were not successful. Therefore, keeping in mind that MLL is essential for cell viability and growth [26,27], we generated and successfully obtained knock-outs in HEK-293 cell lines using doxycycline-inducible Cas9 expression vectors ([52]; Fig 3A). MLL levels were drastically reduced in the 2 independent MLL-knock-out clones shown here (MLL iKO#11 and iKO#20) and both KOs showed cell cycle profile similar to Control cells (S3A Fig). In the case of chromatin-binding proteins, often cellular levels show reduction but not the chromatin-bound fraction. We, therefore, confirmed that MLL chromatin binding was indeed reduced in our inducible knock-out cell lines by ChIP assays (Figs 3B and S3B), both on centromere and HOXA9 promoter”.
Inducible Cas9-mediated knockout provides an opportunity to generate irreversible knockouts, allowing sufficient time to deplete stable proteins and reveal delayed phenotypes or compensatory responses [PMID: 29036671]. However, this advantage has to be balanced with the potential secondary effects arising from prolonged depletion. Indeed, inducible Cas9 knockouts have been generated after similar induction periods by other laboratories to deplete chromatin-bound proteins [PMID: 28216383; PMID: 37702151], especially for members of the KMT2 family [PMID: 40846851, PMID: 27535106; PMID: 37535603 ]. Further, we have not observed any discrepancy with published data for KMT2A.
In mapping the interactions of KMT2A across the rDNA by ChIP they use 18 PCR amplicons, some of which the authors inaccurately state as targeting specific rDNA elements. For example, they state Primers (that is, Amplicons) #1-3 target the spacer and core 47S promoters. In fact, amplicon #2 overlaps neither of these promoters and lies centrally within the 1kbp of enhancer repeats between them. Exactly why emphasis is put on KMT2A chipping at these two promoters is anyhow questionable since KMT2A levels (Figure 1) appear equally high at multiple IGS amplicons.
As the enhancer repeats (ER) are clearly shown in the schematic of Fig. 1A and SF1B, and the same is stated in the legend of SF1B, rather than an inaccuracy, it is a matter of oversight. We thank the Reviewer for noticing this and bringing it to our attention. We will correct it in the text.
We thank the Reviewer for raising this point regarding our focus on the promoters in the present study and the binding of KMT2A to the IGS region. KMT2A’s role in regulating RNA polymerase II transcription by regulating gene promoters is well established. However, its role in regulating RNA polymerase I transcription at the rDNA promoter has not been characterized. In this study, we provide evidence for a previously unrecognized role of KMT2A in regulating rDNA transcription and hence extend its function to regulation of RNA polymerase I.
We agree that our ChIP data show KMT2A occupancy at multiple regions within the IGS, in addition to the promoter regions. For this reason, we felt it was important to present these data and perform ChIP-seq analyses of the rDNA locus to reveal the complete picture.
Our emphasis on the promoter regions is aimed at understanding the potential role of KMT2A in regulating RNA polymerase I transcription initiation. However, we recognize that the enrichment of KMT2A at multiple IGS regions raises the possibility that it may have additional functions beyond promoter regulation. We are currently investigating the role of KMT2A at the IGS, and in our future studies we hope to address the functional significance of this occupancy in greater detail.
Discrepancies with published data are particularly apparent in their mapping of H3K4me2, H3K4me3 and H3K9ac, all directly relevant to their conclusions. These modifications have been shown in several publications to map to a single major site a few hundred bp upstream of the spacer promoter in multiple human and mouse cell types. Since the authors fail to monitor H3K4 methylation and H3K9 acetylation at this dominant site they also fail to provide significant evidence that KMT2A in fact affects H3 methylation or H3 acetylation on the rDNA.
We thank the reviewer for raising this important point about the distribution of histone modifications at the rDNA locus and how they depend on KMT2A. Previous studies have established that active histone modifications are enriched at specific regions of rDNA. In particular, landmark studies from two groups have pioneered the use of genome assemblies to map these histone modifications. The Scacheri group [Zentner et al. 2011, PMID: 21355038] reported enrichment of H3K4me2, H3K4me3, and H3K9ac at the human rDNA promoter, along with additional active-chromatin-associated enrichment within the intergenic spacer. Subsequently, the same group demonstrated that the distribution of histone modifications in mouse embryonic stem cells differs from that in human embryonic stem cells and other differentiated mouse cell types [Zentner et al. 2014, PMID: 24347625]. These findings underscore the importance of examining the relevant regulatory regions in the appropriate biological system.
Similarly, studies from the Moss group radically redefine our understanding of how histone modifications are distributed across rDNA in mammals. They demonstrated that active histone modifications (including H3K4me2/3, H3K9ac, H3K27ac, and H3K36me3) are not present at the core 47S promoter or along the transcribed gene body of active rDNA repeats, but are instead strictly sequestered to a highly localized "Enhancer Boundary Complex" located a few hundred base pairs upstream of the spacer promoter. Consequently, this precise upstream hub represents the sole site of active histone modifications on active rDNA genes [Herdman et al., 2017, PMID: 28715449 ; Mars et al., 2018, PMID: 29158335]. These findings challenge the classical models of histone modification distribution at rDNA.
However, an extensive body of classical literature presents a complementary view where active histone modifications directly decorate the core 47S promoter and proximal transcribed regions to dynamically coordinate RNA Polymerase I transcription. Work from the Grummt and Santoro laboratories established that unmethylated, active rDNA repeats possess prominent H3K4me3 and hyperacetylated histones directly at the core 47S promoter, serving as a functional "poised" platform for pre-initiation complex (PIC) assembly [Santoro and Grummt, 2002, PMID: 12368916; Santoro and Grummt, 2005, PMID: 15767661; Mayer et al., 2006, PMID: 16678107; Schmitz et al., 2010, PMID: 20952535]. Furthermore, tracking chromatin remodelling complexes—such as the CSB-PCAF complex investigated by the different groups—has shown that active depletion of histone acetyltransferases leads to a direct loss of H3K9ac specifically at the primary 47S initiation site, causing a failure in Pol I loading [Shen et al., 2013, PMID: 23667505; Vintermist et al., 2011, PMID: 21559432]. Besides these reports, a wealth of independent functional data highlights that key chromatin regulators dynamically target and modulate the core 47S promoter region to dictate ribosomal transcription initiation. Some examples are referred to here [Xei et al., 2012, PMID: 22570494; Wang et al., 2011, PMID: 21960006; Zhu et al., 2010, PMID: 20531378; Frescas et al., 2007, PMID: 17994099].
Our investigation of KMT2A directly builds upon this functional promoter-centric model. While we appreciate that mapping variations exist between custom rDNA reference models in the literature, our data show clear and reproducible levels of H3 methylation and acetylation dynamics across the primary regulatory sites (including the 47S promoter and adjacent transcribed unit) upon KMT2A depletion. These changes correlate directly with functional changes in 47S pre-rRNA output, supporting a model where KMT2A-mediated histone modifications operate directly at the core transcription initiation machinery to regulate overall nucleolar output.
We acknowledge that the distribution of histone modifications across the rDNA repeat and their functional dependence on KMT2A represent distinct questions. Accordingly, we interpret the ChIP-seq profiles in conjunction with our site-specific ChIP-qPCR and functional experiments rather than relying exclusively on the genome-wide mapping data. We therefore conclude that our results support a role for KMT2A in regulating the acetylation-associated chromatin environment and transcriptional activity of the rDNA promoter, without claiming that KMT2A depletion reduces all active histone modifications across the locus.
We will clarify the relevant analysis and the scope of our conclusions in the revised manuscript.
The KMT2A deletion drastically reduces PolI and UBF (UBTF) engagement across the rDNA, and consistent with this, SL1 and RRN3 engagement is reduced as are 47S levels. Thus, whatever the 7-day KMT2A deletion is doing to the cells it is certainly inducing a very significant shut-down of active rDNA. How such an extreme experimental model can tell anything of KMT2A functions is unclear.
We feel that this concern is on similar lines as that stated in comment 1. We have already explained our inducible knockout model above. The cell-cycle profile of KMT2A-inducible knockout (iKO) cells reported in Malik et al. (2023), PMID: 37379335. provides relevant context for interpreting these effects.
We further wish to highlight that contrary to expectation, the H3K4me2/3 levels are largely unaffected on the rDNA in KMT2A iKO cells, which is surprising but not unexpected given the role of KMT2A in selectively trimethylating specific gene loci (PMID: 19703992). However, the loss of acetylation on H3 is very specific to the study at hand.
Unchanged H3K4me2/3 levels, along with selectively decreased acetylation, have not been reported for a general shutdown of active rDNA under extreme conditions like serum starvation etc. In case of a non-specific effect, both active methylation and acetylation marks are expected to go down. The presence of the H3K4me2/3 mark in our iKO cells acts as an internal control, confirming that the loss of H3 acetylation is a direct consequence of KMT2A depletion.
Furthermore, in the current manuscript, the effects of KMT2A deletion on the Pol I transcriptional machinery can be rescued by the KMT2A catalytic point mutant carrying the N3906A mutation but not by the mutant with TAD deletion. This rescue provides important evidence that the phenotype is linked to a specific function of KMT2A rather than being a nonspecific consequence of KMT2A deletion.
Our findings, considered alongside Lone et al. (2026) PMID: 42096476, support distinct roles for KMT2A and KMT2F in regulating rDNA transcription. In particular, the effects of KMT2A loss occur without a substantial reduction in rDNA H3K4me3, distinguishing its role from the H3K4me3-dependent function of KMT2F in promoting Pol I pre-initiation complex formation.
In the last results section, the authors further claim "Loss of KMT2A results in a stalled RNA Pol I complex at the spacer promoter" (Figure 4 and S4). To support this the authors try to map spacer promoter transcripts but mistakenly claim that the amplicon #1 can detect these transcripts. In fact, this amplicon overlaps the Spacer Promoter initiation site and so could not detect transcripts coming from this promoter. Published data, not cited, have demonstrated that PolI stalling at the spacer promoter occurs as a normal phenomenon on the active rDNA. Its loss is associated with suppression, not the opposite as inferred in this manuscript.
We thank the Reviewer for raising this point. We have double-checked that amplicon #1 is downstream of the spacer promoter start site. However, since the reviewer has raised this point, we will look into it in greater detail and remap our amplicons. We will also revisit the previous literature and compare our results with the published findings.
Reviewer #2 (Public review):
Summary:
This important manuscript presents solid evidence that the major contribution of KMT2A to ribosomal gene transcription is through promoting histone acetylation rather than H3K4 methylation. It is suggested this occurs in part through interactions of KMT2A with PCAF.
Strengths:
This manuscript advances an important concept that chromatin-modifying enzymes can also function to promote transcription through interactions with other transcriptional regulators independently of their catalytic functions. The data are overall supportive of this conclusion for KDM2A.
Weaknesses:
(1) Care needs to be taken regarding interpreting the lack of effect of depleting KMTs or HATs on histone acetylation or methylation levels. There are several overlapping HATS or KMTs that generate these modifications. The most that can be said from these negative results is that the depleted HAT or KMT is not the sole enzyme generating these modifications at these loci.
We thank the Reviewer for this important comment. We agree that histone modifications are regulated by multiple enzymes with overlapping or partially redundant activities, and that the absence of a detectable change following depletion of an individual KMT or KAT does not exclude a contribution from that enzyme to the regulation of the modification.
Specifically, the lack of a detectable reduction in H3K4me3 following KMT2A depletion indicates that KMT2A depletion alone is insufficient to produce a detectable decrease in H3K4me3 under the conditions examined. The persistence of this mark may reflect the activity of other KMTs and hence functional redundancy. In contrast, our observation that KMT2A depletion reduces H3K9 acetylation, together with our findings implicating PCAF in KMT2A-dependent rDNA transcription, supports a role for KMT2A in regulating an acetylation-associated mechanism at this locus.
Nevertheless, we acknowledge that these findings do not, by themselves, establish that PCAF is the sole enzyme responsible for H3K9 acetylation or that it accounts for all KMT2A-dependent effects on rDNA transcription. We will revise the interpretation of our results to avoid attributing the maintenance or loss of a histone modification exclusively to a single enzyme.
(2) The biochemistry supporting an interaction between KDM2A and PCAF is under-developed and not strong enough to support this conclusion at this point.
We thank the reviewer for their comment. Based on these comments and those from Reviewer 3, we plan to add more experiments to strengthen the KMT2A-PCAF interaction, including a reverse pull-down.
(3) It is concluded that acetylation causes increased rDNA transcription. However, acetylation may be a consequence of transcription instead, especially over transcribed regions.
We thank the reviewer for raising this important point regarding the relationship between histone acetylation and rDNA transcription. We agree that increased histone acetylation over transcriptionally active regions may either contribute to transcriptional activation or result from ongoing transcription. These possibilities are not mutually exclusive.
Our study provides several lines of evidence supporting a functional relationship between KMT2A and PCAF-associated histone acetylation, which indicate that acetylation is the cause and not a consequence of rDNA transcription. KMT2A depletion reduces rDNA transcription and is accompanied by a reduction in H3K9 acetylation. In addition, we present a model in which KMT2A facilitates the transfer of RNA Pol I PIC from the spacer promoter to the 47S promoter following its assembly at the spacer promoter by recruiting PCAF to rDNA through its TA domain. Together, these observations support that acetylation supports efficient rDNA transcription
Nevertheless, we acknowledge that these findings do not completely exclude the possibility that some of the observed reduction in histone acetylation occurs downstream of reduced transcription and will modify the discussion accordingly.
(4) Some conclusions, for example implicating PCAF, are based on overexpression experiments that are very artificial and difficult to draw conclusions from.
We thank the reviewer for their comment. We agree that overexpression experiments can produce non-physiological protein levels and potentially influence experimental readouts. However, our interpretation of PCAF involvement is based on a broader set of observations presented in this study, including the effects of KMT2A depletion on rDNA transcription and histone acetylation, together with our experiments examining the contribution of individual histone acetyltransferases to rRNA synthesis. These findings identify PCAF as a key co-activator of KMT2A-dependent transcriptional activation.
Therefore, the overexpression experiments have been used to provide mechanistic support rather than definitive evidence that endogenous PCAF is the sole or essential mediator of this process.
(5) Several figures are generated using publicly available data sets that were reanalyze using updated reference genomes, sometimes from different cell lines/types. This seems to be a useful exercise but the methods should include information regarding the robustness of these data sets.
We thank the Reviewer for their concern; we will update this information in the Methods.
Reviewer #3 (Public review):
Summary:
This manuscript examines a key mechanism by which KMT2A regulates RNA polymerase I transcription at ribosomal DNA. The authors suggest that KMT2A facilitates the recruitment of PCAF to rDNA. This process enhances histone acetylation and supports assembly of the RNA polymerase I transcription machinery. The study addresses an intriguing question by proposing a catalytic-independent role for KMT2A in rDNA transcription, potentially linking the KMT2A-dependent pathway to histone acetylation. However, several aspects of the proposed mechanism require further experimental validation. Notably, while the current evidence demonstrates associations among KMT2A, PCAF occupancy, histone acetylation, and rRNA transcription, it does not fully support the proposed objectives.
Strengths:
This could be the first report of a lysine methyltransferase regulating an acetyltransferase in Pol I-mediated gene expression.
Weaknesses:
(1) The causal relationship between KMT2A and the recruitment of PCAF needs to be demonstrated. Simply showing reduced PCAF occupancy after depleting KMT2A does not, on its own, prove direct recruitment. The authors should investigate whether KMT2A and PCAF physically interact, and whether this interaction depends on DNA or RNA.
As we have shown that KMT2A and PCAF interact by endogenous IP (Fig. S4D) and pull-down (Fig. S4E) experiments with the KMT2A C-terminal fragment containing the TAD domain, does the Reviewer mean in vitro interaction? Both proteins are large and may be difficult to produce in bacteria.
We do plan to perform reverse IP as suggested by Reviewer 2, and during these experiments, we can plan to test if this interaction is promoted by DNA or RNA by testing it in the presence or absence of DNase or RNase.
(2) The main prediction of the model is that restoring PCAF activity should reverse the molecular effects caused by the depletion of KMT2A. The authors should test whether PCAF restoration can mitigate the consequences of KMT2A loss. They should also assess PCAF occupancy, H3K9 acetylation levels, Pol I occupancy, and nascent rRNA synthesis.
We agree with the Reviewer that this is the central hypothesis of our study. Ideally, a simple global restoration or overexpression of PCAF should be able to rescue the effects caused by the depletion of KMT2A. However, based on our model and existing literature, we expect the following issues:
(1) We propose that KMT2A serves as a recruiter, a physical scaffold, to target PCAF to the rDNA. Therefore, mere overexpression of PCAF, in the absence of KMT2A, will not be able to ensure its localisation to the rDNA locus.
(2) Transcription activation by KMT2A involves tight synergy between its TA domain and specific KATs (PMID: 24253305; PMID: 31515273). The KMT2A TAD contains intrinsically disordered regions that must directly engage co-activator docking sites—specifically interacting with domains like the KIX domain of p300/CREB-binding protein acetyltransferase complexes—to transition into a stable secondary structure and drive downstream chromatin remodelling. Because this functional synergy requires the physical presence of both the KMT2A TAD and the KAT complex at the promoter, in our opinion, restoring PCAF activity in a KMT2A-depleted background cannot rescue localized H3K9 acetylation at the rDNA promoter. While we are not sure how PCAF engages with the KMT2A TAD domain, based on the above literature, the physical presence of KMT2A may be required.
We will test this hypothesis by overexpressing PCAF in the absence (or presence) of KMT2A, monitoring rRNA expression, and then plan to undertake future experiments accordingly.
(3) The catalytic-independent function of KMT2A should be directly investigated. The absence of a significant change in H3K4me3 levels following KMT2A depletion does not confirm that KMT2A's catalytic activity is not essential. Rescue experiments comparing wild-type KMT2A with a catalytically inactive variant would provide a much stronger assessment of the proposed non-catalytic mechanism.
We thank the reviewer for their comment. We completely agree that it is important to rule out whether catalytic activity of KMT2A is essential for rRNA transcription. We have shown in Fig. 4G that the catalytic point mutant carrying the N3906A mutation is able to recruit PCAF and RNA Pol I to the rDNA but not the KMT2A∆TAD mutant. This rescue experiment supports our hypothesis that the catalytic activity of KMT2A is not involved in mediating transcription at rDNA, but TA domain-dependent recruitment of PCAF is critical.
(4) The manuscript highlights H3K9ac; however, various acetyltransferases and histone acetylation marks are linked to active rDNA. The authors should investigate whether KMT2A specifically regulates H3K9ac or leads to broader changes in histone acetylation at rDNA.
We thank the reviewer for their comment. This is indeed an insightful suggestion. As shown in Fig 3. Multiple histone acetylation marks decorate the rDNA, and it is not possible for us to test them all. We propose to test some of these marks during revision to gain insight into the range of KMT2A-mediated regulation.
(5) Due to the highly repetitive nature of rDNA, the manuscript must include detailed information about the reference assembly, mapping strategy, management of multimapping reads, normalisation processes, and biological replicates. The authors should demonstrate that any observed changes in occupancy are not due to technical differences in rDNA mapping or variations in copy number.
We thank the reviewer for their comment. As indicated in the manuscript, we have used the pipeline from George et al. al, 2023, PMID: 37121547, which describes all these parameters in detail. Hence, we have omitted a repetition here but added a brief sentence in the Methods now.
(6) KMT2A regulates a wide range of transcriptional programs, and its depletion can affect proliferation, cell-cycle distribution, or nucleolar function. The authors should include appropriate controls to demonstrate that the rDNA phenotype is not a result of changes in cellular proliferation or a generalised response to transcriptional or nucleolar stress.
We thank the reviewer for highlighting this important point. The transcriptional profiling recently published by our lab [Lone et al. (2026), PMID: 42096476; Malik et al., 2023, PMID: 37379335] provides clear evidence that helps distinguish direct regulatory effects from generalized stress responses. Our global transcript analysis indicates that KMT2A depletion specifically downregulates transcription at the rDNA repeats without inducing hallmark markers of nucleolar stress or broad cell-cycle arrest.
(7) The CSB and B-WICH complex have previously been linked to the recruitment of PCAF and the acetylation of H3K9 at active rDNA. The authors should investigate whether KMT2A operates independently of these factors or if KMT2A functions within an established pathway for remodeling rDNA chromatin.
We agree with the Reviewer that CSB and the B-WICH complex, PCAF recruitment, and H3K9ac deposition are indeed a well-documented axis at active rDNA. In the present study, our focus is on establishing the functional role of KMT2A at the rDNA locus. Given that KMT2A traditionally acts as a histone methyltransferase that often precedes or cooperates with acetyltransferases like PCAF, it is highly plausible that KMT2A operates sequentially or in concert with the B-WICH pathway to remodel chromatin.
However, mapping these biochemical interactions is beyond our current aims. Nonetheless, we agree this is a crucial conceptual link, and we will expand this link in our Discussion.
(8) The distinction between active and silent rDNA should be taken into account. The authors need to determine whether KMT2A/PCAF occupancy is preferentially associated with active rDNA copies. Sequential ChIP analysis or the use of established markers for active, poised, and silent rDNA would help clarify the chromatin context in which the proposed mechanism operates.
We appreciate the Reviewer’s suggestion to determine if KMT2A/PCAF occupancy is restricted to active rDNA copies. While this is an important factor to consider, making a clear distinction is currently not possible due to several constraints. First, the highly repetitive nature of rDNA repeats makes it difficult to differentiate active from silent fractions both experimentally and bioinformatically. Second, performing sequential ChIP to definitively map this co-occupancy is technically challenging and establishing and optimising this protocol within the revision timeframe will be extremely difficult.
Furthermore, the exact histone landscapes defining active, poised, and silent rDNA are not yet fully characterized; much of the existing consensus in epigenetic literature relies heavily on observations drawn from RNA Polymerase II systems rather than Pol I repeats. As demonstrated by Xie et al., 2012 [PMID: 22570494], rDNA promoters frequently feature bivalent marks—bearing both active H3K4me3 and repressive H3K27me3 modifications—that characterize a poised, non-transcribing state. This biological ambiguity makes absolute active/silent categorization extremely difficult in our case.
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