The mechanisms by which the HuSH complex is recruited to genomic regions are still poorly understood, and significant questions remain regarding how this complex effectively silences retrotransposable elements and pseudogenes. Notably, the subunits that constitute the HuSH complex lack apparent enzymatic activity. Instead, the prevailing model proposes that the complex primarily functions as a central hub, enabling important protein-protein interactions necessary for its silencing activities7. To identify proteins associated with the HuSH complex, which may contribute to its recruitment and silencing functions, and potentially discover novel core members, we conducted a comprehensive study using a rigorous immunoprecipitation (IP) approach combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Specifically, we used a stably expressed epitope-tagged transgenes of the known HuSH core complex members, namely TASOR, MPP8, and PPHLN1, in HEK293 cells (Fig. 1B-D).
As expected, our analyses, which involved the use of LC-MS/MS, immunoblotting, and silver staining, validated robust associations between MPP8, PPHLN1, and TASOR. However, we made an intriguing and unexpected discovery of an association between MPP8 and PPHLN1 with an uncharacterized protein, fam208b, also known as TASOR2, which is a paralog of TASOR. By utilizing mass spectrometry analysis on samples obtained from FLAG-PPHLN1 and FLAG-MPP8 immunoprecipitation, we were able to attain a comprehensive peptide coverage across TASOR2 (Figure S1C-D). While TASOR and TASOR2 orthologs possess N-terminal pseudo-PARP domains, our mass spectrometry data did not reveal any peptides originating from the TASOR2 pseudo-PARP in our immunoprecipitation experiments. Notably, multiple annotated TASOR2 isoforms that lack the exons encompassing the N-terminal pseudo-PARP domain are documented. These findings indicate that TASOR2 lacking the pseudo-PARP domain is the dominant isoform within HEK293 and K-562 cells and is the isoform that associates with PPHLN and MPP8 (Fig. 1A; Figure S1A).
To validate and further investigate the association of TASOR2 with the HuSH complex subunits, we generated stable cell lines expressing FLAG epitope-tagged TASOR and TASOR2 transgenes in K-562 cells, followed by FLAG immunoprecipitation. Our findings revealed that TASOR2 specifically associated with PPHLN1 and MPP8, while it does not co-immunoprecipitate with TASOR (Fig. 1C). Conversely, TASOR co-IP experiments clearly demonstrated an association with PPHLN1 and MPP8, but no such association was observed with TASOR2 (Fig. 1B-C). Additionally, an anti-MPP8 antibody efficiently co-immunoprecipitated TASOR2 in an MPP8-dependent manner. Similarly, using an anti-TASOR antibody enabled the co-immunoprecipitation of MPP8 in a TASOR-dependent manner, but no interaction was observed with TASOR2 (Fig. 1E). These experiments collectively illustrate the existence of mutually exclusive HuSH assemblies for TASOR and TASOR2, suggesting the potential for distinct roles of these proteins within HuSH complexes.
In addition to TASOR2, our immunoprecipitation experiments revealed previously undescribed proteins that associate with HuSH assemblies. Specifically, we identified subunits of the human nuclear exosome targeting (NEXT) complex10, subunits of the EHMT1/2 complex11, and proteins involved in DNA damage response12 as new HuSH-associating proteins. Additionally, we observed associations between HuSH and RNA binding proteins, as well as lysine deacetylases (Figure S1B). Interestingly, some complexes involved in gene repression showed distinct associations with either TASOR or TASOR2. For example, the EHMT1/2 complex associated with HuSH, while subunits in lysine deacetylase complexes were associated with HuSH2. However, most associated proteins showed no distinct preference between the two HuSH complexes.
Prior work has proposed that TASOR serves as the core subunit for the HuSH complex, primarily based on the inability of MPP8 to co-IP PPHLN1 in overexpression experiments in cells7,13,14. Our immunoprecipitation experiments revealed that TASOR and TASOR2 exhibit mutual exclusivity in their associations with MPP8 and PPHLN1. To study the function of TASOR2 and other HuSH subunits, we employed CRISPR/Cas-9 technology to create knockout cell lines for MPP8, PPHLN1, TASOR, TASOR2, as well as a dual knockout of TASOR1/2. We found that epitope tagged PPHLN1 can associate with MPP8 in cell lines in which either TASOR or TASOR2 has been knocked out. However, the association between PPHLN1 and MPP8 was completely abrogated in the double TASOR1/2 mutant cells (Fig. 1F). These findings indicate that TASOR and TASOR2 facilitate the association between MPP8 and PPHLN1, and that TASOR2 may serve as the core protein for a second HuSH complex. However, we observed a discernible reduction in the amount of MPP8 associating with PPHLN1 in co-IP experiments from TASOR knockout cells, when compared to TASOR2 knockout cells. This observation suggests that HuSH might be more abundant than HuSH2 within the K562 cells, also suggested by stoichiometry observed in a silver stain of both complexes (Fig. 1D).
The results obtained from the immunoprecipitation experiments provide solid evidence for the existence of two distinct HuSH complexes, each organized around one of the two TASOR paralogs. We sought to investigate whether these two HuSH complexes bind to the same genomic loci. Prior research on HuSH has revealed its association with both pseudogenes and retrotransposable elements2,4,8,6. To address the question of whether the two HuSH complexes associate with identical or distinct genomic loci, we performed MPP8 chromatin immunoprecipitation sequencing (ChIP-seq) experiments. We chose the human K-562 chronic myelogenous leukemia (CML) cell line for our studies as it shows high levels of TASOR2 expression and average levels of TASOR expression relative to other commonly used cell lines (Figure S2A-B). We generated knockout cell lines for each HuSH subunit and conducted ChIP-seq experiments using antibodies specific for TASOR, MPP8, and PPHLN1. This allowed us to identify the genomic binding sites of the HuSH complexes and evaluate the influence of individual subunits on their localization (Fig. 2A-B, E; S3A).
Our ChIP-seq data revealed a strong correlation between the localization of MPP8 and PPHLN1 and the concurrent presence of either TASOR or TASOR2. By analyzing MPP8 and PPHLN1 ChIP-seq peaks in TASOR and TASOR2 knockout cell lines, we were able to identify specific genomic regions clearly bound by either HuSH or HuSH2 (Fig. 2C; S3A). Because of the unavailability of reliable TASOR2 antibodies, we have defined HuSH2 peaks as genomic regions characterized by the co-localization of both MPP8 and PPHLN1 while excluding the presence of TASOR. Additionally, these HuSH2 sites exhibit a loss of MPP8 and PPHLN1 ChIP signal in TASOR2 knockout cells, further suggesting that these sites are dependent upon the presence of TASOR2 for HuSH2 recruitment (Fig. 2A, C-D; S3A). We observed the co-localization of all three subunits in HuSH – MPP8, TASOR, and PPHLN1 – at more than 260 genomic sites. In contrast, our analysis revealed the existence of over 400 distinct HuSH2 peaks distributed across different genomic regions. Notably, these two distinct groups exhibit absolutely no overlap in their designated genomic (Figure S4A). Additionally, our ChIP-seq analyses have revealed a robust correlation between HuSH and LINE1 elements, with nearly 40% of these HuSH ChIP peaks located in close proximity to LINE1 elements. This correlation is especially pronounced at the relatively young L1HS and L1PA families of primate LINE1 elements (Figure S4B-D). Intriguingly, this distinctive pattern of HuSH localization presents a stark juxtaposition when compared to HuSH2, which exhibit only minimal overlap with LINE1 elements. Instead of spreading throughout the genome as seen in retrotransposons such as LINE1, HuSH2 peaks are notably concentrated in close proximity to gene promoters and enhancers (Figure S4B).
Interestingly, we observed a significant increase in the enrichment of MPP8 and PPHLN1 at HuSH2 sites in TASOR knockout cells. Conversely, there was minimal change in the enrichment of TASOR, MPP8, and PPHLN1 at HuSH sites in TASOR2 knockout cells (Fig. 2A, C-D, S3A). These findings substantiate the results from our co-IP experiments, suggesting higher levels of HuSH compared to HuSH2 in K-562 cells (Fig. 1D, F). Based on the insights gained from our co-IP and ChIP-seq experiments, we propose a model in which MPP8 and/or PPHLN1 serve as critical and limiting factors, influencing the formation of the HuSH complex around either TASOR or TASOR2. To test our hypothesis regarding the competitive interaction between TASOR proteins and their influence on the localization of the two complexes, we conducted MPP8 ChIP experiments in cell lines overexpressing epitope-tagged TASOR or TASOR2 proteins (Fig. 3A-C). Notably, the overexpression of either TASOR or TASOR2 in wildtype parental cells led to a reduction in MPP8 levels at the opposing genomic sites. These data support the proposed model, which suggests that MPP8, and potentially PPHLN1, play pivotal roles as limiting members in the assembly of functionally competent HuSH complexes.
Our ChIP-seq data provide evidence for the existence of a second HuSH complex centered around TASOR2, which exhibits distinct localization patterns within the human genome. Except for the pseudo-PARP domain, which is absent in the dominant TASOR2 isoform found in K-562 cells (Figure S1A), all other known domains in TASOR are present in TASOR2 (Fig. 1A). Previous studies have suggested that the pseudo-PARP plays an important role for HuSH in LINE-1 silencing7,15. We aimed to explore the importance of the TASOR pseudo-PARP domain in localization of HuSH and silencing of LINE-1 in K-562 cells. We also aimed to test whether TASOR lacking its pseudo-PARP domain can relocalize to HuSH2 sites. To address these hypotheses, we introduced a wildtype TASOR transgene or a TASOR transgene lacking the pseudo-PARP domain in TASOR knockout cells and performed ChIP-seq experiments (Fig. 4A). We performed immunoblot analysis using an antibody specific to the LINE-1 ORF1 protein (ORF1p) as a key marker for LINE-1 silencing. Our findings revealed that TASOR knockout led to the loss of TASOR ChIP-seq signal at HuSH sites, accompanied by an increase in the LINE-1 ORF1p protein (Fig. 4B-C). Furthermore, the introduction of a wildtype TASOR transgene into TASOR knockout cells restored the ChIP-seq peaks associated with HuSH (Fig. 4C). Remarkably, we observed that a TASOR transgene lacking the pseudo-PARP domain was unable to effectively silence LINE-1 elements in a TASOR knockout, in contrast to the wildtype transgene which demonstrated a robust decrease in ORF1p levels (Fig. 4B). Notably, no TASOR peaks were identified in the absence of its pseudo-PARP domain, and the ChIP-seq data resembled that of a TASOR knockout. These results support the important role of the pseudo-PARP domain of TASOR in HuSH localization. However, the absence of the pseudo-PARP domain did not cause a relocalization of TASOR to HuSH2 sites. These data imply that additional domains specific to TASOR2 play an important role in the localization to gene promoters and other chromosomal regions displaying HuSH2 localization.
To investigate the roles of HuSH2 and TASOR2 in LINE-1 silencing, we evaluated ORF1p levels in HuSH subunit knockout cells (Fig. 5A). We observed a notable upregulation of ORF1p in the PPHLN1, MPP8, and TASOR knockout cell lines, suggestive of LINE-1 silencing derepression, consistent with findings from prior research. Notably, there were no discernible changes in ORF1p expression when comparing TASOR2 knockout cells to the wildtype parental cells, consistent with the lack of enrichment of HuSH2 ChIP-seq peaks at LINE-1 elements. This finding suggests that TASOR2 may not play a direct role in the repression of LINE-1 elements within the regulatory network of the HuSH complex. In our immunoblot analysis, we detected a decrease in the steady-state levels of MPP8 and PPHLN1 in TASOR knockout cells. Conversely, the deletion of PPHLN1 or MPP8 resulted in a reduction of TASOR levels, but TASOR2 knockout cells did not exhibit a similar decrease, again suggesting lower amounts of HuSH2 in cells (Fig. 5A).
We introduced wildtype transgenes of PPHLN1, MPP8, and TASOR to investigate their potential to rescue the silencing of LINE-1 in the respective knockout models. Notably, our findings demonstrate that each of these transgenes effectively restored LINE-1 silencing in their corresponding knockout cell lines and stabilize levels of other HuSH complex members (Fig. 5B-C, F). Furthermore, we observed that TASOR was also capable of rescuing LINE-1 silencing in a double TASOR-TASOR2 knockout cell line (Fig. 5D). These results provide robust evidence for the regulatory role of HuSH in silencing LINE-1 elements and underscore the contributions of all subunits in this process (Fig. 5E). In contrast, the expression of a TASOR2 transgene in a TASOR knockout background failed to rescue LINE-1 silencing but did successfully rescue MPP8 protein steady state levels (Fig. 5F). Our co-IP and ChIP-seq findings led us to propose that PPHLN1 and/or MPP8 are limiting HuSH subunits in K-562 cells. Consequently, we suggest that competition for these two subunits could potentially regulate LINE-1 elements silencing by altering the levels of HuSH and HuSH2 in cells. An increase in HuSH2 would lead to a decrease in HuSH and loss of LINE-1 silencing (Fig. 5I). To test this hypothesis, we titrated the levels of a wildtype TASOR2 transgene in TASOR2 knockout cells. We found that high levels of the TASOR2 transgene led to derepression of LINE-1, mimicking the effect observed in TASOR knockout cells (Fig. 5G). Additionally, we found that high levels of a wildtype TASOR2 transgene in wildtype K-562 cell line also led to derepression of LINE-1 elements (Fig. 5H). Importantly, overexpression of a wildtype TASOR transgene did not have this effect. These findings reveal that by manipulating the expression levels of TASOR and TASOR2, either through knockout or overexpression, we can modulate the levels of HuSH and consequently alter the silencing of LINE-1 elements.
Our research findings indicate that MPP8 and/or PPHLN1 serve as limiting factors in the assembly of HuSH complexes. We aimed to gain a deeper understanding of how MPP8 functions in modulating the equilibrium within HuSH complexes. Our hypothesis centered on the notion that disrupting the interaction between MPP8 and either TASOR or TASOR2 would result in changes in cellular levels of HuSH2 or HuSH. To achieve this, we employed AlphaFold predictions to investigate the formation of secondary and tertiary structures during the complex formation between the TASOR proteins and MPP8.
Previous studies have already defined the interaction domains between TASOR and MPP8, which include the MPP8 region located C-terminal to the ankyrin repeats, as well as the DUF3715, SPOC, and DomI domains of TASOR7,16. Although the DomI domains in TASOR and TASOR2 orthologs display substantial dissimilarity in their sequences, AlphaFold predictions suggest that both human TASOR proteins share a similar predicted secondary structure (Fig. 6A, Figure S5) and adopt a structurally analogous fold that encompasses four alpha helices. One of these alpha helices is anticipated to interact with a 137-residue domain situated at the C-terminus of MPP8, comprising of beta-sheets and an alpha helix. We generated TASOR and TASOR2 transgenes with two amino acid substitutions, in which we replaced leucine or isoleucine residues with arginine. Subsequently, we introduced these transgenes into TASOR and TASOR2 knockout cells. Our results revealed that the dual L/I-to-R substitutions in both TASOR proteins disrupted their interaction with MPP8 while leaving the interaction with PPHLN1 unaffected, as demonstrated by co-immunoprecipitation (Fig. 6B, C).
The expression of the mutant TASOR transgene in a TASOR knockout background failed to restore LINE-1 silencing, a function effectively accomplished by expression of a wildtype TASOR transgene (Fig. 6D). When introduced in the context of a TASOR2 transgene, the L/I-to-R substitutions impaired the ability of TASOR2 overexpression to cause derepression of LINE-1, a function attainable with overexpression of wildtype TASOR2 in parental K-562 cells (Fig. 6E). To assess whether the derepression of LINE-1 elements is indeed linked to the inability of MPP8 to interact with and form HuSH, we performed chromatin immunoprecipitation targeting MPP8, followed by quantitative polymerase chain reaction (MPP8 ChIP-qPCR), in the TASOR mutant transgenic K-562 cell lines. TASOR knockout cells expressing the mutant TASOR transgene exhibited no MPP8 interaction at HuSH sites (Fig. 6F). Additionally, TASOR2 knockout cells expressing a TASOR2 mutant transgene resulted in a loss of MPP8 interaction at HuSH2 sites (Fig. 6G). These findings underscore the significance of MPP8 interaction in the functionality of the HuSH complexes.
Collectively, these findings have led us to propose a model for the silencing of LINE-1 elements by the HuSH complex. Under normal conditions in K-562 cells, TASOR acts as a central hub, facilitating the interaction between MPP8 and PPHLN1, which together form the HuSH complex. This complex associates with additional factors that may play a role in regulating its recruitment to chromatin, ultimately contributing to the subsequent silencing of LINE-1 elements. In K-562 cells, LINE-1 expression is typically low, primarily due to the high abundance of HuSH. However, when the levels of HuSH2 increase, either by reducing TASOR or increasing TASOR2 levels, MPP8 and PPHLN1 are titrated away from HuSH sites, thereby decreasing the abundance of HuSH in cells and resulting in the derepression of LINE-1 elements (Fig. 7).