Competition shapes the landscape of X-chromosome-linked genetic diversity

Buenaventura, Teresa; Bagci, Hakan; Patrascan, Ilinca; Graham, Joshua J.; Hipwell, Kelsey D.; Oldenkamp, Roel; King, James W. D.; Urtasun, Jesus; Young, George; Mouzo, Daniel; Gomez-Cabrero, David; Rowland, Benjamin D.; Panne, Daniel; Fisher, Amanda G.; Merkenschlager, Matthias

doi:10.1038/s41588-024-01840-5

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Published: 26 July 2024

Competition shapes the landscape of X-chromosome-linked genetic diversity

Nature Genetics volume 56, pages 1678–1688 (2024)Cite this article

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Abstract

X chromosome inactivation (XCI) generates clonal heterogeneity within XX individuals. Combined with sequence variation between human X chromosomes, XCI gives rise to intra-individual clonal diversity, whereby two sets of clones express mutually exclusive sequence variants present on one or the other X chromosome. Here we ask whether such clones merely co-exist or potentially interact with each other to modulate the contribution of X-linked diversity to organismal development. Focusing on X-linked coding variation in the human STAG2 gene, we show that Stag2^variant clones contribute to most tissues at the expected frequencies but fail to form lymphocytes in Stag2^WT Stag2^variant mouse models. Unexpectedly, the absence of Stag2^variant clones from the lymphoid compartment is due not solely to cell-intrinsic defects but requires continuous competition by Stag2^WT clones. These findings show that interactions between epigenetically diverse clones can operate in an XX individual to shape the contribution of X-linked genetic diversity in a cell-type-specific manner.

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Main

Eutherian mammals such as humans and mice compensate for differences in X-linked gene dosage between males and females by X chromosome inactivation¹ (XCI; Fig. 1a). In XX embryos, each cell randomly chooses one of its two X chromosomes for inactivation, which results in the silencing of the majority of genes on that chromosome^1,2,3,4. XX embryos therefore resemble mixtures of clones expressing genes from either their maternal or paternal X chromosome. The identities of the active (Xa) and inactive (Xi) X chromosomes are clonally propagated through organismal development by epigenetic mechanisms^5,6. Hence, XX individuals are clonally heterogeneous as a result of XCI and its propagation.

**Fig. 1: Sequence variation in the human X-linked *STAG2* gene disrupts cohesin–CTCF binding.**

Human population shows extensive genetic diversity, including single-nucleotide polymorphisms⁷ (SNPs), which occur at comparable frequencies on autosomes and X chromosomes⁸ (Supplementary Table 1). The human X chromosome harbors >600 protein-coding genes annotated in OMIM, the Online Catalog of Human Genes and Genetic Disorders⁹. Together, these genes contain ~400k nonsynonymous SNPs that change their coding potential¹⁰, indicating extensive variation between human X chromosomes. This variation, combined with XCI and its epigenetic propagation, gives rise to intra-individual clonal diversity in XX individuals.

Given that X-linked intra-individual diversity is widespread among XX individuals, it is of interest to consider its potential significance for organismal development. What is known so far is that stochastic and selective processes can affect the deployment of intra-individual clonal diversity.

Stochastic X-linked bias can arise from sampling errors early when founder cells are allocated to the three germ layers (ectoderm, endoderm and mesoderm) in embryonic development and can be further amplified by the allocation of cells to particular fates within each germ layer⁴ (Extended Data Fig. 1a). The resulting bias has been exploited to estimate the number of founder cells for cell types and tissues in embryonic development⁴ and the number of hematopoietic stem cells (HSCs) that contribute to the regeneration of blood cells in later life¹¹.

A distinct form of X-linked bias arises from clonal selection against deleterious genetic variants that compromise the ability of variant-expressing clones to expand or survive in a cell-intrinsic fashion (Extended Data Fig. 1b). Clonal selection results in the dominance of clones that have inactivated the X chromosome harboring the deleterious variant and is relevant in the context of human disease, where intra-individual clonal diversity can mean a more favorable outcome in XX than XY individuals^2,12.

Here we ask a different question, namely whether epigenetically diverse clones, which arise from the combined effect of XCI and X-linked genetic variation, merely co-exist in XX individuals, or whether they interact, and, if so, how such interactions may shape the landscape of X-linked clonal diversity. To this end, we generate mouse models of X-linked genetic variation found in the human STAG2 gene and uncover a noncell-autonomous mode of X-linked bias which is distinct from stochastic variation and selection against deleterious variants. We find that clones expressing Stag2 variants fail to adopt a lymphoid fate in the presence of competitor clones that have silenced the variant allele by XCI. Unexpectedly, however, the absence of competitors expressing wild-type (WT) Stag2 restored the full range of cell fate choices to clones expressing Stag2 variants. Our observations reveal that clonal interactions have the potential to shape the contribution of X-linked genetic diversity to specific cell types and tissues in XX individuals.

Results

Sequence variation and XCI combine to generate intra-individual genetic diversity

Analysis of 3,775 X chromosomes across 2,504 individuals from phase 3 of the 1000 Genomes Project¹³ found 13,796 nonsynonymous SNPs (SNPs that alter the amino acid sequence of proteins encoded on the X chromosome). The average number of such missense variants between any two X chromosomes was 138 (minimum = 3 and maximum = 232), omitting genes that escape X-inactivation in humans^3,4. Ninety percent of X chromosome pairs harbored at least 101 missense variants. This analysis shows that sequence variation has the potential to generate intra-individual diversity in XX individuals when combined with XCI and its clonal propagation (Fig. 1a,b).

Sequence variants in the X-linked STAG2 gene disrupt cohesin–CTCF binding

STAG2 is an essential X-linked gene that is evolutionarily highly conserved¹⁴ (Fig. 1c and Extended Data Fig. 2) and encodes a subunit of cohesin, a protein complex that contributes to 3D genome organization as well as DNA replication, DNA repair and the stable propagation of chromosomes through cell division¹⁵. A survey of 125,748 human exomes¹⁰ (gnomAD v2.1) found that STAG2 coding variation was lower than predicted by chance, indicating a level of constraint expected for an essential gene (Fig. 1c,d). Nevertheless, >150 distinct missense variants were observed (Fig. 1c and Extended Data Fig. 2). We focused on gnomAD variant X-123185062—G-C (GRCh37) found in HG02885, an XX individual of African origin who self-reported as healthy, and participated with her husband and daughter in the control (nondisease) cohort of gnomAD v2.1.1. This SNP changes STAG2 arginine 370 to proline (R370P). STAG2 R370 contributes to an interaction interface that is formed jointly by the cohesin subunits STAG1/STAG2 and RAD21 (Fig. 1e). This interface has been described as a ‘conserved essential surface’ and is bound by the following cohesin-interacting proteins that are engaged in a range of DNA-based processes: CTCF in 3D genome organization¹⁶ (Fig. 1e), Shugoshin in sister chromatid cohesion^17,18, MCM3 (minichromosome maintenance protein 3) in DNA replication¹⁹ and likely other cohesin interaction partners²⁰. We used isothermal calorimetry to assess the impact of STAG2^R370P on cohesin–CTCF interactions and found a complete loss of binding (Fig. 1f). Hence, sequence variation in the X-linked STAG2 gene illustrates the potential for clonal heterogeneity within XX individuals.

Stag2 ^variant progenitors fail to form lymphocytes in heterozygous XX individuals

To explore the impact of X-linked sequence variation at the organismal level, we generated mouse models of Stag2 variants in the conserved essential surface between STAG2 and CTCF (Fig. 1e). Stag2^R370Q had a tenfold lower CTCF binding affinity than WT (Fig. 1f). A second variant, Stag2^W334A, abolished the STAG2–CTCF interaction to the same extent as the human R370T variant (Fig. 1f). As expected¹⁶, STAG2–CTCF interface variants retained the ability to form DNA-bound cohesin complexes (Extended Data Fig. 3c). Stag2^R370Q and Stag2^W334A variants showed equivalent phenotypes and are therefore described together.

WT and variant Stag2 were equally represented in genomic DNA (gDNA) from heterozygous X^Stag2-WT and X^{Stag2-variant} female mice, as illustrated for gDNA from blood (Fig. 2a, left). An equivalent representation of Stag2^WT and Stag2^variant genomic sequences was expected, as the presence of gDNA is unaffected by the epigenetic inactivation of one X chromosome in XX individuals¹. We next analyzed a range of cell types and tissues in heterozygous female mice to determine the contribution of clones in which the active X chromosome harbored the Stag2^WT allele (Stag2^WT clones) versus clones in which the active X chromosome harbored the Stag2^variant allele (Stag2^variant clones). We isolated RNA, reverse-transcribed RNA into cDNA and sequenced the complementary DNA (cDNA). Brain, gut and other tissues showed a roughly equal representation of Stag2^WT and Stag2^variant clones (Fig. 2a), while skewing toward Stag2^WT clones was found in skeletal muscle (Fig. 2a). cDNA isolated from peripheral blood mononuclear cells showed a markedly reduced expression of variant Stag2 (Fig. 2a and Extended Data Fig. 4a,b), indicating a near-complete absence of Stag2^variant clones.

**Fig. 2: *Stag2*^variant clones fail to form lymphocytes in *Stag2*^WT *Stag2*^variant individuals.**

To quantify the contribution of Stag2^variant versus Stag2^WT clones, we used allele-specific qRT–PCR (see Extended Data Fig. 4c for calibration). This analysis confirmed reduced representation of Stag2^variant clones in blood mononuclear cells (Fig. 2b) and in skeletal muscle and revealed increased representation of Stag2^variant clones in the heart (Fig. 2b and variants are shown separately in Extended Data Fig. 4d).

T and B lymphocytes are the major mononuclear cell types in blood. CD4 T and B cells isolated from lymph nodes of Stag2^variant Stag2^WT heterozygous females (Fig. 2c(i) and gating strategy in Extended Data Fig. 4e) showed a near-complete absence of Stag2^variant clones as determined by sequencing (Fig. 2c(ii)) and allele-specific qRT–PCR (Fig. 2c(iii)). We developed a reporter system to directly visualize individual cells expressing Stag2^variant or Stag2^WT by inserting a Luc/βGal reporter construct^21,22 into the X-linked Atrx gene, which is subject to XCI and broadly expressed across cell types and tissues, including the hematopoietic system²³. Atrx^Luc/βGal allows the visualization and prospective isolation of live Atrx^Luc/βGal cells by flow cytometry, based on the conversion of nonfluorescent fluorescein di-β-d-galactopyranoside (FDG) into green fluorescent fluorescein isothiocyanate (FITC) by the enzymatic activity of β-galactosidase (βGal). We confirmed that FDG conversion was indeed dependent on the presence of the Atrx^Luc/βGal reporter (Extended Data Fig. 5a–c). In female mice that were heterozygous for the Atrx^Luc/βGal reporter and had two WT alleles of Stag2, FDG to FITC conversion occurred in approximately half of all T and B lymphocytes (Fig. 2c(iv), top) and other hematopoietic cell types examined (Extended Data Fig. 5a–c). This indicates that the reporter itself does not substantially skew X chromosome usage. Sanger sequencing and allele-specific qRT–PCR confirmed the fidelity of the reporter, as well as the monoallelic expression of Stag2 in XX individuals (Extended Data Fig. 5d). In lymphocytes isolated from Stag2^WT Stag2^variant Atrx^Luc/βGal heterozygous females, Stag2^WT clones dominated over Stag2^variant Atrx^Luc/βGal clones (Fig. 2c(iv), bottom, and Extended Data Fig. 5c). Taken together with the sequencing and allele-specific qRT–PCR data, these results indicate that Stag2^variant clones fail to contribute substantially to mature T and B lymphocytes in Stag2^WT Stag2^variant heterozygous females.

Blood cells are continuously replenished by hematopoietic stem and progenitor cells¹¹ (Fig. 2d), allowing the developmental origin of skewed X chromosome usage to be traced. T cell fate specification of bone marrow-derived progenitors occurs in the thymus, and we, therefore, examined the representation of Stag2^variant clones among thymocyte subsets at successive stages of development (Fig. 2e(i) and gating strategy in Extended Data Fig. 4e). Sequencing (Fig. 2e(ii)), allele-specific qRT–PCR (Fig. 2e(iii)) and FDG labeling of Stag2^WT Stag2^variant Atrx^Luc/βGal thymocytes (Fig. 2e(iv) and Extended Data Fig. 5c) showed that Stag2^variant clones were barely detectable among developing T cells. Thymocyte differentiation of Stag2^variant clones was not rescued by provision of rearranged lymphocyte receptor transgenes (Extended Data Fig. 6). Stag2^variant clones were also absent from developing pro-B and pre-B cells in the bone marrow (Extended Data Fig. 7).

We next examined the representation of variant Stag2 RNA in hematopoietic stem (LSK), c-kit⁺ and common lymphoid progenitor (CLP) cells isolated from the bone marrow of heterozygous Stag2^WT Stag2^variant female mice (Fig. 2f(i) and gating strategy in Extended Data Fig. 4e). Sequencing (Fig. 2f(ii)), allele-specific qRT–PCR (Fig. 2f(iii)) and FDG labeling (Fig. 2f(iv) and Extended Data Fig. 5b) revealed skewing against Stag2^variant clones in hematopoietic stem and progenitor cells. In contrast to lymphocytes, the representation of Stag2^variant clones among mature myeloid cells remained comparable to hematopoietic stem and progenitor cells (Extended Data Fig. 7).

In conclusion, the hematopoietic system of Stag2^WT Stag2^variant heterozygous individuals appeared outwardly normal with respect to the number and composition of cell types in bone marrow, thymus and peripheral lymph nodes. However, the clonal composition of the hematopoietic system was skewed toward Stag2^WT clones, and few, if any, Stag2^variant clones contributed to immature and mature lymphocyte subsets. These findings suggested that hematopoietic progenitors with an active X chromosome harboring Stag2 variants were unable to undergo lymphoid specification and differentiation.

Reduced lymphoid priming in Stag2 ^variant hematopoietic progenitors

We isolated lineage-negative, c-kit⁺ Stag2^WT and Stag2^variant cells from the bone marrow of heterozygous females for single-cell RNA-sequencing (scRNA-seq; Fig. 3a, Extended Data Fig. 8a and gating strategy in Extended Data Fig. 4e) and identified progenitors based on established marker genes (Supplementary Data 1). DESeq2 found 1,600 upregulated and 802 downregulated genes in Stag2^variant progenitors (adjusted P < 0.01; Fig. 3b and representative gene ontology terms in Extended Data Fig. 8b). As STAG2 is part of the cohesin complex, we analyzed the relationship between cohesin binding and deregulated gene expression in Stag2^variant progenitors. Leveraging cohesin chromatin immunoprecipitation followed by sequencing (ChIP–seq) from hematopoietic progenitors, we found that genes that were deregulated in Stag2^variant progenitors were highly enriched for cohesin promoter binding compared to non-deregulated genes (Extended Data Fig. 8c), which links transcriptional deregulation in Stag2^variant cells to cohesin.

**Fig. 3: Reduced efficiency of lymphoid priming in *Stag2*^variant hematopoietic progenitors.**

We harnessed scRNA-seq gene expression profiles to identify long-term HSCs and lineage-primed progenitors among Stag2^variant and Stag2^WT progenitors. While the absolute number of Stag2^variant progenitors was reduced compared to Stag2^WT, the progenitors that were present in Stag2^variant showed an increased proportion of HSCs relative to Stag2^WT (Fig. 3c). Analysis of cell cycle markers suggested that Stag2^WT and Stag2^variant HSCs were largely quiescent (~99% G1), while lineage-primed progenitors were cycling in both Stag2^WT and Stag2^variant (Fig. 3c). The proportion of Stag2^variant lymphoid-primed progenitors was reduced, while the proportions of granulocyte/macrophage (G/M)-primed, erythroid (Ery)-primed and megakaryocyte (Mega)-primed progenitors were increased among Stag2^variant progenitors (Fig. 3c). Reduced lymphoid priming of Stag2^variant progenitors was progressive, as indicated by a further reduction in the proportion of Stag2^variant advanced lymphoid-primed progenitors that expressed a greater number of lymphoid genes (AUCell score of ≥0.2; Fig. 3d), although cell cycle profiles of lymphoid-primed progenitors were comparable between Stag2^WT and Stag2^variant progenitors (Fig. 3c,d). Figure 3e summarizes log₂(fold change) in the proportions of Stag2^variant progenitor subsets. Hence, despite the failure of Stag2^variant hematopoietic progenitors to form early B and T cells (pro-B cells and double-negative (DN) thymocytes, respectively), scRNA-seq provided evidence of lymphoid priming, albeit with reduced efficiency compared to Stag2^WT progenitors.

Competition between Stag2 ^variant and Stag2 ^WT clones

Based on these results, we wondered whether the failure of clones expressing variant Stag2 to contribute to lymphoid lineages was entirely due to cell-intrinsic defects that preclude lymphoid cell fate specification. To address this question, we generated Stag2^variant hemizygous males and Stag2^variant homozygous females, which exclusively harbored Stag2^variant cells. To our surprise, we found that in the absence of Stag2^WT, the cellularity and subset distribution of Stag2^variant thymocytes (Fig. 4a) and lymph node cells (Fig. 4b) were indistinguishable from WT controls in Stag2^variant hemizygous males and Stag2^variant homozygous females.

**Fig. 4: Successful lymphoid specification and differentiation of *Stag2*^variant cells in the absence of *Stag2*^WT.**

Cohesin is required for secondary rearrangements at the Tcra locus in immature thymocytes²⁴ and class switch recombination at the Igh immunoglobulin heavy chain locus in B cells^25,26. Unlike Rad21^ko thymocytes, Stag2^variant thymocytes rearrange both proximal (Jα61) and distal (Jα22) Tcra gene segments to a similar extent as Stag2^WT thymocytes (Extended Data Fig. 9a). Similarly, we found WT concentrations of immunoglobulin isotypes in Stag2^variant mice, indicating class switch recombination (Extended Data Fig. 9b). Mature lymphocytes are quiescent, but upon engagement of their receptors for antigen and costimulatory ligands, they undergo a program of activation that culminates in cell cycle entry and cellular proliferation. We activated T cells with antibodies to the T cell receptor at graded concentrations, together with a fixed dose of antibody to the costimulatory receptor CD28. As a readout, we measured the expression of the activation marker CD69 by flow cytometry (Extended Data Fig. 9c, left, and gating strategy in Extended Data Fig. 9d) and assessed T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE), which fluorescently labels cellular proteins that are diluted twofold at each successive cell division (Extended Data Fig. 9c, middle and right). The results showed that Stag2^variant CD4 and CD8 T cells generated in X^{Stag2-variant} hemizygous males were as responsive to activation signals as Stag2^WT cells.

We conclude that Stag2^variant progenitors can generate lymphocytes that are competent to undergo Tcra rearrangement, Igh class switch recombination and in vitro activation. However, Stag2^variant progenitors fail to realize their lymphoid potential in the presence of Stag2^WT cells. The impact of Stag2^WT cells on Stag2^variant progenitors is reminiscent of a form of cell competition whereby cells are eliminated only when they differ from their neighbors^27,28,29.

Stag2 ^variant progenitors retain lymphoid potential in the face of competition

As described above, Stag2^variant clones are detectable in the hematopoietic progenitor pool of heterozygous Stag2^variant Stag2^WT individuals and undergo at least limited lymphoid priming, but fail to substantially contribute to lymphoid specification and differentiation. Given that Stag2^variant clones were potentially exposed to competition throughout embryonic development, they may already be wounded or damaged beyond rescue by the time they enter the hematopoietic progenitor pool in heterozygous Stag2^variant Stag2^WT females. To gain additional insights into the rules of X-linked competition, we generated heterozygous Stag2^variant Stag2^lox female mice. The Stag2^lox allele encodes normal levels of WT STAG2 protein, but when deleted by Cre recombinase, it curtails differentiation of Stag2^ko progenitors into lymphocytes³⁰. We used VavCre³¹ to delete Stag2 upon entry into the hematopoietic progenitor pool (Fig. 5a). In this experimental setting, clones expressing variant Stag2 face competition from Stag2^WT cells until VavCre expression in hematopoietic progenitors. VavCre converts Stag2^lox into Stag2^ko, effectively releasing Stag2^variant progenitors from competition by Stag2^WT cells (Fig. 5a). We used the Atrx^Luc/βGal reporter integrated into the X chromosome harboring the Stag2^R370Q variant to determine the abundance of Stag2^variant clones. Stag2^variant clones continued to be outnumbered in the hematopoietic stem and progenitor compartment of VavCre⁺ Stag2^ko Stag2^R370Q bone marrow (Fig. 5b), as observed in Stag2^WT Stag2^variant mice. Lymph nodes of VavCre^pos Stag2^ko Stag2^R370Q heterozygous females showed similar cellularity as VavCre^neg Stag2^lox Stag2^R370Q (Fig. 5b). However, in stark contrast to control VavCre^neg Stag2^lox Stag2^R370Q lymph node CD4 T and B cells, Sanger sequencing and the Atrx^Luc/βGal reporter indicated dominance of Stag2^variant transcripts in cDNA of VavCre⁺ Stag2^ko Stag2^R370Q lymph node CD4 T and B cells (Fig. 5c) and thymocytes (Fig. 5d) following deletion of Stag2 ^lox by VavCre. As expected, Stag2^variant clones generated few—if any—lymphocytes in VavCre⁻ Stag2^lox Stag2^R370Q mice (Fig. 5c), where they competed against clones expressing WT STAG2 protein encoded by Stag2^lox. These data show that the removal of Stag2^WT competition at the hematopoietic progenitor stage is sufficient to reveal the lymphoid potential of Stag2^variant progenitor cells.

**Fig. 5: *Stag2*^variant progenitors in heterozygous females retain the potential to support lymphoid specification and differentiation.**

Hence, Stag2^variant cells are capable of generating normal numbers of lymphocytes, either in the complete absence of Stag2^WT (that is, in hemizygous Stag2^variant males or homozygous Stag2^variant females) or on release from competition by selective removal of Stag2^WT cells from the hematopoietic progenitor pool.

X-linked competition in humans

Mouse models revealed that clones expressing Stag2 variants failed to contribute to the formation of lymphocytes of XX females. To test the relevance of this finding for human biology, we examined the representation of the human STAG2 rs777011872 R370P variant described in Fig. 1. As expected, both WT and rs777011872 variant sequences were represented in the gDNA of polyclonal B cells derived from the blood of HG02885 (marked by a red rectangle in Fig. 6a, top). By contrast, only WT sequences were detected in cDNA, while the rs777011872 variant was absent (marked by a red rectangle in Fig. 6a, bottom). Consistent with the mouse models, clones expressing variant STAG2 were therefore underrepresented in human B lymphocytes, indicating that the STAG2 R370P variant skews the clonal composition of human blood. As a control, we analyzed polyclonal B cells derived from the blood of HG00690 with a synonymous variant (T to C substitution at F367), which does not alter the STAG2 protein sequence. Both the WT and the variant were readily detectable in cDNA (red rectangle in Fig. 6b, bottom) as well as in gDNA (red rectangle in Fig. 6b, top). This indicates that not all sequence variation in STAG2 necessarily affects the representation of variant-expressing clones in human B lymphocytes.

**Fig. 6: X-linked human genetic variation associated with skewed X chromosome usage in blood-derived polyclonal B lymphoblastoid cells.**

Discussion

X-linked genetic variation is ubiquitous in XX individuals and gives rise to intra-individual epigenetic diversity as a result of XCI and its clonal propagation. Here we report how X-linked genetic variation can alter organismal development. Stag2^variant clones were found enriched in the heart but excluded from the lymphoid compartment. Notably, and in contrast to certain X-linked disease mutations^2,12, the impact of genetic variation on lymphoid specification and differentiation was due not to an intrinsic inability of Stag2^variant clones to expand or survive. Instead, it was driven by interactions between WT and variant clones. In the absence of Stag2^WT cells—namely in hemizygous Stag2^variant males and homozygous Stag2^variant females—Stag2^variant progenitors generated normal numbers of lymphocytes.

Although Stag2 variants reduce or abolish cohesin–CTCF interactions, Stag2^variant T and B cells showed WT levels of secondary Tcra rearrangements and Igh class switch recombination, both of which are cohesin-dependent genomic processes^24,25,26. Future work will address whether cohesin–ligand interactions are dispensable for Tcra recombination and Igh class switch recombination or whether the presence of WT Stag1 compensates for variant Stag2 in these processes.

The finding that Stag2^WT cells exclude Stag2^variant clones from the lymphoid compartment is reminiscent of classical cell competition paradigms where cells are eliminated not because they have low absolute levels of fitness but rather due to fitness differentials between neighboring cells^27,28,29. Current models suggest that cell competition amplifies the impact of small fitness differentials, which can manifest in the expression of ribosomal or mitochondrial genes^27,32. Stag2^variant progenitors display deregulated gene expression, including genes related to ribosomal and mitochondrial (dys)function. While genes deregulated in Stag2^variant cells overlap gene sets implicated in cell competition^27,32, they are also highly enriched for cohesin binding. To what extent these changes are caused directly by disruption of cohesin–ligand interactions remains to be determined.

The sensing of fitness differentials in cell competition may involve dedicated receptor–ligand systems²⁹ or interactions with support systems such as epithelia²⁹ or stem cell niches^29,33,34. The outcome of cell competition is typically that loser cells die by apoptosis and do not contribute to the adult organism^27,28,29. By contrast, STAG2^variant clones contributed to adult cell types and tissues, and their contribution varied from >50% in the heart, ~50% in the brain and <50% in skeletal muscle, to essentially nil in the lymphoid system. Hence, in the scenario examined here, X-linked competition does not eliminate X-linked genetic diversity but determines how this diversity is deployed in organismal development.

Strikingly, Stag2^variant clones retained their lymphoid potential in the face of competition. Removal of Stag2 from WT clones at the hematopoietic stem and progenitor cell stage allowed Stag2^variant clones to progress through lymphoid specification and differentiation and to dominate the lymphoid compartment. Interestingly, in the same individual mice where Stag2^variant clones dominated the lymphoid compartment, Stag2^variant clones continued to be outnumbered within the hematopoietic stem and progenitor compartment and hence appeared to be on a loser trajectory. This ‘loser takes all’ behavior was unexpected, as in other forms of cell competition, loser cells are stereotypically eliminated by apoptosis^27,28,29. In our experimental setting, therefore, Stag2^WT cells were continually required to exclude X-linked variants from the lymphoid compartment.

What mechanisms might underlie X-linked competition in hematopoiesis? Stem cells need niches that provide resources such as the stem cell factor (SCF) and the chemokine CXCL12. If such niches are limiting, competition may serve as a mechanism of control³³. Indeed, leukemic stem cells may outcompete normal HSCs for niche access in the bone marrow³⁴. Of note, mRNA for the SCF receptor c-kit and the CXCL12 receptor CXCR4 was reduced in Stag2^variant progenitors (Supplementary Data 2), which—we speculate—may limit their competitiveness for niche-derived factors in the presence of Stag2^WT. Interestingly, HSCs and lymphoid progenitors may depend on distinct niches^35,36,37, which could potentially explain the difference in severity of X-linked competition among stem cells and lymphoid progenitors.

In agreement with our findings in mouse models, STAG2^variant clones were undetectable in blood-derived human B cells heterozygous for the R370P STAG2 missense variant rs777011872, suggesting that genetic variation can drive X-linked competition in humans. In support of this conclusion, female patients with mutations in STAG2 or the X-linked cohesin regulator HDAC8 typically show heavy skewing of X chromosome usage toward STAG2^WT clones in blood^{38,39,40,41,42,43,44}.

In conclusion, noncell-autonomous mechanisms shape the contribution of X-linked clonal diversity across cell types and tissues as the result of clonal interactions. As X-linked genetic variation is common in humans, clonal interactions that shape the deployment of X-linked diversity may be widespread in XX individuals.

Methods

This study complies with all relevant ethical regulations. The protocols used were approved by the Imperial College London Animal Welfare and Ethical Review Body and were performed according to the Animals (Scientific Procedures) Act under a Project License issued by the UK Home Office.

Human sequence analysis

We interrogated gnomAD (v2.2.2) for human sequence variation and used dbSNP to identify gnomAD variant X-123185062 as rs777011872. ENSEMBL data slicer (http://www.ensembl.org/Homo_sapiens/Tools/DataSlicer/Edit?db=core;tl=0ZVTRpmGovxkhbJc) was used to query position of X chromosome (chrX): 124051212–124051212 in the 1000 Genomes high coverage variants (http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/1000G_2504_high_coverage/working/20201028_3202_raw_GT_with_annot/20201028_CCDG_14151_B01_GRM_WGS_2020-08-05_chrX.recalibrated_variants.vcf.gz). A single instance of rs777011872 was found. Scanning donors with GT of 0/1 or 1/1 (that is, with an alternative allele) identified HG02885 as the donor of this variant. She is part of a trio with daughter HG02886 and husband HG02884, and neither husband nor daughter has the variant. A search of the Coriell repository (https://www.coriell.org/Search?q=HG02885) indicates the availability of DNA and LCLs for HG02885.

Isothermal calorimetry

STAG2–RAD21 complexes were isolated as described previously¹⁶. Isothermal calorimetry was performed using a MicroCal iTC 200 (Malvern Panalytical) at 25 °C. STAG21–RAD21 and CTCF peptide ligands were dialyzed overnight at 4 °C against 20 mM Tris (pH 7.7), 150 mM NaCl and 0.5 mM tris(2-carboxyethyl)phosphine. For each titration, 300 μl of 50 μM STAG2–RAD21 was added to the calorimeter cell. CTCF peptide was adjusted to a concentration of 500 μM and injected into the sample cell as 16× 2.5-μl syringe fractions. Results were analyzed and displayed using Origin 7.0 software package supplied with the instrument. Data were analyzed using the one-site binding model.

Mice

Experiments on mice were performed under a UK Home Office project license and according to the Animals (Scientific Procedures) Act. Mice carrying Stag2 variants were generated by zygotic co-injections of Cas9 mRNA (GeneArt, Invitrogen), ssDNA donor template (IDT) and tracrRNA/crRNA (IDT; see Supplementary Table 1 for guide sequences) and maintained on a mixed C57BL/129/CD1 background. The Atrx^Luc/βGal reporter allele was generated as described^21,22. Stag2^lox (Stag2tm1c(EUCOMM)Wtsi; JAX stock, 030902 (ref. ³⁰)) and VavCre (B6.Cg-Tg(VAV1-cre)1Graf/MdfJ; JAX stock, 035670 (ref. ³¹)) and OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J; JAX stock, 003831 (ref. ⁴⁵)) mice have been described.

Antibody staining, flow cytometry analysis and cell sorting

Mouse bone marrow cells were stained for lineage markers using biotinylated CD4, CD8, B220, CD19, NK1.1, CD11b, Ter119 and Gr-1 antibodies, incubated with streptavidin magnetic beads (Miltenyi Biotec, 130-048-102) and depleted using MACS LS columns (Miltenyi Biotec, 130-042-401). To analyze and sort LSKs, c-kit⁺ cells and CLPs, lineage-negative cells were stained with Sca-1-BV510 (BD Biosciences, 565507; 1:50), cKit-PE-Cy7 (Thermo Fisher Scientific, 25-1171-82; 1:100), FLT3-PE (Thermo Fisher Scientific, 12-1351-82; 1:50), CD127 (Thermo Fisher Scientific, 17-1271-82; 1:50) and streptavidin-eFluor 450 (eBioscience, 48-4317-82; 1:100). To isolate B cell progenitors, bone marrow cells were depleted of Ter119, CD11b and Gr-1 and stained with B220-FITC (BD Biosciences, 553088; 1:100), PE antimouse CD19 (BD Biosciences, 557399; 1:100), IgM-BV421 (BioLegend, 406517; 1:100) and CD43-APC (BD Biosciences, 560663; 1:100) antibodies. Mature monocytes and granulocytes were sorted from total bone marrow stained with CD11b-APC (BioLegend, 101212; 1:100) and Ly6-G-FITC (BD Biosciences, 561105; 1:100) antibodies. Thymocytes were stained with anti-CD4-BV421 (BioLegend, 100438; 1:300), CD8-APC (BioLegend, 17-0081-83; 1:300), CD25-PE (BioLegend, 102007; 1:100) and TRCβ-FITC (BD Biosciences, 553171; 1:100). Lymph node cells were stained with B220-BV421 (BioLegend, 103240; 1:100) and CD4-PE (BioLegend, 100512; 1:300) or CD4-APC (Thermo Fisher Scientific, 17-0041-83; 1:300). Cell populations were analyzed using a Fortessa Flow Cytometer (BD Biosciences) and sorted using a BD Aria Fusion or Aria III (see Supplementary Table 2 for details).

Live-cell reporter assays

Thymocytes, lymphocytes and bone marrow cells were isolated, and bone marrow was depleted of cells expressing the lineage markers CD4, CD8, B220, CD19, NK1.1, CD11b, Ter119 and GR-1. To detect βGal activity, 1 mM of nonfluorescent FDG (Thermo Fisher Scientific, F1179) substrate was delivered into the cells by hypotonic loading at 37 °C. In total, 2 × 10⁶–2 × 10⁷ cells in 100 μl PBS, 2% FBS and 10 mM HEPES (Merck, H0887) were prewarmed to 37 °C, and 100 μl of prewarned FDG solution was added to 100 μl of cells for 1 min. To stop FDG loading, samples were placed on ice, and 2 ml of ice-cold PBS, 2% FBS and 10 mM HEPES were added. Following 45-min incubation on ice, cells were stained for surface markers as described above, and the conversion of FDG into FITC was detected by flow cytometry. All experiments included cells lacking the Atrx^Luc/βGal reporter as negative controls.

Cell line culture and genetic engineering of HAP1 cells

Epstein-Barr Virus-transformed B lymphoblastoid cells (Coriell Institute for Medical Research) were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) medium supplemented with 15% foetal calf serum (FCS), 2 mM l-glutamine and 1% penicillin–streptomycin (Pen–Strep). HAP1 cells⁴⁶ were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen) supplemented with 10% FCS (Clontech), 1% Pen–Strep (Invitrogen) and 1% UltraGlutamin (Lonza). Mutant cells were generated by CRISPR–Cas9 technology. Guide RNAs were annealed into pX330. To mutate the locus of interest, we cotransfected the repair oligonucleotide with the desired mutation as well as a silent mutation (see Supplementary Table 1 for primer sequences).

T cell culture and cell proliferation assay

Round-bottom 96-well plates were coated overnight at 4 °C with purified anti-TCRβ chain clone H57 (BD Biosciences, 553167) in PBS with MgCl₂ and CaCl₂ (Sigma, D8662-1L). Thymocyte cell suspensions were incubated in the plates for 16–18 h in IMDM media (Gibco, 12440-053) with 10% FBS, 1% l-glutamine, 1% Pen–Strep, 1% sodium pyruvate, 0.1% 2-mercaptoethanol and 2 µg ml⁻¹ soluble anti-CD28 (BioLegend, 102102). Thymocyte cell proliferation was tracked using CellTrace CFSE Cell Proliferation Kit (Thermo Fisher Scientific, C34554) according to the manufacturer’s instructions after 3 days of incubation. Cells were stained with PE anti-CD4 (BioLegend, 100512; 1:300), APC anti-CD8a (Thermo Fisher Scientific, 17-0081-83; 1:300) and BV421 anti-CD69 (BD Biosciences, 562920; 1:50). Single cells were sorted using the FACSAria Fusion Flow Cytometer (BD Biosciences). Flow cytometry FCS files were analyzed with FlowJo v10 (TreeStar).

RNA extraction, reverse transcription and allele-specific qPCR

RNA was extracted from sorted cells using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturer’s instructions. Tissue samples were lysed in Trizol and homogenized using a TissueLyser II (Qiagen) and 5 mm stainless steel beads (Qiagen) for 4 min at 24,000 rpm. Tissue homogenates were extracted with chloroform. RNA isolation from tissue homogenates was performed using the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific) following the manufacturer’s instructions, with 10 µM random primers. Allele-specific qPCR assays were performed with TaqMan Fast Universal Master Mix (Thermo Fisher Scientific) and run on a CFX96 real-time PCR machine (Bio-Rad). Allele-specific primers and fluorescent TaqMan probes were used to discriminate between WT and variant alleles. Real-time PCR data were collected and analyzed using CFX Maestro 1.1 Software (Bio-Rad). Percentages of WT and variant mRNA were calculated based on the normalized ΔC_T values between amplification with WT and variant TaqMan probes (see Supplementary Table 1 for primer sequences and TaqMan probes).

Sanger sequencing

gDNA was isolated from sorted cells and tissue samples using the DNeasy Blood and Tissue Kit (Qiagen), following the manufacturer’s instructions. gDNA and cDNA were amplified by PCR followed by Sanger sequencing (see Supplementary Table 1 for primer sequences).

scRNA-seq

Bone marrow cells were depleted of lineage-positive cells, loaded with FDG as described above, and then stained with antibodies against Sca-1-BV510, cKit-PE-Cy7, FLT3-PE CD127-APC and streptavidin-eFluor 450. FITC⁺ and FITC⁻ progenitor cells were sorted and loaded on the 10X Genomics Chromium System. scRNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kits User Guide (v2 Chemistry), sequenced on a NextSeq 2000 (100 cycles; Illumina), and 10X Genomics CellRanger (v5.0.1) was used for barcode splitting, UMI (unique molecular identifier) counting and alignment to the mouse genome (GRCm38, Ensembl 107 annotations). Quality control and subsequent analysis were conducted in R using Seurat (v4.3.0.1)⁴⁷. Cells with aberrant feature counts or mitochondrial sequence fractions were discarded using data-driven filter criteria (two median absolute deviations on either side of the median values). For each sample individually, the structure was assessed using a subset of 2,000 variable genes (identified using the FindVariableFeatures function) that were used to identify the principal component analysis (PCA) dimensionality for downstream Uniform Manifold Approximation and Projection (UMAP) analysis⁴⁸. Samples were integrated using genes identified by the Seurat FindIntegrationAnchors function. Progenitors were identified using gene lists from scType⁴⁹ supplemented with markers for bone marrow progenitors (Supplementary Data 1). Differential expression analysis was conducted using DESeq2 (v1.42.0) with a threshold of adjusted P < 0.01. Gene ontology analyses were conducted using clusterProfiler (v4.10.0)⁵⁰ with a threshold of adjusted P < 0.05. Annotation of the lineage-primed clusters was performed using AUCell⁵¹ combined with manual annotation using marker genes provided in Supplementary Data 4. The observed versus expected numbers of WT and variant cells in each cluster were tested by bootstrapped permutation tests (1,000 iterations) using scProportionTest in R⁵². Classification of cell cycle stages was implemented in R using Seurat (v4.1.0)⁴⁷.

ChIP–seq and analysis of cohesin binding

STAG1 and STAG2 gene editing and chromatin immunoprecipitation using mouse anti-RAD21 (Millipore, 05-908; 10 μg per ChIP) were done as was described¹⁶. DNA was sheared using Biorupter Pico (Diagenode), five cycles of 15-s on and 90-s off. Reads were trimmed using TrimGalore (v.0.6.0)⁵³, mapped to hg19 using Bowtie 2 (v.2.3.4)⁵⁴ with default settings. Bigwig files were generated with DeepTools (v.3.1.3)⁵⁵ with the following settings: minimum mapping quality of 15, bin length of 10 bp, extending reads to 200 bp and reads per kilobase per million reads normalization. Heatmaps were generated using DeepTools on previously called RAD21 peaks¹⁶. Reads for cohesin SMC1 ChIP–seq from hematopoietic progenitors⁵⁶ (GSM3790131) were trimmed with cutadapt (https://doi.org/10.14806/ej.17.1.200) and aligned to mm10 with Bowtie 2 (ref. ⁵⁴). Duplicates were removed with Picard 2.27.5 (https://broadinstitute.github.io/picard/) and peaks called with MACS3 3.0.0b1 (ref. ⁵⁷). Promoters with SMC1 peaks <2 kb from the transcription start site were called cohesin-associated. Heatmaps were produced using the genomation toolkit⁵⁸. Odds ratios and P values were calculated using Fisher’s exact test.

Analysis of Tcra locus rearrangement and serum immunoglobulin isotypes

gDNA from sorted double-positive (DP) thymocytes was isolated using DNeasy Blood and Tissue Kits (Qiagen). Threefold serial dilutions of gDNA were amplified using a forward Vα8 primer and reverse primers for Jα61 or Jα22 as described previously²⁴. Cd14 was the genomic control (see Supplementary Table 1 for primer sequences). Concentrations of serum immunoglobulin isotypes in adult unimmunized mice were determined by enzyme-linked immunosorbent assay as advised by the manufacturers (Thermo Fisher Scientific; IgM: 88-50470-22, IgG2a: 88-50420-22, IgG2b: 88-50430-22 and IgG3: 88-50440-22).

Statistics and reproducibility

No statistical method was used to predetermine the sample size. No data were excluded from the analyses. The experiments were not randomized, as sample allocation into different groups was defined by genotype. The investigators were not blinded to allocation during experiments and outcome assessment. ChIP–seq peaks were called in MACS3, and odds ratios and P values were calculated by Fisher’s exact test. Flow cytometry statistics were done in FlowJo. Statistical analysis of differential gene expression in scRNA-seq experiments was performed by DESeq2. Statistical analysis of cell frequencies was done by bootstrapped permutation tests using scProportionTest in R⁵². Statistical analysis of allelic representation was done in Prism.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

High-throughput sequencing data generated in this study are available from the NCBI Gene Expression Omnibus (GEO) under accession GSE261622. Source data are provided with this paper.

Code availability

No custom code was generated for this study.

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Acknowledgements

We thank Z. Webster and her team for oocyte injections, J. Elliott and B. Patel for cell sorting, L. Game and I. Andrew for sequencing and N. Brockdorff (University of Oxford), P. Sarkies (University of Oxford), J. Merkenschlager (Rockefeller University), K. Small (King’s College London), H. Rehm (Harvard Medical School), J. Ware, H. Leitch, T. Rodriguez and members of our laboratories for advice and discussion. This work was supported by the Medical Research Council UK and the Wellcome Trust (investigator award 099276/Z/12/Z to M.M.).

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Authors and Affiliations

MRC LMS, Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK
Teresa Buenaventura, Hakan Bagci, Ilinca Patrascan, James W. D. King, Jesus Urtasun, George Young, Amanda G. Fisher & Matthias Merkenschlager
Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, Leicester, UK
Joshua J. Graham, Kelsey D. Hipwell & Daniel Panne
Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands
Roel Oldenkamp & Benjamin D. Rowland
Translational Bioinformatics Unit, Navarrabiomed, Universidad Pública de Navarra (UPNA), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain
Daniel Mouzo & David Gomez-Cabrero
Bioscience Program, Biological and Environmental Sciences and Engineering Division (BESE), King Abdullah University of Science and Technology KAUST, Thuwal, Saudi Arabia
David Gomez-Cabrero
Department of Biochemistry, University of Oxford, Oxford, UK
Amanda G. Fisher

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Teresa Buenaventura
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Contributions

T.B., H.B., I.P., B.R., D.P., D.G.C., A.G.F. and M.M. conceptualized the study. T.B., H.B., I.P., K.H., J.J.G. and R.O. generated data. T.B., H.B., I.P., K.H., J.J.G., R.O., J.W.D.K., J.U., G.Y., D.M., D.G.C., B.R., D.P. and M.M. analyzed and visualized data. All authors contributed to writing the manuscript.

Corresponding author

Correspondence to Matthias Merkenschlager.

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Nature Genetics thanks Elphège Nora, Panagiotis Ntziachristos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Stochastic and cell-intrinsic mechanisms can affect the representation of X-linked variation.

a, Stochastic sampling of founder cells can result in skewed X chromosome usage that varies between cell lineages and tissues. b, Deleterious variation in X-linked genes can skew X chromosome usage by cell-intrinsic failure of clones to expand or survive.

Extended Data Fig. 2 Sequence variation of the X-linked STAG2 gene.

STAG2 protein sequence alignment of human (black, sp|Q8N3U4|STAG2_HUMAN), mouse (blue, sp|O35638|STAG2_MOUSE) and sequence variation in the human population (red, gnomAD v2.1.1), excluding disease-associated variants from ClinVar or other patient databases. Alignment was performed by CLUSTAL O (1.2.4) based on Uniprot STAG2 ENSEMBL transcript ENST000003218089.9 (1231aa). gnomAD variant X-123185062—G-C (GRCh37) is highlighted. The variant has a site quality value = 3.46e+2 and genotype quality of 95–100% (allele number = 164895, allele frequency = 0.0000061 and changes STAG2 arginine 370 to proline (R370P) on one X chromosome in an XX individual).

Extended Data Fig. 3 Characterization of STAG2 variants.

a, Schematic of CTCF, STAG2 and RAD21. The regions of each protein used for in vitro binding assays are highlighted. b, Characterization of protein preparations used for isothermal calorimetry experiments. GST-CTCF pull-down. I, input; B, bound fraction; M, molecular weight marker. c, Variants in the conserved essential surface form chromatin-associated cohesin complexes. Cohesin complex formation and chromatin association of variants were tested by chromatin immunoprecipitation of the cohesin subunit RAD21 in HAP1 cells. Both STAG2 W334A and STAG1 W337A were mutated to rule out complementation of variant STAG2 by WT STAG1. Note that a moderate reduction in the association of cohesin with chromatin is expected in STAG1^W337A STAG2^W334A cells, as cohesin is no longer stabilized by CTCF interactions¹⁶.

Extended Data Fig. 4 Representation of Stag2^variant clones in heterozygous XX individuals.

a, Analysis of the representation of clones with active Stag2^wild-type versus Stag2^variant in blood mononuclear cells by Sanger sequencing in females heterozygous for Stag2^R370Q. b, Analysis as in a, but for Stag2^W334A. c, Calibration of allele-specific qRT–PCR of Stag2^wild-type and Stag2^variant cDNA. Left: ratio of Stag2^wild-type and Stag2^variant mRNA (y-axis) extracted from bone marrow progenitors containing the indicated proportions of cells (x-axis). Right: comparison of expected vs observed Stag2^variant mRNA ratios. Mean ± SD of 2 biological replicates. d, Allele-specific qRT-PCR for Stag2^WT and Stag2^variant clones in heterozygous females as shown in Fig. 2, with the exception that data for Stag2^R370Q and Stag2^W334A are displayed separately. e, Gating strategies used to isolate lymph node cells, thymocytes, and bone marrow progenitors. Expression of lineage markers on bone marrow cells (Lin SA-EF450) is shown before (dark gray) and after depletion with streptavidin beads (light gray).

Source data

Extended Data Fig. 5 Live-cell reporter assay for the representation of Stag2^wild-type and Stag2^variant clones among hematopoietic cell populations at the single-cell level.

a, Hematopoietic stem and progenitor cells. Live-cell reporter assay for the representation of Stag2^wild-type (FITC-negative) and STAG2^variant (FITC-positive) clones in hematopoietic stem (LSK) and progenitor (c-kit) cells from bone marrow. See b for details. b, Thymocyte subsets. Live-cell reporter assay for the representation of Stag2^wild-type (FITC-negative) and STAG2^variant (FITC-positive) clones in thymocyte subsets. See b for details. c, Mature lymph node T and B cells. Live-cell reporter assay for the representation of Stag2^wild-type (FITC-negative) and STAG2^variant (FITC-positive) clones in lymph node CD4 T and B cells. Top: Stag2^wild-type female lacking the Atrx^Luc/βGal reporter. Middle: Stag2^wild-type female heterozygous for Atrx^Luc/βGal. Bottom: heterozygous Stag2^wild-type Stag2^variant female with Atrx^Luc/βGal reporter allele located on the same X chromosome as the STAG2^variant allele. d, Fidelity of the Atrx^Luc/βGal reporter allele and selective expression of Stag2 alleles. Lineage-negative BM cells from female mice that were heterozygous for the Stag2 variant R370Q and the Atrx^Luc/βGal reporter allele on the same chromosome were labeled with FDG and sorted into FITC-negative and FITC-positive cells by flow cytometry. Sanger sequencing of cDNA and allele-specific qRT–PCR were performed to determine the expression of wild-type and variant Stag2. Note that FITC-negative cells expressed exclusively Stag2^wild-type, and FITC-positive cells expressed exclusively Stag2^variant. Two independent biological replicates.

Extended Data Fig. 6 Rearranged T cell receptor transgenes fail to rescue the differentiation of Stag2 variant progenitor cells.

Allele-specific qRT–PCR of Stag2^wild-type and Stag2^variant cDNA isolated from bone marrow progenitors (BM), thymocyte subsets (thymus) and CD8 lymph node T cells (LN) isolated from a heterozygous Stag2^wild-type Stag2^variant female harboring an OT-I T cell receptor transgene. One replicate (see Extended Data Fig. 4e for gating strategy).

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Extended Data Fig. 7 B cell development and myeloid cells in the bone marrow.

Allele-specific qRT–PCR of Stag2^wild-type and Stag2^variant cDNA isolated from the indicated subsets of B cells (top) and myeloid cells (bottom) isolated from Stag2^wild-type Stag2^W334A heterozygous females. Mean ± SD of 2 biological replicates per population and genotype. B cell progenitors were defined as follows: pro-B (B220^lo CD19⁺ IgM⁻ CD43⁺), pre-B (B220^lo CD19⁺ IgM⁻ CD43⁻) and immature B (B220^lo CD19⁺ IgM⁺). Granulocytes (Cd11b⁺ Ly6-G⁺). Monocytes (Cd11b⁺ Ly6-G⁻).

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Extended Data Fig. 8 Isolation of Stag2^wild-type and Stag2^variant hematopoietic progenitors and analysis of differentially expressed genes.

a, Isolation of lineage-negative c-kit⁺ bone marrow progenitors (see Extended Data Fig. 4d for the gating strategy). Lineage markers are shown before and after depletion of lineage-positive cells. FDG staining of lineage-negative bone marrow cells from heterozygous females that harbor Stag2^wild-type on one X chromosome and the Stag2^R370Q on the other, along with the Atrx^Luc/βGal reporter. b, Representative gene ontology terms ‘biological function’ of genes found upregulated (right) or downregulated (left) in Stag2^R370Q versus Stag2^wild-type hematopoietic progenitor cells isolated from Stag2^wild-type Stag2^R370Q Atrx^Luc/βGal mice. The horizontal axis displays Benjamini–Hochberg adjusted P-values and is truncated at P < 10E−30. Significance was determined by one-sided Fisher's exact test implemented in ClusterProfiler (see Supplementary Data 3 for a full list of GO terms). c, Heatmaps of cohesin binding at gene promoters in hematopoietic progenitor cells. Promoters were classified as upregulated, downregulated or not deregulated according to the status of the associated transcripts in Stag2^variant versus Stag2^wild-type progenitors. Odds ratios and P-values were calculated by two-sided Fisher’s exact test. The nominal values for P-values given as P < 2.2 10e-16 are 1.05e-192 for upregulated vs non-DE and 1.73e-149 for downregulated vs non-DE.

Extended Data Fig. 9 Stag2^variant lymphocytes are competent to undergo secondary Tcra rearrangements, Igh class switch recombination and in vitro activation.

a, Threefold dilutions of genomic Vα8-Jα PCR products obtained from DP thymocytes sorted from Stag2^variant compared to Stag2^wild-type males. Cd14 was used as a genomic control. One of three similar biological replicates. b, Concentrations of the indicated immunoglobulin isotypes were determined by enzyme-linked immunosorbent assay in the sera of unimmunized adult Stag2^wild-type and Stag2^variant males. Four independent biological replicates were analyzed per genotype. P-values were determined by unpaired two-tailed t-test. c, Lymph node cells were activated using plate-bound H57 anti-TCRβ antibodies at the indicated concentrations, together with 2 μg/ml soluble anti-CD28. Left: CD69 expression was assessed by flow cytometry after 1 day of activation. Middle: the fraction of cells that completed the indicated number of cell divisions as determined by flow cytometric assessment of CFSE dilution. Right: representative CFSE traces. Mean ± SEM of 3 biological replicates per genotype. d, Gating strategy used in c.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Data 1

Marker genes for the annotation of multipotent and lineage-restricted progenitors.

Supplementary Data 2

Differentially expressed genes in multipotent and lineage-restricted progenitors.

Supplementary Data 3

Gene ontology terms enriched among upregulated and downregulated genes in merged progenitors.

Supplementary Data 4

Marker genes for the annotation of lineage-primed progenitors.

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Source Data Extended Data Fig. 9a

Unprocessed and processed gel.

Source Data Extended Data Fig. 9b,c

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Buenaventura, T., Bagci, H., Patrascan, I. et al. Competition shapes the landscape of X-chromosome-linked genetic diversity. Nat Genet 56, 1678–1688 (2024). https://doi.org/10.1038/s41588-024-01840-5

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Received: 21 September 2023
Accepted: 21 June 2024
Published: 26 July 2024
Issue Date: August 2024
DOI: https://doi.org/10.1038/s41588-024-01840-5

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Abstract

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Main

Results

Sequence variation and XCI combine to generate intra-individual genetic diversity

Sequence variants in the X-linked STAG2 gene disrupt cohesin–CTCF binding

Stag2 variant progenitors fail to form lymphocytes in heterozygous XX individuals

Reduced lymphoid priming in Stag2 variant hematopoietic progenitors

Competition between Stag2 variant and Stag2 WT clones

Stag2 variant progenitors retain lymphoid potential in the face of competition

X-linked competition in humans

Discussion

Methods

Human sequence analysis

Isothermal calorimetry

Mice

Antibody staining, flow cytometry analysis and cell sorting

Live-cell reporter assays

Cell line culture and genetic engineering of HAP1 cells

T cell culture and cell proliferation assay

RNA extraction, reverse transcription and allele-specific qPCR

Sanger sequencing

scRNA-seq

ChIP–seq and analysis of cohesin binding

Analysis of Tcra locus rearrangement and serum immunoglobulin isotypes

Statistics and reproducibility

Reporting summary

Data availability

Code availability

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Stag2 ^variant progenitors fail to form lymphocytes in heterozygous XX individuals

Reduced lymphoid priming in Stag2 ^variant hematopoietic progenitors

Competition between Stag2 ^variant and Stag2 ^WT clones

Stag2 ^variant progenitors retain lymphoid potential in the face of competition