# FOXP1 contributes to murine hematopoietic stem cell functionality

Françoise Levavasseur, Samia Oussous, Alessandro Framarini, Ismael Boussaid, Panhong Gou, Zubaidan Tuerdi, Iman Litchy Boueya, Helyette Hoffner, Marta de Almeida, Morgane Le Gall, et al.

► To cite this version:

Françoise Levavasseur, Samia Oussous, Alessandro Framarini, Ismael Boussaid, Panhong Gou, et al. FOXP1 contributes to murine hematopoietic stem cell functionality. Experimental Hematology, 2025, 149, pp.104815. 10.1016/j.exphem.2025.104815. hal-05125883

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# FOXP1 contributes to murine hematopoietic stem cell functionality

Françoise Levavasseur<sup>a, *, *</sup>, Samia Oussous<sup>a, *, *</sup>, Alessandro Framarini<sup>a, *, *</sup>, Ismael Boussaid<sup>a, b</sup>, Panhong Gou<sup>c, d</sup>, Zubaidan Tuerdi<sup>a</sup>, Iman Litchy Boueya<sup>c</sup>, Helyette Hoffner<sup>a</sup>, Marta De Almeida<sup>a</sup>, Morgane Le Gall<sup>f</sup>, Haley Tucker<sup>g</sup>, Stéphane Giraudier<sup>c, d</sup>, Didier Bouscary<sup>a, b</sup>, Michaela Fontenay<sup>a, b</sup>, Diana Passaro<sup>c</sup>, Isabelle Dusanter-Fourt<sup>a, #</sup>, and Evelyne Lauret<sup>a, # #</sup>

<sup>a</sup>Normal and Pathological Hematopoiesis laboratory, Université Paris Cité, Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche médicale (INSERM), Institut Cochin, Paris, France; <sup>b</sup>Assistance Publique–Hôpitaux de Paris. Centre-Université Paris Cité, Hôpital Cochin, Laboratory of Hematology, Paris, France; <sup>c</sup>Institut National de la Santé et de la Recherche médicale (INSERM) Unité mixte de recherche (UMR)-S 1131, Hôpital Saint-Louis, Paris, France. Université de Paris Cité, Paris, France; <sup>d</sup>Service de Biologie Cellulaire, Hôpital Saint Louis, Assistance Publique-Hopitaux de Paris, Paris, France; <sup>e</sup>Leukemia and Niche Dynamics laboratory, Institut Cochin, Université Paris Cité Unité mixte de recherche (UMR)-S1016, Institut National de la Santé et de la Recherche médicale (INSERM) U1016, CNRS UMR8104, Paris, France; <sup>f</sup>Plateforme Proteom'IC, Université Paris Cité, CNRS, Institut National de la Santé et de la Recherche médicale (INSERM), Institut Cochin, Paris, France; <sup>g</sup>Departments of Molecular Biosciences, The University of Texas at Austin, Austin, TX

Transcription factor forkhead box P1 (FOXP1) is a key regulator of immune cell functions. We have shown that FOXP1 contributes to the expansion of human hematopoietic stem/progenitor cell (HSPC) and acute myeloid leukemia cells. Here, we investigated the role of FOXP1 in early adult mouse hematopoiesis in vivo. We showed that loss of hematopoietic-specific FOXP1 expression leads to attrition of the hematopoietic stem cell (HSC) and multipotent progenitor (MPP)-1 compartment in parallel with enhancement of myeloid-biased MPP3 in adult bone marrow and fetal liver. Transplantation experiments confirmed that FOXP1-deficient bone marrow had an intrinsic reduced HSC compartment. FOXP1-deficient MPP compartments also showed enhanced proliferation with G0 phase reduction. Transcriptome analyses revealed that FOXP1-deficient HSC exhibited reduced stemness and enhanced expression of cell proliferation pathways. Thus, our current results revealed that FOXP1 plays a critical role in early murine hematopoiesis by maintaining HSCs, limiting the expansion of all MPP compartments, and restricting early myeloid commitment in vivo. © 2025 The Author(s). Published by Elsevier Inc. on behalf of International Society for Experimental Hematology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

## HIGHLIGHTS

* FOXP1 maintains hematopoietic stem cell (HSC) pool.

* FOXP1 limits HSC and multipotent progenitor proliferation.

* FOXP1 restrains myeloid commitment.

Cells of the blood system provide oxygen-carrying capacity, hemostasis, and innate and adaptive immunity. In the adult, these mature effector cells are generated from rare quiescent hematopoietic stem cells (HSCs) that reside in the bone marrow (BM), where they sit at the top of the hematopoietic hierarchy and self-renew for the lifetime of the organism. To maintain blood cell production, HSCs differentiate into multipotent progenitors (MPPs), which despite their limited

self-renewal potential, are the true workhorses of the hematopoietic system. They proliferate and differentiate through an amplifying cascade of increasingly committed progenitors, ultimately resulting in all mature blood cell types [1–3]. Elucidating the molecular regulation of how HSCs and multipotent cells engage in every mature blood lineage informs normal stem cell biology and how these cells become dysregulated in pathological conditions.

Hematopoietic stem and progenitor cells' (HSPCs) self-renewal and fate are tightly controlled by both intrinsic molecular pathways and extrinsic signals from the microenvironment. We identified the transcription factor forkhead box P1 (FOXP1) as a novel essential regulator of both human HSPC maintenance and human acute myeloid leukemia (AML) cell growth and survival [4]. FOXP1 belongs to the forkhead box (FOX) evolutionarily conserved family of transcription factors involved in critical functions during development and

<footnote>∗FL, SO, and AF are co-first authors.</footnote>

<footnote>#IDF and EL co-senior authors.</footnote>

<footnote>Addresses correspondence to Evelyne Lauret, Normal and Pathological Hematopoiesis laboratory, Université Paris Cité, CNRS, INSERM, Institut Cochin, F-75014 Paris, France; E-mail: evelyne.lauret@inserm.fr</footnote>

<footnote>0301-472X/© 2025 The Author(s). Published by Elsevier Inc. on behalf of International Society for Experimental Hematology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)</footnote>

<footnote>https://doi.org/10.1016/j.exphem.2025.104815</footnote>
adulthood [5,6]. FOXO family factors that are members of the same family, are essential for the maintenance and function of HSPC pool, as demonstrated in murine models [7–9]. Conditional deletion of FOXO1/3/4 or deletion of FOXO3a alone in murine hematopoiesis compromised the lineage-c-Kit+Sca1+ (LSK) cell maintenance and their long-term repopulation capacity [7, 10, 11].

FOXP1 is known to play important roles in immune responses, development, and tumor growth. It regulates monocyte-derived macrophage differentiation [12], multiple stages of B cell development, quiescence, and activation of T cells [13–15]. More recently, the involvement of FOXP1 in murine FOXP3⁺ regulatory T (T<sub>Reg</sub>) cell production was emphasized by two studies showing a reduction of FOXP3 DNA binding activity in the absence of FOXP1, leading to attrition of BM T<sub>Reg</sub> cells and an impaired function [16,17]. Besides, FOXP1 controls multiple cell-differentiation pathways, including embryonic stem cell pluripotency [18], hair follicle stem cell quiescence [19], mesenchymal stem cell commitment [20], adipocyte differentiation [21], lung epithelial cell-fate determination [22,23], cardiomyocyte proliferation [24,25] and neuronal activity [26,27]. However, the role of FOXP1 in cancer progression remains unclear because of its dual function as tumor suppressor and oncogene [5].

Given the above data, we aimed to delineate the functional role of FOXP1 in early hematopoiesis in vivo, through a conditional deficiency of FOXP1 in murine hematopoietic cells using the pan-hematopoietic vav-iCRE transgene. Overall, our results identify FOXP1 as an important player in HSC maintenance, by both inhibiting HSC/MPP proliferation and limiting early myeloid commitment.

## EXPERIMENTAL PROCEDURES

Detailed Materials and Methods are presented in Supplementary file.

## RESULTS

### FOXP1 Deficiency Impaired Adult Medullar Hematopoiesis at Steady State

FOXP1-null mice die during mid-term embryogenesis (at embryonic day 14.5) because of heart valve and outflow tract anomalies [24]. Therefore, to investigate the role of FOXP1 in adult mouse hematopoiesis, we developed a conditional knockout (KO) mouse model by crossing *Foxp1*-floxed mice (*Foxp1tm1.1Pwt*, Jackson Laboratory) [13] (*Figure 1A* and *Supplementary Figure E1A*) to Vav1-iCre mice [28], which constitutively express the codon-improved Cre driven by Vav1 regulatory elements (*Foxp1*-vKO). iCRE is expressed from mid-gestation in HSC and thereafter in their progeny, resulting in HSC-gene deletion at mid-gestation. Deletion of exons 11–12 in *Foxp1*-vKO cells, as confirmed using polymerase chain reaction (PCR) analysis (*Supplementary Figure E1B*) [13], led to an efficient reduction in FOXP1 protein expression in the lineage-negative (Lin⁻) cells isolated from *Foxp1*-vKO BM of 8-week-old mice (*Supplementary Figure E1C*). *Foxp1*-vKO mice displayed normal Mendelian ratios at birth (*Supplementary Figure E1D*), indicating that Vav1-dependent *Foxp1* deletion in hematopoietic cells did not induce a damaging phenotype during early mouse development.

To explore the role of FOXP1 in adult hematopoiesis, we first analyzed the blood hematologic parameters at steady state in 8–12-week-old mice. A decreased number of white blood cells (WBCs) was evidenced in *Foxp1*-vKO mice, primarily due to a decrease in the

number of B lymphocytes (*Supplementary Figure E1E*), in agreement with the reported key role of FOXP1 in B cell differentiation [15,29,30]. *Foxp1*-vKO mice also displayed a reduction in the number of basophils (*Supplementary Figure E1F*). Regarding other blood cell types, *Foxp1*-vKO had no impact on the number of red blood cells (RBCs), platelets, neutrophils, eosinophils, or monocytes (*Supplementary Figure E1G*). We noticed that heterozygous mice displayed the same phenotype as wild-type (WT) mice (*Supplementary Figure E1E–G*).

We next assessed the consequences of *Foxp1*-vKO deficiency on hematopoietic cells in the BM. *Foxp1*-vKO had no impact on the total number of BM mononuclear cells (*Supplementary Figure E1H*). We then examined the BM HSPC compartments using the gating strategy defined by Cabezas-Wallscheid et al. [31] in 2014, which is illustrated in *Figure 1B* [32]. In the BM of *Foxp1*-vKO mice (*Figure 1C*), we observed a twofold decrease in the number of LSK CD150⁺ CD48⁻ (HSC-MPP1), which we subsequently divided into HSC (LSK CD150⁺ CD48⁻ CD34⁻) and MPP1 (LSK CD150⁺ CD48⁻ CD34⁺). Both compartments were diminished in the BM of *Foxp1*-vKO mice. The reduced number of BM HSC-MPP1 was associated with a twofold increase in the number of MPP3 (LSK CD150⁻ CD48⁺ CD135⁻). The other MPP compartments (MPP2, LSK CD150⁺ CD48⁺, and MMP4, LSK CD150⁻ CD48⁻ CD135⁺) did not change. Again, MPP repartition in heterozygous mice was similar to the WT mice (*Figure 1C*). Neither the number of more committed progenitors, granulocyte-macrophagic progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), nor common lymphoid progenitors (CLPs) changed following *Foxp1* gene disruption in BM cells (*Supplementary Figure E1I*). We did find a reduction in the number of colony-forming cells (CFCs) in the BM of *Foxp1*-vKO mice, mostly due to a slight reduction in the number of granulocyte-macrophage colony-forming cells (GM-CFCs). We observed no modification in burst-forming unit-erythroid (BFU-E) and mixed colonies (*Figure 1D*). Together these data indicate that hematopoiesis of *Foxp1*-vKO mice displayed a reduction in size of the most immature HSC and MPP1 compartments and an expansion of the myeloid-biased MPP3 compartment, together with minor alterations of GM progenitors in colony assay within the BM at steady state. For the rest of the study, because the HSC and MPP1 compartments behave in the same way, we only considered the overall HSC-MPP1 population.

### FOXP1 Deficiency Perturbed Steady-State Splenic Hematopoiesis

We then expanded our analysis to the spleen as a secondary hematopoietic organ in mice [33] and compared spleen characteristics of *Foxp1*-vKO mice to those of WT mice. We first noticed that spleens of *Foxp1*-vKO mice tended to have lower weights (*Supplementary Figure E2A*), likely because of the loss of B cells compared with WT (*Supplementary Figure E2B*), consistent with the previously demonstrated negative impact of FOXP1 disruption on B cells [15]. Regarding the immature cell populations, we observed an increase in the size of the LSK compartment in the spleen of *Foxp1*-vKO mice, mainly due to an increase in the number of MPP3, without any significant change in the other MPP compartments (*Figure 2A*). With respect to more committed compartments, we observed an increased number of splenic GMP without a significant change in the number of MEP (*Figure 2B*). Analysis of the clonogenic potential of *Foxp1*-
<figure><img src="image_3.png" /><figcaption>**Figure 1** FOXP1 deficiency perturbs steady-state adult bone marrow (BM) hematopoiesis. (A) Schematic of the *FoxP1* deletion in the embryo and hematopoietic analysis strategy in fetal liver (FL) and adult blood, BM, and spleen. (B) Cytometric strategy to assess the different BM hematopoietic compartments in alive Zombie-Lin- cells. (C) The total number of BM HSC-MPP1, HSC, MPP1, MPP2, MPP3, and MPP4 per two legs from wild-type (WT), Het, and *Foxp1-vKO*. *n* = 8–12 mice per genotype. (D) Number of CFC (GM-CFC, BFU-E, and mixed colonies) per 10⁴ BM mononucleated cells (BMNCs). *n* = 4–10 mice per genotype. White bars (WT), gray bars (Het), and red bars (vKO). *n* = 4–10 mice per genotype. Data are presented as mean ± SEM. Each point represents an individual</figcaption></figure>
vKO mice progenitors revealed an enhanced number of GM-CFC at the expense of BFU-E (Figure 2C). Thus, splenic hematopoiesis of *Foxp1*-vKO mice revealed a myeloid bias at the expense of erythroid compartment.

Collectively, our results in BM and spleen indicate that hematopoie-
sis of Foxp1-vKO mice exhibits a myeloid bias of MPPs, suggesting
that FOXP1 restricts expansion of myeloid MPP3 in BM and spleen,
of committed GMP and GM-CFC in spleen and, on the contrary,
supports the expansion of splenic BFU-E.

FOXP1 Deficiency Perturbed Fetal Hematopoiesis

We then questioned whether the alterations observed in adult hema-
topoiesis were already present before birth (Figure 1A). The fetal liver
(FL) is the main hematopoietic organ between E12.5 and P0, a critical
period characterized by expansion/differentiation of the pool of FL
progenitors [34]. A comparison of *Foxp1*-vKO and WT FL at E14.5
showed that they both displayed a similar number of mononucleated
cells (MNCs) (Supplementary Figure E2D). We next analyzed the
size of HSC/MPP compartments in *Foxp1*-vKO and WT mice
employing a gating strategy similar to the previous one with slight
modifications regarding the Lin<sup>-</sup> cocktail [35] (Supplementary Figure
E2C). *Foxp1*-vKO FL displayed a profile similar to the one observed
in the adult BM; namely, an enhanced number of MPP3 at the
expense of the HSC-MPP1 compartment. We noticed in the FL of
*Foxp1*-vKO mice an enlarged MPP4 compartment (Figure 2D). Yet
the GMP and MEP compartments were unmodified in *Foxp1*-vKO
FL (Supplementary Figure E2E).

These data, in line with those obtained in adult BM, indicated that
FOXP1 deficiency affects the most immature progenitors already
present in FL; they revealed that FOXP1 contributes to the establish-
ment of the HSC-MPP1 compartment at very early stages of devel-
opmental hematopoiesis.

FOXP1-deficient BM Cells Displayed a Reduced Repopulation
Capacity

The alterations in the HSC and MPP compartments of *Foxp1*-vKO mice could be due to changes in the niche. Vav-Cre mice strains have been shown to target hematopoietic and endothelial cells, with occasional Cre recombinase activity also reported either in the testes or the ovaries [36]. Moreover, FoxP1 deficiency led to a specific reduction of T<sub>REG</sub> number in the BM [16,17]. Interestingly, a reduced number of BM T<sub>REG</sub> cells was associated with an increase in LSK and HSC-MPP1 compartment size [37,38]. On the contrary, we observed a decrease of immature compartments in *Foxp1*-vKO mice, thus invalidating a role for T<sub>REG</sub> cells in the *Foxp1*-vKO niche and suggesting that FOXP1 deletion may affect HSC-MPP1 compartment intrinsically. To further investigate this hypothesis, we assessed the long-term in vivo repopulating capacity of *Foxp1*-vKO versus WT HSC in WT mice in competition with WT cells as described in Supplementary Figure E3A. Using congenic mouse strains with the CD45.1 or CD45.2 variants, we transplanted ~10<sup>6</sup> BM MNCs from *Foxp1*-vKO (or WT for control) mice expressing CD45.2 together

with ~10⁶ BM MNCs from a WT mouse with the CD45.1 cell sur-
face variant into CD45.1⁺ irradiated mice. Four months later, ~10⁶
BM MNCs from the primary recipient were injected into secondary
irradiated CD45.1⁺ recipient mice. As shown in *Supplementary
Figure E3B*, blood parameters of the primary recipients showed a
decrease in the number of lymphocytes (as expected) [13–15]
and circulating monocytes from their *Foxp1*-vKO BM grafts,
whereas neutrophils were equally represented. Regarding the
BM, the Lin⁻ cell compartment from *Foxp1*-vKO BM cells was sig-
nificantly decreased in primary recipients compared with WT,
mostly due to a decrease in the Sca-1⁻/c-Kit⁻ (14% ± 2% vs.
35% ± 5% among engrafted cells, *Foxp1*-vKO vs. WT) and the
Sca1⁺/c-kit⁻ (0.4% ± −0.1% vs. 7% ± 1% among engrafted
cells, *Foxp1*-vKO vs. WT) populations. The MPP2 compartment
was also affected (Figure 3A). In secondary recipients, blood
parameters were all reduced (Supplementary Figure E3B); BM
Lin⁻, LK, and LSK compartments were decreased in mice
engrafted with *Foxp1*-vKO BM; the HSC-MPP1 compartment
was similarly decreased (Figure 3B).

These data indicated that FOXP1 intrinsically contributes to the
maintenance of the HSC-MPP1 compartment.

FOXP1 Deficiency Accelerated the Reconstitution of
Hematopoiesis Following 5-Fluorouracil Treatment

To delineate FOXP1's impact on HSC functionality in vivo, we inves-
tigate the effects of FOXP1 deletion on hematopoietic reconstitution.
We administered to *Foxp1*-vKO and WT mice one single injection of
5-fluorouracil (5-FU) dose [39] (Figure 4A). 5-FU kills proliferating
cells, including progenitors, and thus stimulates HSCs to proliferate
and replenish the hematopoietic system. After 7 days, an ~15-fold
reduction in the number of BM MNCs was observed in WT mice
(Supplementary Figure E1H). Surprisingly, an ~100-fold reduction in
BM cells was observed in *Foxp1*-vKO mice (Figure 4B), attesting to a
far greater sensitivity of *Foxp1*-vKO cells to 5-FU. This reduction was
accompanied by a decrease in the number of LK cells. In contrast, at
day 10, rapid recovery of LSK cells in the *Foxp1*-vKO population was
observed: i.e., ~ 7–8 times more LSK cells within the BM of *Foxp1*-
deficient mice compared with WT mice.

The higher sensitivity of *Foxp1*-vKO cells to 5-FU, together with the
enhanced recovery potential of *Foxp1*-vKO LSK compared with WT
LSK, strongly suggests that FOXP1 significantly restricts LSK prolifera-
tion.

FOXP1 Impairs Cell Cycling of HSC/MPP

To decipher the underlying mechanism(s) leading to HSC-MPP1
decrease and MPP3 increase in *Foxp1*-vKO mice, we examined apo-
ptosis in all compartments using Annexin V labeling. Our results pre-
sented in Supplementary Figure E4A revealed no significant
modification in the rate of apoptosis of HSC-MPP1-4 and GMP/
MEP compartments between *Foxp1*-vKO and WT mice. Our findings
indicated that apoptosis was not the major mechanism underlying
changes in the size of hematopoietic compartments in absence of

mouse. Data were analyzed using one-way analysis variance (ANOVA) (C, D). \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001. BFU-E=Burst-forming unit-erythroid; CFC=Colony-forming cell; HSC=Hematopoietic stem cell; GM=Granulocyte-macrophage; KO=Knockout; MPP=Multipotent progenitor.
<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

**Figure 2** FOXP1 deficiency perturbs adult splenic and fetal hematopoiesis. (A) The number of splenic LSK, HSC-MPP1, MPP2, MPP3, and MPP4 from wild-type (WT) and *Foxp1-vKO* mice. *n* = 5–10 mice per genotype. (B) The number of splenic GMP and MEP from WT and *Foxp1-vKO* mice. *n* = 5–10 mice per genotype. (C) The number of GM-CFC and BFU-E per 10⁵ spleen cells. (D) The number of fetal liver HSC-MPP1, MMP2, MPP3, and MPP4 from WT and *Foxp1-vKO* mice. White bars WT and red bars *Foxp1-vKO*. *n* = 6–8 mice per genotype. Data are presented as mean ± SEM. Each point represents an individual mouse. Data were analyzed using the Student *t* test. \* *p* < 0.05, \*\* *p* < 0.01. *BFU-E=Burst-forming unit-erythroid; CFC=Colony-forming cell; HSC=Hematopoietic stem cell; GM=Granulocyte-macrophage; GMP=Granulocyte-macrophage progenitor; KO=Knockout; LSK=Lineage-c-Kit+Sca1+; MEP=Megakaryocyte/erythrocyte progenitor; MPP=Multipotent progenitor.*
A

Primary recipient

<figure><img src="image_8.png" /><figcaption>**Figure 3** FOXP1 deficiency compromises the intrinsic long-term hematopoietic reconstitution. (A) The number of depicted donor-derived cells in 10⁷ bone marrow (BM) cells (left) and % of donor cells (right) in BM of primary recipient mice (n = 4–5 mice per genotype). (B) The number of depicted donor-derived cells in 10⁷ BM cells (left) and % of donor cells (right) in BM of secondary recipient mice (n = 3 mice per genotype). Each point represents an individual mouse. Data are mean ± SEM and were analyzed using the Student t test. \*p < 0.05, \*\*p < 0.01.</figcaption></figure>

FOXP1 in adult mice. In contrast, assessment of in vivo proliferation via BrdU incorporation analysis demonstrated a significant enhancement of cycling cells in *Foxp1-vKO* LSK cells relative to WT in all compartments, including HSC-MPP1, MPP2, MPP3, and MPP4 (Figure 5A). Proliferation rates of the more committed progenitors (GMP and MEP) were not significantly modified (Supplementary Figure E4B). Hoechst/Ki-67 analysis showed a lower proportion of cells in G0 phase in HSC-MPP1 and MPP3 compartments of *Foxp1-vKO* relative to WT mice (Figure 5B and Supplementary Figure E4C), further revealing that FOXP1 restrains MPP proliferation through a significant increase in the G0 stage. The overall results

show that FOXP1 displays the novel capacity to restrain proliferation of HSC and MPP compartments.

## FOXP1 Deficiency Altered Gene Expression Profiles of MPP Compartments

To identify potential molecular mediators of FOXP1 activities, we performed genome-wide expression profiling on sorted WT and *Foxp1-vKO* HSC-MPP1 and MPP3 populations at steady state (GSE268789). Gene set enrichment analysis (GSEA) performed on HSC-MPP1 compartment revealed the repression of genes involved
<figure><img src="image_9.png" /><figcaption>Figure 3 Continued.</figcaption></figure>

in the "Ivanova Hematopoiesis Stem Cell Long-Term" pathway, such as *Vdr*, *Prdm16*, and *Hlf*, known to regulate HSC functions [40–42] (Figure 6B). Among the 50 upregulated pathways in *Foxp1*-vKO HSC-MPP1, we noticed pathways specific to hematopoietic differentiation (7/50), such as *IVANOVA_HEMATOPOIESIS_MATURE_-CELL, and others involved in mitochondria/oxidative phosphorylation (18/50), cell cycle (16/50), and ribosome/translation (5/10) (Figure 6A, C, and Table 1). Among dysregulated genes, the volcano plot (Figure 6D) revealed the upregulation of *KDR* and *OsmR*, as well as *MeiE2* [43] (involved in HSC proliferation) and *Neo1* (associated with myeloid-biased HSC) [44]. Their expression was validated using reverse transcription-quantitative PCR (RT-qPCR) analysis (Figure 6E). Conversely, we noticed the downregulation of numerous pathways involved in immune functions (27/50) in *Foxp1*-vKO HSC/MPP1 (Figure 6A, F, and Table 1).

GSEA analysis performed on the MPP3 populations revealed that most of the upregulated gene sets in *Foxp1*-vKO cells were related to cell cycle and DNA repair (27/50) (Supplementary Figure E5A, B, and Table 2). Among the upregulated genes, we validated using RT-qPCR *GFRA2*, *Kdr*, and *N-myc* as markers of proliferative MPP [45,46], and *HoxB5* as an HSC stress marker [47] (Supplementary Figure E5C–E). Worth noting, as previously observed with HSC-MPP1 compartment data sets, numerous pathways associated with immune functions (15/50) were also downregulated in *Foxp1*-deficient MPP3, with a few transcripts validated using RT-qPCR analysis, namely *Ctsf* and *Ndrg2* (Supplementary Figure E5C and Table 2).

Taken together, these transcriptome analyses identified *FOXP1* as a new member of the regulatory gene network that dictates HSC functionality: *FOXP1* regulates major HSC-gene players, restricts
A

<figure><img src="image_10.png" /></figure>

B

<figure><img src="image_11.png" /></figure>

**Figure 4** FOXP1-deficient bone marrow (BM) cells display a different sensitivity to drug stress. (A) The schematic diagram of 5-fluorouracil (5-FU) treatment (one single injection of 150 mg/kg). Mice were sacrificed, and BM was harvested at 7 or 10 days. (B) The number of BM cells (BMCs), Lin⁻-c-kit⁺ (LK), and Lineage⁻c-Kit⁺Sca1⁺ (LSK) cells/two legs is 7 or 10 days after 5-FU injection. Each point represents an individual mouse (n = 7–8 mice per genotype). Data are presented as mean ± SEM and were analyzed using the Student t test. \*p < 0.05, \*\*p < 0.001, \*\*\*p < 0.0001. ns=not significant.
<figure><img src="image_12.png" /><figcaption>**Figure 5** FOXP1 deficiency promotes hematopoietic stem cell (HSC) and multipotent progenitor (MPP) proliferation. (A) Cell cycle analysis of different bone marrow (BM) progenitor compartments (Lineage-c-Kit+Sca1+ [LSK], HSC-MPP1, MPP2, MPP3, MPP4) through BrdU incorporation of wild-type [WT] and *Foxp1-vKO* mice (*n*=5 mice per genotype). (B) Cell cycle analysis of HSC-MPP1 and MPP3 cells through Hoechst/Ki67 labeling in WT and *Foxp1-vKO* mice (*n* = 6 mice per genotype). Frequencies of cell cycle distribution are shown. Each point represents an individual mouse. Data are presented as mean ± SEM and were analyzed using the Student *t* test. \*p* < 0.05, \*\*p* < 0.01. KO=Knockout.</figcaption></figure>

very early progenitor proliferation, and limits early myeloid commitment.

DISCUSSION

In this study, we investigated the role of FOXP1 in early hematopoie-
sis by developing a mouse model in which Foxp1 was depleted by
Vav-1-iCre-mediated deletion, which targets the very earliest HSPCs.
Here, we identified novel features associated with FOXP1 deletion in
early hematopoiesis. These mice were characterized by attrition of
the BM HSC-MPP1 compartment, in parallel with an expansion of
the BM and spleen myeloid-biased MPP3 compartment. These prop-
erties of the HSC/MPP compartments were already effective in the
FL. We performed transplantation experiments to disprove the
hypothesis that FOXP1 has an indirect niche effect (such as T<sub>REG</sub> cell
activity or inappropriate iCRE expression in niche cells) and to assess
the specific effects of FOXP1 deletion in HSCs. Our reconstitution
experiments confirmed the cell-intrinsic role of FOXP1 in maintain-
ing HSC pool, independent of microenvironmental interactions.

Further experiments are required to more precisely assess the self-
renewal properties of FOXP1 KO HSC by injecting the same num-
ber of HSC (WT or FoxP1 KO) into two groups of primary recipients.

Another conclusion of this study is that FOXP1 regulates the main-
tenance of the murine HSC-MPP1 compartment through restriction
of their proliferation/entry into cell cycle. In fact, FOXP1 deletion led
to an enhanced proliferation of all MPP compartments via a marked
decrease in quiescence for HSCs and MPP3. These proliferative char-
acteristics were particularly evidenced upon 5-FU treatment of
FOXP1-vKO mice, which induces leucopenia by enhancing the flux
from HSCs to MPPs. FOXP1-deficient cells are dramatically sensitive
to 5-FU treatment at day 7 because of their high proliferative rate. In
contrast, at day 10, LSK replenishment was more effective in FOXP1
deficient mice, as compared with WT mice in agreement with a
higher proliferation. Taken together, these data indicate that FOXP1
intrinsically restrains HSC and MPP proliferation.

Attrition of the HSC-MPP1 compartment in *Foxp1*-vKO mice
might result from this enhanced proliferation, which then leads to a
reduction of this upstream compartment. Gene enrichment analysis
performed on these populations revealed downregulation of genes
<figure><img src="image_13.png" /></figure>

**Figure 6** FOXP1 deficiency changes the gene expression profiles. (A) Volcano plot representation of fast gene set enrichment analysis (fGSEA) performed with the complete Mouse MSigDB Collection in *Foxp1*-vKO vs. wild-type (WT) HSC-MPP1 cells. (B) GSEA plot related to "IVANOVA HEMATOPOIESIS STEM CELL LONG TERM" from MSigDB c2.cgp.v7.4 shows a significant enrichment in the WT phenotype. Heatmap showing expression of "IVANOVA HEMATOPOIESIS STEM CELL LONG TERM" molecular signature in WT and Foxp1-deficient HSC-MPP1. Genes are ordered depending on a hierarchical clustering. (C) GSEA plots related to "IVANOVA HEMATOPOIESIS MATURE CELL," "OXIDATIVE PHOSPHORYLATION," "REACTOME MITOTIC METAPHASE AND ANAPHASE," and "LARGE RIBOSOMAL SUBUNIT." (D) Volcano plot depicting DEGs between *Foxp1*-vKO and WT HSC-MPP1 cells. (E) Relative expression levels of Neo1, MeiE2, Osm, Kdr, and Ccl4 in WT and *Foxp1*-vKO HSC-MPP1 BM, as measured using real-time polymerase chain reaction (PCR). messenger RNA (mRNA) levels were normalized to ACTR8, CERE2, and RTCB (n = 6-7 mice per genotype). Each point represents an individual mouse. (F) GSEA plots related to "ALPHA BETA T CELL ACTIVATION" and "LARGE PRE BII LYMPHOCYTE DN." Data are presented as mean ± SEM and were analyzed using the Student t test. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001. DEGs=Differential expressed gene; HSC=Hematopoietic stem cell; MPP=Multipotent progenitor.
D

HSC-MPP1

<figure><img src="image_14.png" /></figure>

E

Neo1

<figure><img src="image_15.png" /></figure>

Meis2

<figure><img src="image_16.png" /></figure>

OsmR

<figure><img src="image_17.png" /></figure>

Kdr

<figure><img src="image_18.png" /></figure>

Ccl4

<figure><img src="image_19.png" /></figure>

F

Enrichment plot:
GOBP ALPHA BETA T CELL ACTIVATION

<figure><img src="image_20.png" /></figure>

Enrichment plot:
MORI_LARGE_PRE_BII_LYMPHOCYTE_DN

<figure><img src="image_21.png" /><figcaption>Figure 6 Continued.</figcaption></figure>
<table><caption>Table 1 List of the 50 most down- or upregulated pathways in Foxp1-vKO HSC-MPP1 vs. WT.</caption><thead><tr><th>Downregulated pathways in Foxp1-vKO HSC-MPP1</th><th>NES</th><th>log10padj</th></tr></thead><tbody><tr><td>ZEMEK Immune Checkpoint Blockade Ovarian Cancer Overlap UP</td><td>-2.49564105507822</td><td>7.44E+00</td></tr><tr><td>ZEMEK Immune Checkpoint Blockade Ovarian Cancer Renca UP</td><td>-2.4938088753722</td><td>7.35E+00</td></tr><tr><td>Mori Large Pre-Bi LYMPHOCYTE DN</td><td>-2.46307404447591</td><td>7.07E+00</td></tr><tr><td>GOBP ACTIVATION OF GTPase Activity</td><td>-2.31205655977765</td><td>5.48E+00</td></tr><tr><td>CHYLA CBFA2T3 TARGETS UP</td><td>-2.2803941711885</td><td>1.54E+01</td></tr><tr><td>GOBP Alpha Beta T Cell Activation</td><td>-2.28005994821314</td><td>8.61E+00</td></tr><tr><td>GOBP SYNAPTIC TRANSMISSION GLUTAMATERGIC</td><td>-2.25924704263039</td><td>4.97E+00</td></tr><tr><td>GOMF PHOSPHATIDYLINOSITOL 3-KINASE BINDING</td><td>-2.25711247728203</td><td>3.93134815</td></tr><tr><td>GOBP RESPONSE TO TYPE II INTERFERON</td><td>-2.24674035599095</td><td>6.47E+00</td></tr><tr><td>GOBP CD4 POSITIVE ALPHA BETA T CELL DIFFERENTIATION</td><td>-2.227296413159</td><td>4.90E+00</td></tr><tr><td>YU MYC TARGETS DN</td><td>-2.19868763749708</td><td>3.98074224</td></tr><tr><td>GOBP POSITIVE REGULATION OF ALPHA BETA T CELL ACTIVATION</td><td>-2.19486696654636</td><td>4.43E+00</td></tr><tr><td>MARSON FOXP3 TARGETS DN</td><td>-2.1942280509767</td><td>3.81252457</td></tr><tr><td>GOCC MHC PROTEIN COMPLEX</td><td>-2.19272503265823</td><td>3.19601904</td></tr><tr><td>GOBP CELLULAR RESPONSE TO TYPE II INTERFERON</td><td>-2.18144541319954</td><td>5.49E+00</td></tr><tr><td>WP EBV LMP1 SIGNALING</td><td>-2.1800463438942</td><td>2.86688775</td></tr><tr><td>VILIMAS NOTCH1 TARGETS UP</td><td>-2.17599391720408</td><td>3.70349474</td></tr><tr><td>GOBP POSITIVE T CELL SELECTION</td><td>-2.16280215609989</td><td>3.18100203</td></tr><tr><td>GOMF PEPTIDE ANTIGEN BINDING</td><td>-2.15371058224711</td><td>3.12821548</td></tr><tr><td>IVANOVA HEMATOPOIESIS STEM CELL LONG TERM</td><td>-2.14849199232753</td><td>7.69E+00</td></tr><tr><td>GOBP ANTIGEN PROCESSING AND PRESENTATION OF EXOGENOUS ANTIGEN</td><td>-2.14801399771842</td><td>3.30018359</td></tr><tr><td>ZENG GU POST ICB METAGENE 31</td><td>-2.13262000785477</td><td>6.74E+00</td></tr><tr><td>WANG IMMORTALIZED BY HOXA9 AND MEIS1 DN</td><td>-2.13190216894544</td><td>2.68178355</td></tr><tr><td>ZENG GU ICB CONTROL METAGENE 8 PRECICTIVE ICB RESPONSE</td><td>-2.12724928064273</td><td>6.11E+00</td></tr><tr><td>BIOCARTA IL12 PATHWAY</td><td>-2.12682124379642</td><td>2.74466918</td></tr><tr><td>GOBP ALPHA BETA T CELL DIFFERENTIATION</td><td>-2.12381838169628</td><td>5.12E+00</td></tr><tr><td>HALLMARK TNFA SIGNALING VIA NFKB</td><td>-2.12063864477282</td><td>6.48E+00</td></tr><tr><td>GOBP PINOCYTOSIS</td><td>-2.11996615438178</td><td>2.61725914</td></tr><tr><td>GOBP CD8 POSITIVE ALPHA BETA T CELL ACTIVATION</td><td>-2.10648172743591</td><td>3.27962312</td></tr><tr><td>DESCARTES ORGANOGENESIS EPITHELIAL CELLS</td><td>-2.10447169923571</td><td>4.64E+00</td></tr><tr><td>ICHIBA GRAFT VERSUS HOST DISEASE D7 UP</td><td>-2.10411166106365</td><td>4.84E+00</td></tr><tr><td>GOMF NUCLEOSIDE TRIPHOSPHATASE REGULATOR ACTIVITY</td><td>-2.10382568920727</td><td>1.11E+01</td></tr><tr><td>GOMF GUANYL NUCLEOTIDE EXCHANGE FACTOR ACTIVITY</td><td>-2.09992482938135</td><td>7.01E+00</td></tr><tr><td>GOBP REGULATION OF NATURAL KILLER CELL PROLIFERATION</td><td>-2.08720766179445</td><td>2.28072864</td></tr><tr><td>GOBP POSITIVE REGULATION OF LEUKOCYTE PROLIFERATION</td><td>-2.08410838208926</td><td>5.45E+00</td></tr><tr><td>BIOCARTA NKT PATHWAY</td><td>-2.0639804451344</td><td>2.20636819</td></tr><tr><td>GOBP REGULATION OF CD4 POSITIVE ALPHA BETA T CELL DIFFERENTIATION</td><td>-2.05631067924243</td><td>2.84646252</td></tr><tr><td>REACTOME NUCLEAR RECEPTOR TRANSCRIPTION PATHWAY</td><td>-2.05572429036343</td><td>2.76289843</td></tr><tr><td>GOBP T CELL DIFFERENTIATION</td><td>-2.05517213740099</td><td>7.86E+00</td></tr><tr><td>GOMF ANTIGEN BINDING</td><td>-2.05284750523131</td><td>3.19701144</td></tr></tbody></table>

(continued)
<table><caption>Table 1 (Continued)</caption><thead><tr><th>Downregulated pathways in Foxp1-vKO HSC-MPP1</th><th>NES</th><th>log10padj</th></tr></thead><tbody><tr><td>GOMF_GTPASE_ACTIVATOR Activity</td><td>-2.05179734943819</td><td>6.80E+00</td></tr><tr><td>GOBP_GLYCOSPHINGOLIPID_METADATA_PROCESS</td><td>-2.05174280088562</td><td>2.90644785</td></tr><tr><td>RASHI_NFKB1_TARGETS</td><td>-2.04946550494726</td><td>2.36494992</td></tr><tr><td>MIR_7087_3P</td><td>-2.04640561375939</td><td>3.89137727</td></tr><tr><td>MORI_MATURE_B_LYMPHOCYTE_UP</td><td>-2.04608809467682</td><td>3.65280286</td></tr><tr><td>GOBP_T CELL_ACTIVATION_INVOLVED_IN_IMMUNE_RESPONSE</td><td>-2.04137727067267</td><td>3.92907019</td></tr><tr><td>GOBP_NATURAL_KILLER CELL_PROLIFERATION</td><td>-2.03879315892871</td><td>2.20208353</td></tr><tr><td>GOBP_ADAPTIVE_IMMUNE_RESPONSE</td><td>-2.03806604403141</td><td>8.95E+00</td></tr><tr><td>GOBP_REGULATION_OF_SYNAPTIC_TRANSMISSION_Glutamatergic</td><td>-2.03703990665228</td><td>2.81451087</td></tr><tr><td>WP_NUCLEAR_RECEPTORS</td><td>-2.03295497863175</td><td>2.48456913</td></tr><tr><th colspan="3">Upregulated pathways in Foxp1-vKO HSC-MPP1</th></tr><tr><td>WP_OXIDATIVE_PHOSPHORYLATION</td><td>2.90776203</td><td>1.16E+01</td></tr><tr><td>REACTOME_COMPLEX_I_BIOGENESIS</td><td>2.91524511</td><td>1.18E+01</td></tr><tr><td>REACTOME_G1_S_TRANSITION</td><td>2.91871798</td><td>1.53E+01</td></tr><tr><td>KONG_E2F3_TARGETS</td><td>2.92260032</td><td>1.47E+01</td></tr><tr><td>GOCC_OXIDOREDUCTASE_COMPLEX</td><td>2.92280116</td><td>1.58E+01</td></tr><tr><td>MORI_LARGE_PRE_BII_LYMPHOCYTE_UP</td><td>2.93570826</td><td>1.45E+01</td></tr><tr><td>GOCC_MITOCHONDRIAL_LARGE_RIBOSOMAL_SUBUNIT</td><td>2.94302687</td><td>1.27E+01</td></tr><tr><td>REACTOME_CDK_MEDIATED_PHOSPHORYLATION_AND_REMOVAL_OF_CDC6</td><td>2.95038875</td><td>1.19E+01</td></tr><tr><td>REACTOME CELL Cycle_CHECKPOINTS</td><td>2.95167677</td><td>2.87E+01</td></tr><tr><td>REACTOME_Siesthase</td><td>2.95321967</td><td>1.89E+01</td></tr><tr><td>REACTOME_SEPARATION_OF_SISTER_CHROMATIDS</td><td>2.9565356</td><td>2.23E+01</td></tr><tr><td>REACTOME_ASSEMBLY_OF_THE_PRE_REPLICATIVE_COMPLEX</td><td>2.95790927</td><td>1.41E+01</td></tr><tr><td>REACTOME_ORC1_REMOVAL_FROM_CHROMATIN</td><td>2.95919262</td><td>1.35E+01</td></tr><tr><td>GOBP_AEROBIC_RESPIRATION</td><td>2.96057839</td><td>2.23E+01</td></tr><tr><td>HALLMARK_HEME_METADATA</td><td>2.96284416</td><td>2.32E+01</td></tr><tr><td>GOCC_LARGE_RIBOSOMAL_SUBUNIT</td><td>2.97234119</td><td>1.88E+01</td></tr><tr><td>REACTOME_MITOTIC_METADATA_AND_ANAPHASE</td><td>2.98032692</td><td>2.46E+01</td></tr><tr><td>YAO_TEMPORAL_RESPONSE_TO_PROGESTERONE_CLUSTER_13</td><td>3.00192411</td><td>2.34E+01</td></tr><tr><td>REACTOME_SWITCHING_OF_ORIGINS_TO_A_POST_REPLICATIVE_STATE</td><td>3.00296264</td><td>1.65E+01</td></tr><tr><td>REACTOME_ACTIVATION_OF_APC_C_AND_APC_C_CDC20_MEDIATED_DEGRADATION_OF_MITOTIC_PROTEINS</td><td>3.00620009</td><td>1.39E+01</td></tr><tr><td>TABULA_MURIS_SENIS_MARROW MACROPHAGE_AGEING</td><td>3.00997256</td><td>2.38E+01</td></tr><tr><td>MORI Immature B_LYMPHOCYTE_DN</td><td>3.01121813</td><td>1.66E+01</td></tr><tr><td>GOBP_MITOCHONDRIAL_TRANSLATION</td><td>3.02218104</td><td>2.01E+01</td></tr><tr><td>GOBP_ATP_SYNTHESIS_COUPLED_ELECTRON_TRANSPORT</td><td>3.03145788</td><td>1.43E+01</td></tr><tr><td>ISHIDA_E2F_TARGETS</td><td>3.03686488</td><td>1.47E+01</td></tr><tr><td>HALLMARK_OXIDATIVE_PHOSPHORYLATION</td><td>3.04688047</td><td>2.62E+01</td></tr><tr><td>REACTOME_DNA_REPLICATION_PRE_INITIATION</td><td>3.05342846</td><td>1.70E+01</td></tr><tr><td>GOBP_OXIDATIVE_PHOSPHORYLATION</td><td>3.06238691</td><td>2.13E+01</td></tr></tbody></table>

(continued)
<table><caption>Table 1 (Continued)</caption><thead><tr><th>Downregulated pathways in Foxp1-vKO HSC-MPP1</th><th>NES</th><th>log10padj</th></tr></thead><tbody><tr><td>IVANOVA_HEMATOPOIESIS_MATURE CELL</td><td>3.06301106</td><td>3.21E+01</td></tr><tr><td>BURTON ADIPOGENESIS 3</td><td>3.06529199</td><td>1.73E+01</td></tr><tr><td>REACTOME_THE_CITRIC_ACID_TCA Cycle AND RESPIRATORY ELECTRON_TRANSPORT</td><td>3.07053588</td><td>2.38E+01</td></tr><tr><td>GOMF STRUCTURAL Constituent OF RIBOSOME</td><td>3.07962926</td><td>2.45E+01</td></tr><tr><td>HALLMARK E2F TARGETS</td><td>3.09039802</td><td>2.75E+01</td></tr><tr><td>DESCARTES_ORGANOGENESIS_PRIMITIVE_ERYTHROID_LINEAGE</td><td>3.09237491</td><td>4.71E+01</td></tr><tr><td>TABULA MURIS SENIS LUNG CLASSICAL MONOCYTE AGEING</td><td>3.11059838</td><td>4.42E+01</td></tr><tr><td>GOCC_ORGANELLAR RIBOSOME</td><td>3.13252747</td><td>1.93E+01</td></tr><tr><td>REACTOME_DNA REPLICATION</td><td>3.13590881</td><td>2.16E+01</td></tr><tr><td>GOCC RIBOSOMAL_SUBUNIT</td><td>3.13770938</td><td>2.86E+01</td></tr><tr><td>LAZARO GENETIC MOUSE MODEL_HIGH GRADE_LARGE CELL NEUROENDOCRINE LUNG CARCINOMA_UP</td><td>3.14294673</td><td>4.08E+01</td></tr><tr><td>REACTOME_MITOCHONDRIAL_TRANSLATION</td><td>3.15363304</td><td>1.99E+01</td></tr><tr><td>GOBP PROTON MOTIVE FORCE DRIVEN ATP SYNTHESIS</td><td>3.15562531</td><td>1.77E+01</td></tr><tr><td>REACTOME RESPIRATORY ELECTRON TRANSPORT</td><td>3.16982339</td><td>2.02E+01</td></tr><tr><td>REACTOME TRANSLATION</td><td>3.16991207</td><td>3.18E+01</td></tr><tr><td>WP ELECTRON TRANSPORT CHAIN</td><td>3.17883835</td><td>2.08E+01</td></tr><tr><td>ZHANG UTERUS C13_EPITHELIAL1 CELL</td><td>3.18517333</td><td>4.21E+01</td></tr><tr><td>GOCC RESPIRASOME</td><td>3.1953818</td><td>2.11E+01</td></tr><tr><td>REACTOME RESPIRATORY ELECTRON TRANSPORT_ATP SYNTHESIS_BY CHEMIOSMOTIC COUPLING_AND HEAT PRODUCTION_BY_UNCOUPLING_PROTEINS</td><td>3.22808936</td><td>2.36E+01</td></tr><tr><td>TABULA MURIS SENIS SPLEEN MEGAKARYOCYTE ERYTHROID PROGENITOR CELL AGEING</td><td>3.25819753</td><td>1.82E+01</td></tr><tr><td>CHYLA_CBFA2T3_TARGETS_DN</td><td>3.28815571</td><td>3.83E+01</td></tr><tr><td>GOCC INNER MITOCHONDRIAL MEMBRANE PROTEIN COMPLEX</td><td>3.33202369</td><td>2.99E+01</td></tr><tr><td>GOCC MITOCHONDRIAL PROTEIN_CONTAINING COMPLEX</td><td>3.54068831</td><td>5.54E+01</td></tr></tbody></table>

HSC=Hematopoietic stem cell; KO=Knockout; MPP=Multipotent progenitor; WT=wild-type.

involved in LT-HSC function and a positive enrichment of transcripts regulating the cell cycle. Notably, the expression of several HSC-related genes was disrupted, including the upregulation of the Neogene-1 (Neo1) messenger RNA (mRNA), a marker of a less regenerative, myeloid-biased fraction of LT-HSCs [44]. Furthermore, the enhanced Meis2 expression observed in FOXP1-deficient cells is consistent with previous data showing that MEIS2 overexpression enhances progenitor proliferation [43].

One of our unexpected results was that FOXP1 deficiency leads to an enlargement of the myeloid-biased MPP3 compartment. As for the HSC-MPP1 compartment, we identified positive enrichment of gene set characteristics of cell cycle in MPP3, with an enhanced expression of N-myc and HoxB5. MYC activity (both c-MYC and N-MYC) controls crucial aspects of HSC function, including proliferation, differentiation, and survival [45]. Despite much controversy, MYC is thought to control the balance between HSC self-renewal and differentiation [48]. Regarding Hoxb5 overexpression in Foxp1-deficient MPP3, it has been suggested that one of the physiologic

roles of HOXB5 in the HSC/MPP compartments is to provide a fail-safe system to protect them from exhaustion throughout life by conferring tolerance to DNA replication stress [47].

Interestingly, transcriptome analysis of Foxp1 vKO HSC-MPP1 and MPP3 compartments revealed a significant reduction in numerous lymphoid signaling pathways. In previous studies conducted in mice, Foxp1 has been identified as a key factor in several lymphocyte-mediated mechanisms, including facilitation of B cell development, suppression of follicular helper T cell differentiation, maintenance of naive T cell quiescence [13–15], and BM T<sub>REG</sub> cells [16,17]. The transcriptome data of the Foxp1 vKO HSC-MPP1 and MPP3 compartments suggest that, although there is no visible change in the phenotype of lymphoid progenitors, Foxp1 also contributes to very early lymphoid differentiation, leading to the myeloid differentiation bias toward MPP3 observed in *Foxp1-vKO* BM.

Although our study primarily focuses on the stem and progenitor compartments and only marginally addresses the mature
<table><caption>Table 2 List of the 50 most down- or upregulated pathways in Foxp1-vKO MPP3 vs. WT.</caption><thead><tr><th>Downregulated pathways in Foxp1-vKO MPP3</th><th>NES</th><th>log10padj</th></tr></thead><tbody><tr><td>BOYLAN_MULTIPLE_MYELOMA_PCA1_UP</td><td>-2.5505614</td><td>9.5342769</td></tr><tr><td>BOYLAN_MULTIPLE_MYELOMA_C_D_DN</td><td>-2.4738652</td><td>13.7948835</td></tr><tr><td>CHYLA_CBFA2T3_TARGETS_DN</td><td>-2.3594709</td><td>10.0429539</td></tr><tr><td>DESCARTES_ORGANOGENESIS_MELANOCYTES</td><td>-2.3588318</td><td>4.07276663</td></tr><tr><td>REACTOME_ROLE_OF_PHOSPHOLIPIDS_IN_PHAGOCYTOSIS</td><td>-2.3146727</td><td>5.0820833</td></tr><tr><td>PASQUALUCCI_LYMPHOMA_BY_GC_STAGE_UP</td><td>-2.2749962</td><td>9.77088065</td></tr><tr><td>YAO_TEMPORAL_RESPONSE_TO_PROGESTERONE_CLUSTER_3</td><td>-2.2511859</td><td>2.9194014</td></tr><tr><td>REACTOME_BINDINGS_AND_UPTAKE_OF_LIGANDS_BY_SCAVENGER_RECEPTORS</td><td>-2.2385266</td><td>4.20565967</td></tr><tr><td>REACTOME INITIAL_TRIGGERING_OF_COMPLEMENT</td><td>-2.2343469</td><td>4.0205063</td></tr><tr><td>chr6C1</td><td>-2.2340139</td><td>4.72995657</td></tr><tr><td>REACTOME_ROLE_OF_LAT2_NTAL_LAB_ON CALCIUM MOBILIZATION</td><td>-2.1583897</td><td>3.32603683</td></tr><tr><td>REACTOME_COMPLEMENT_CASCADE</td><td>-2.1579338</td><td>3.23471481</td></tr><tr><td>GOMF_ANTIGEN_BINDINGS</td><td>-2.123775</td><td>3.98871857</td></tr><tr><td>MIR_376B_5P_MIR_376C_5P</td><td>-2.113376</td><td>2.59091794</td></tr><tr><td>BOYLAN_MULTIPLE_MYELOMA_C_DN</td><td>-2.1073529</td><td>3.12784602</td></tr><tr><td>GOBP_ANTIGEN_PROCESSING_AND_PRESENTATION_OF_PEPTIDE_OR_POLYSACCHARIDE_ANTIGEN_VIA_MHC_CLASS_II</td><td>-2.0916507</td><td>1.90591988</td></tr><tr><td>VANLOO_SP3_TARGETS_DN</td><td>-2.0761586</td><td>2.71128225</td></tr><tr><td>GOMF GlUTATHIONE_TRANSF ERASE ACTIVITY</td><td>-2.0571196</td><td>1.73864421</td></tr><tr><td>CAIRO_LIVER_DEVELOPMENT_DN</td><td>-2.033733</td><td>4.2084733</td></tr><tr><td>ZHANG_UTERUS_C15_B CELL</td><td>-2.025831</td><td>2.59091794</td></tr><tr><td>REACTOME_NEUROTRANSMITTER_RELEASE Cycle</td><td>-2.0225995</td><td>1.59224436</td></tr><tr><td>KATSANOU_ELAVL1_TARGETS_UP</td><td>-2.0191161</td><td>3.48537172</td></tr><tr><td>REACTOME_FCERI_MEDIATED_CA_2 MOBILIZATION</td><td>-2.0109702</td><td>2.46740307</td></tr><tr><td>MT</td><td>-2.010795</td><td>1.83514882</td></tr><tr><td>MIR_6932_5P</td><td>-2.0048074</td><td>1.70946545</td></tr><tr><td>chr1E2</td><td>-1.9975318</td><td>1.73125341</td></tr><tr><td>YAO_TEMPORAL RESPONSE_TO_PROGESTERONE_CLUSTER_0</td><td>-1.983977</td><td>2.33644349</td></tr><tr><td>chr7E3</td><td>-1.9765928</td><td>2.33632529</td></tr><tr><td>WP_METAPATHWAY_BIOTRANSFORMATION</td><td>-1.9653849</td><td>2.13152534</td></tr><tr><td>GOBP_ANTIGEN_PROCESSING_AND_PRESENTATION_OF_EXOGENOUS_PEPTIDE_ANTIGEN_VIA_MHC_CLASS_II</td><td>-1.9492304</td><td>1.5045383</td></tr><tr><td>GOBP_POSITIVE_REGULATION_OF_T_HELPER_1_TYPE ImmUNE RESPONSE</td><td>-1.9428506</td><td>1.50420023</td></tr><tr><td>REACTOME ImmUNOREGULATORY_INTERACTIONS BETWEEN_A_LYMPHOID_AND_A_NON_LYMPHOID CELL</td><td>-1.9311231</td><td>3.16857231</td></tr><tr><td>GOBP ImmUNOGLOBULIN_PRODUCTION</td><td>-1.9282665</td><td>3.50119542</td></tr><tr><td>GOMF_FRIZZLED_BINDINGS</td><td>-1.9236591</td><td>1.34169002</td></tr><tr><td>GOMF_PROTEIN_LIPID_COMPLEX_BINDINGS</td><td>-1.9161274</td><td>1.36989606</td></tr><tr><td>GOMF ImmUNOGLOBULIN_BINDINGS</td><td>-1.9156235</td><td>1.40421786</td></tr><tr><td>GOBP_ACYLGLYCEROL_HOMEOSTASIS</td><td>-1.9146455</td><td>1.30368867</td></tr><tr><td>REACTOME_FCGAMMA_RECEPTOR_FCGR_DEPENDENT PHAGOCYTOSIS</td><td>-1.913553</td><td>2.74792772</td></tr></tbody></table>

(continued)
<table><caption>Table 2 (Continued)</caption><thead><tr><th>Downregulated pathways in Foxp1-vKO MPP3</th><th>NES</th><th>log10padj</th></tr></thead><tbody><tr><td>GOCC_IMMUNOGLOBULIN_COMPLEX</td><td>-1.9097874</td><td>1.40134454</td></tr><tr><td>ICHIBA_GRAFT_VERSUS_HOST_DISEASE_D7_UP</td><td>-1.9075151</td><td>2.75438631</td></tr><tr><td>KOHOUTEK_CCNT1_TARGETS</td><td>-1.9044956</td><td>1.61712405</td></tr><tr><td>GOBP_RESPONSE_TO_DIETARY_EXCESS</td><td>-1.9036743</td><td>1.26508854</td></tr><tr><td>REACTOME CELL SURFACE INTERACTIONS AT THE VASCULAR WALL</td><td>-1.903112</td><td>2.6404916</td></tr><tr><td>GOBP_PYROPTOSIS</td><td>-1.894862</td><td>1.5045383</td></tr><tr><td>BERENJENO_TRANSFORMED_BY_RHOA_FOREVER_UP</td><td>-1.8904777</td><td>1.30368867</td></tr><tr><td>WANG_IMMORTALIZED_BY_HOXA9_AND_MEIS1_UP</td><td>-1.8891722</td><td>1.34437893</td></tr><tr><td>GOBP B CELL MEDIATED IMMUNITY</td><td>-1.8880248</td><td>3.23475443</td></tr><tr><td>chr6A1</td><td>-1.88579780350966</td><td>1.54256206</td></tr><tr><td>MIR_7065_5P</td><td>-1.88497376332794</td><td>1.32170844</td></tr><tr><td>REACTOME FCERI MEDIATED MAPK ACTIVATION</td><td>-1.88405688436105</td><td>2.01733662</td></tr><tr><td colspan="3"><strong>Up-regulated pathways in Foxp1-vKO MPP3</strong></td></tr><tr><td>REACTOME_HDMS_DEMETHYLATE_HISTONES</td><td>2.03254729</td><td>2.09436791</td></tr><tr><td>REACTOME_DNA_REPAIR</td><td>2.03548518</td><td>6.57675914</td></tr><tr><td>chr12A3</td><td>2.03740483</td><td>1.72686985</td></tr><tr><td>GOBP_BEHAVIORAL RESPONSE TO PAIN</td><td>2.04056688</td><td>1.8658845</td></tr><tr><td>GOBP_SPINDLE_ORGANIZATION</td><td>2.04423249</td><td>4.55339137</td></tr><tr><td>HOFFMANN_LARGE_TO_SMALL_PRE_BII_LYMPHOCYTE_UP</td><td>2.04996591</td><td>3.98871857</td></tr><tr><td>HOFFMANN Immature TO Mature B LYMPHOCYTE_DN</td><td>2.05237027</td><td>2.07111883</td></tr><tr><td>BOYLAN MULTIPLE MYELOMA C_UP</td><td>2.06927241</td><td>2.24088054</td></tr><tr><td>GOBP_DOUBLE_STRAND_BREAK_REPAIR VIA NONHOMOLOGOUS END JOINING</td><td>2.07308422</td><td>2.37243772</td></tr><tr><td>REACTOME FORMATION OF SENESCENCE ASSOCIATED HETEROCHROMATIN FOCI SAHF</td><td>2.0763465</td><td>2.15749683</td></tr><tr><td>GOBP_MITOTIC_SPINDLE_ORGANIZATION</td><td>2.08178028</td><td>3.72433434</td></tr><tr><td>REACTOME CELL CYCLE CHECKPOINTS</td><td>2.11150903</td><td>7.04026465</td></tr><tr><td>LAZARO Genetic MOUSE MODEL HIGH GRADE LARGE CELL NEUROENDOCRINE LUNG CARCINOMA_UP</td><td>2.12701513</td><td>8.66611374</td></tr><tr><td>REACTOME_DNA_DOUBLE_STRAND_BREAK_REPAIR</td><td>2.12761233</td><td>4.98200328</td></tr><tr><td>GOBP_NUCLEOSOME_ASSEMBLY</td><td>2.12925793</td><td>2.70636515</td></tr><tr><td>HALLMARK E2F TARGETS</td><td>2.13760833</td><td>5.96171781</td></tr><tr><td>GOBP_CHROMOSOME_CONDENSATION</td><td>2.13899782</td><td>2.25061474</td></tr><tr><td>GOBP_MICROTUBULE CYTOSKELETON_ORGANIZATION INVOLVED IN MITOSIS</td><td>2.1392785</td><td>4.88863096</td></tr><tr><td>LE EGR2 TARGETS_UP</td><td>2.14500713</td><td>3.73096886</td></tr><tr><td>chr13A3</td><td>2.15351186</td><td>4.07276663</td></tr><tr><td>REACTOME HOMOLOGY DIRECTED REPAIR</td><td>2.15755589</td><td>4.5806768</td></tr><tr><td>KONG E2F3_TARGETS</td><td>2.18973307</td><td>4.2084733</td></tr><tr><td>REACTOME M PHASE</td><td>2.19150687</td><td>9.97414254</td></tr><tr><td>GOBP_BASE_EXCISION_REPAIR</td><td>2.2086643</td><td>2.77006959</td></tr><tr><td>REACTOME_DNA_DOUBLE_STRAND_BREAK RESPONSE</td><td>2.24930517</td><td>3.43519973</td></tr></tbody></table>

(continued)
compartment and restricts the proliferation of all MPP compartments. This interaction is particularly important given the major functional role of the MPP compartment in controlling blood cell production under native conditions [52,53].

## Conflict of Interest Disclosure

The authors do not have any conflicts of interest to declare in relation to this work.

## Acknowledgments

We thank Dr. Frédéric Pendino (Institut Cochin, Paris) for providing us with the tav-1-iCRE mice, Dr. Bruno Lucas for the CD45.1 mice, Dr. Muriel Andrieu (the Cytometry and Immunobiology Facility of Institut Cochin/INSERM U1016), Dr. Franck Letourneur (Platform Genom'IC, Institut Cochin), Isabelle Rombeau and Marcio Do Cruzeiro (Animal facility, Institut Cochin), for technical help, and Pr. Michaela Fontenay and Pr. Didier Bouscary (Institut Cochin) for valuable discussions. This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Université de Paris Cité, and Ligue Nationale contre le Cancer (ELFUZ17337). The funders had no role in study design, data collection and analysis, publishing decisions, or manuscript preparation.

## Author Contributions

EL, IDF, and DP designed the study. FL, SO, DP, IDF, EL, and IB developed the methods.

FL, SO, AF, PG, ILB, HL, MDA, and ZT performed the experiments. HT provided the FOXP1 mice. SG provided tools and animal facilities. IB, SO, and MLG performed computational analyses. DB, MF, IDF, and EL acquired/provided funding. EL, IDF, and DP wrote the manuscript. All authors reviewed the manuscript.

## SUPPLEMENTARY MATERIALS

Supplementary material associated with this article can be found in the online version at https://doi.org/10.1016/j.exphem.2025.104815.

## REFERENCES

1. Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 2008;132:631-44.

2. Shang F, Rodewald HR. Toward the dissection of hematopoietic stem cell fates and their determinants. Curr Opin Genet Dev 2022;75:101945.

3. Wilkinson AC, Igarashi KJ, Nakauchi H. Haematopoietic stem cell self-renewal in vivo and ex vivo. Nat Rev Genet 2020;21:541-54.

4. Naudin C, Hattabi A, Michelet F, et al. PUMILIO/FOXP1 signaling drives expansion of hematopoietic stem/progenitor and leukemia cells. Blood 2017;129:2493-506.

5. Kim JH, Hwang J, Jung JH, Lee HJ, Lee DY, Kim SH. Molecular networks of FOXP family: dual biologic functions, interplay with other molecules and clinical implications in cancer progression. Mol Cancer 2019;18:180.

6. Koon HB, Ippolito GC, Banham AH, Tucker PW. FOXP1: a potential therapeutic target in cancer. Expert Opin Ther Targets 2007;11:955-65.

7. Miyamoto K, Araki KY, Naka K, et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell 2007;1:101-12.

8. Tothova Z, Gilliland DG. FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem Cell 2007;1:140-52.

9. Tothova Z, Kollipara R, Huntly BJ, et al. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 2007;128:325-39.

10. Bigarella CL, Li J, Rimmelé P, Liang R, Sobol RW, Ghaffari S. FOXO3 transcription factor is essential for protecting hematopoietic stem and progenitor cells from oxidative DNA damage. J Biol Chem 2017;292:3005-15.

11. Liang R, Arif T, Kalmykova S, et al. Restraining lysosomal activity preserves hematopoietic stem cell quiescence and potency. Cell Stem Cell 2020;26:359-376.e7.

12. Shi C, Sakuma M, Mooroka T, et al. Down-regulation of the forkhead transcription factor Foxp1 is required for monocyte differentiation and macrophage function. Blood 2008;112:4699-711.

13. Feng X, Ippolito GC, Tian L, et al. Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development. Blood 2010;115:510-8.

14. Feng X, Wang H, Takata H, Day TJ, Willen J, Hu H. Transcription factor Foxp1 exerts essential cell-intrinsic regulation of the quiescence of naive T cells. Nat Immunol 2011;12:544-50.

15. Hu H, Wang B, Borde M, et al. Foxp1 is an essential transcriptional regulator of B cell development. Nat Immunol 2006;7:819-26.

16. Ghosh S, Roy-Chowdhur S, Kang K, Im SH, Rudra D. The transcription factor Foxp1 preserves integrity of an active Foxp3 locus in extrathymic Treg cells. Nat Commun 2018;9:4473.

17. Konopacki C, Pritykin Y, Rubtsov Y, Leslie CS, Rudensky AY. Transcription factor Foxp1 regulates Foxp3 chromatin binding and coordinates regulatory T cell function. Nat Immunol 2019;20:232-42.

18. Gabut M, Samavarchi-Tehrani P, Wang X, et al. An alternative splicing switch regulates embryonic stem cell pluripotency and reprogramming. Cell 2011;147:132-46.

19. Leishman E, Howard JM, Garcia GE, et al. Foxp1 maintains hair follicle stem cell quiescence through the regulation of Fgf18. Development 2013;140:3809-18.

20. Li H, Liu P, Xu S, et al. FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging. J Clin Invest 2017;127:1241-53.

21. Liu P, Huang S, Ling S, et al. Foxp1 controls brown/beige adipocyte differentiation and thermogenesis through regulating β3-AR desensitization. Nat Commun 2019;10:5070.

22. Li S, Wang Y, Zhang Y, et al. Foxp1/4 control epithelial cell fate during lung development and regeneration through regulation of anterior gradient 2. Development 2012;139:2500-9.

23. Shu W, Lu MM, Zhang Y, Tucker PW, Zhou D, Morrisey EE. Foxp2 and Foxp1 cooperatively regulate lung and esophagus development. Development 2007;134:1991-2000.

24. Wang B, Weidenfeld J, Lu MM, et al. Foxp1 regulates cardiac outflow tract, endocardial cushion morphogenesis and myocyte proliferation and maturation. Development 2004;131:4477-87.

25. Zhang Y, Li S, Yuan L, et al. Foxp1 coordinates cardiomyocyte proliferation through both cell-autonomous and nonautonomous mechanisms. Genes Dev 2010;24:1746-57.

26. Adams KL, Rousso DL, Umbach JA, Novitch BG. Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells. Nat Commun 2015;6:6778.

27. Araujo DJ, Anderson AG, Berto S, et al. FoxP1 orchestration of ASD-relevant signaling pathways in the striatum. Genes Dev 2015;29:2081-96.

28. de Boer J, Williams A, Skavdis G, et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol 2003;33:314-25.

29. van Keimpema M, Grüneberg LJ, Mokry M, et al. FOXP1 directly represses transcription of proapoptotic genes and cooperates with NF-κB to promote survival of human B cells. Blood 2014;124:3431-40.
30. van Keimpema M, Grüneberg LJ, Mokry M, et al. The forkhead transcription factor FOXP1 represses human plasma cell differentiation. Blood 2015;126:2098-109.

31. Cabezas-Wallscheid N, Klimmeck D, Hansson J, et al. Identification of regulatory networks in HSCs and their immediate progeny via integrated proteome, transcriptome, and DNA methylome analysis. Cell Stem Cell 2014;15:507-22.

32. Eich M, Trumpp A, Schmitt S. OMIP-059: identification of mouse hematopoietic stem and progenitor cells with simultaneous detection of CD45.1/2 and controllable green fluorescent protein expression by a single staining panel. Cytometry A 2019;95:1049-52.

33. Wolber FM, Leonard E, Michael S, Orschell-Traycoff CM, Yoder MC, Srour EF. Roles of spleen and liver in development of the murine hematopoietic system. Exp Hematol 2002;30:1010-9.

34. Ganuza M, Hall T, Myers J, et al. Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors. Nat Cell Biol 2022;24:1475-86.

35. Manesia JK, Xu Z, Broekaert D, et al. Highly proliferative primitive fetal liver hematopoietic stem cells are fueled by oxidative metabolic pathways. Stem Cell Res 2015;15:715-21.

36. Joseph C, Quach JM, Walkley CR, Lane SW, Lo Celso C, Purton LE. Deciphering hematopoietic stem cells in their niches: a critical appraisal of genetic models, lineage tracing, and imaging strategies. Cell Stem Cell 2013;13:520-33.

37. Hirata Y, Kakiuchi M, Robson SC, Fujisaki J. CD150high CD4 T cells and CD150high regulatory T cells regulate hematopoietic stem cell quiescence via CD73. Haematologica 2018;104:1136.

38. Hirata Y, Furuhashi K, Ishii H, et al. CD150high bone marrow Tregs maintain hematopoietic stem cell quiescence and immune privilege via adenosine. Cell stem cell 2018;22:445-53.

39. Cheng T, Rodrigues N, Shen H, et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 2000;287:1804-8.

40. Cortes M, Chen MJ, Stachura DL, et al. Developmental vitamin D availability impacts hematopoietic stem cell production. Cell Rep 2016;17: 458-68.

41. Gudmundsson KO, Nguyen N, Oakley K, et al. Prdm16 is a critical regulator of adult long-term hematopoietic stem cell quiescence. Proc Natl Acad Sci U S A 2020;117:31945-53.

42. Lehnertz B, Chagraoui J, MacRae T, et al. HLF expression defines the human hematopoietic stem cell state. Blood 2021;138:2642-54.

43. Cai M, Langer EM, Gill JG, et al. Dual actions of Meis1 inhibit erythroid progenitor development and sustain general hematopoietic cell proliferation. Blood 2012;120:335-46.

44. Gulati GS, Zukowska M, Noh JJ, et al. Neogenin-1 distinguishes between myeloid-biased and balanced Hoxb5+ mouse long-term hematopoietic stem cells. Proc Natl Acad Sci U S A 2019;116:25115-25.

45. Laurenti E, Varnum-Finney B, Wilson A, et al. Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell 2008;3:611-24.

46. Reavie L, Della Gatta G, Crusio K, et al. Regulation of hematopoietic stem cell differentiation by a single ubiquitin ligase-substrate complex. Nat Immunol 2010;11:207-15.

47. Sakamaki T, Kao KS, Nishi K, et al. Hoxb5 defines the heterogeneity of self-renewal capacity in the hematopoietic stem cell compartment. Biochem Biophys Res Commun 2021;539:34-41.

48. Delgado MD, León. Myc roles in hematopoiesis and leukemia. Genes Cancer 2010;1:605-16.

49. Eijkelenboom A, Burgering BMT. FOXOs: signalling integrators for homeostasis maintenance. Nat Rev Mol Cell Biol 2013;14:83-97.

50. Levavasseur F, Oussous S, Zubaidan T, et al. FOXP1 regulates oxidative stress, SIRT1 expression, and resistance to chemotherapies in acute myeloid leukemia cells. Blood Adv 2023;7:3265-75.

51. Liao BY, Zhang J. Null mutations in human and mouse orthologs frequently result in different phenotypes. Proc Natl Acad Sci U S A 2008;105:6987-92.

52. Sun J, Ramos A, Chapman B, et al. Clonal dynamics of native haematopoiesis. Nature 2014;514:322-7.

53. Busch K, Klapproth K, Barile M, et al. Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 2015;518:542-6.