Conditional deletion of Ahr alters gene expression profiles in hematopoietic stem cells

The aryl hydrocarbon receptor (AHR) is a ligand activated bHLH transcription factor that belongs to the Per-Arnt-Sim (PAS) superfamily of proteins involved in mediating responses to cellular environment regulating normal physiological and developmental pathways. The AHR binds a broad range of naturally derived and synthetic compounds, and plays a major role in mediating effects of certain environmental chemicals. Although our understanding of the physiological roles of the AHR in the immune system is evolving, there is little known about its role in hematopoiesis and hematopoietic diseases. Prior studies demonstrated that AHR null (AHR-KO) mice have impaired hematopoietic stem cell (HSC) function; they develop myeloproliferative changes in peripheral blood cells, and alterations in hematopoietic stem and progenitor cell populations in the bone marrow. We hypothesized mice lacking AHR expression only within hematopoietic cells (AHRVav1 mice) would develop similar changes. However, we did not observe a complete phenocopy of AHR-KO and AHRVav1 animals at 2 or 18 months of age. To illuminate the signaling mechanisms underlying the alterations in hematopoiesis observed in these mice, we sorted a population of cells highly enriched for HSC function (LSK cells: CD34-CD48-CD150+) and performed microarray analyses. Ingenuity Pathway and Gene Set Enrichment Analyses revealed that that loss of AHR within HSCs alters several gene and signaling networks important for HSC function. Differences in gene expression networks among HSCs from AHR-KO and AHRVav1 mice suggest that AHR in bone marrow stromal cells also contributes to HSC function. In addition, numerous studies have suggested a role for AHR in both regulation of hematopoietic cells, and in the development of blood diseases. More work is needed to define what these signals are, and how they act upon HSCs.


Introduction
All mature lineages of blood cells are generated from hematopoietic stem cells (HSCs), which reside primarily in bone marrow (BM) of adult mice and humans. One of the most important aspects of HSC biology is the precise regulation of their proliferation, differentiation, and selfrenewal. This balance can be shifted due to genetic mutations, environmental exposures to toxicants, and age [1][2][3][4][5]. For example, exposure to environmental toxicants which activate the aryl hydrocarbon receptor (AHR) have been linked to blood diseases in humans.
The aryl hydrocarbon receptor (AHR) is an environment sensing transcriptional regulator that is expressed in hematopoietic and non-hematopoietic cells. While the normal, physiological role of AHR is not fully understood, it regulates aspects of HSC function, immune system development, and hematopoietic diseases [3,[6][7][8][9][10][11]. Several proposed physiological functions of AHR in non-hematopoietic tissues have been suggested from studies using AHR-null-allele (AHR-KO) mouse models [9,12,13]. We have summarized these previous data in Table 1. While these models have generated much information on possible roles of the receptor in a variety of tissues and cell types, few studies have sought to describe the role of AHR as an intrinsic regulator of BM stem cell functions. Hematopoietic cells, including HSCs, exist in the BM in close proximity to a variety of other cell types. Multiple studies that have described the role of these non-hematopoietic cells in the regulation of HSC function have led to the development of models that describe a hematopoietic "niche", the cells of which can have significant regulatory effects on HSCs and greatly alter their function and output [14][15][16][17][18][19].
In order to better understand the role of AHR signaling intrinsic to HSCs, we used a conditional knockout (AHR Vav1 ) model that utilizes a Cre-loxP system to target loss of AHR expression to the hematopoietic system [20,21]. Targeted deletion of AHR allows for better discrimination of genes that are regulated intrinsically, and avoids the potential confounding effects in global knockout because all cells, including BM stromal cells, lack AHR expression. Here we report the characterization of the morphology and hematopoietic phenotype of AHR Vav1 mice, as well as the gene expression profiles of long-term HSC (LT-HSC) from these mice at 8 weeks or 18 months of age. LT-HSCs are the most primitive multipotent BM stem cell, and are responsible for long term multi-lineage cell generation. These data provide insight into the genes that AHR may intrinsically regulate within HSCs, and guide new approaches to elucidate how modulation or loss of AHR signaling can lead to diseases or dysfunction of the hematopoietic system.

Mice
Breeding pairs of B6.Cg-Tg A2Kio/J (Vav1-Cre) and AHR tm3.1Bra /J (AHR-KO) mice were obtained from Jackson Laboratories (Bar Harbor, Maine). Initial breeding stocks for Ahr fx/  [20]. All mice were maintained in micro-isolator housing according to approved protocols at the University of Rochester. Males expressing Vav1-Cre were crossed with females homozygous for the floxed Ahr allele. This breeding scheme produced males heterozygous for both transgenes, which were then crossed with female Ahr fx/fx mice, to produce female Vav1-cre +/-Ahr fx/fx (AHR Vav1 ) mice. These female mice were used in the experiments presented. Female Ahr fx/fx (AHR FX ) mice were used as experimental controls. The CD45.1 + (B6.SJL-Ptprc<a>/BoAiTac) mice used in the repopulation experiments were purchased from Taconic Farms (Rensselaer, New York).

Ethics statement
Animals were used at 2 or 18 months of age. All handling and experimental procedures were carried out in accordance with University of Rochester approved protocols. Our study was approved by both the University Committee on Animal Resources (UCAR) as well as the Institutional Biosafety Committee (IBC). Mice are housed in cages with air-handling systems designed to reduce ammonia build-up. Food and water are changed frequently by dedicated animal care technicians and were provided ab libitum for all studies described here. Mice receive nesting materials and red plastic houses to minimize distress and to encourage natural behaviors such as nesting and familial socialization. Prior to experiments, all mice were sacrificed using CO 2 asphyxiation and cervical dislocation to ensure death and absence of pain due to tissue harvests. Mice are monitored daily by dedicated vivarium staff for health and wellbeing. These checks monitor appearance, behavior and food and water levels to ensure the health and comfort of all experimental animals.

Cell isolation and detection of Ahr excision by PCR
Cells were isolated from the bone marrow, spleen, lymph nodes, and lung as previously described [11,22,23]. After hypotonic lysis to remove erythrocytes, single cell suspensions were prepared. CD45 negative (non-hematopoietic cells) were isolated from the lung. To do so, 1% low melting point agarose was inserted into the lung via the trachea, and the tissue was then digested with dispase. CD45 negative cells were isolated from the resulting cell suspension using a magnetic enrichment beads conjugated to an anti-mouse CD45 antibody (Miltenyi Biotec, Auburn, CA). Genomic DNA was isolated from cell suspensions using a QIAamp DNA Mini Kit (QIAGEN), and PCR to detect the excised Ahr allele was performed as previously described [20,23,24].

Tissue and cell collection and hematological profiles
Mice were euthanized with CO 2 and peripheral blood was collected from the retroorbital plexus vein into BD microtainer tubes containing EDTA (Beckton Dickinson and Company). Hematological profiles were analyzed using a HESKA Hematology Analyzer (HESKA Corporation, Colorado). Organs were harvested to collect wet weights. BM cells from both femurs of each mouse were harvested as previously described [6]. The number of cells obtained was determined using a hemocytometer.

Spleen colony forming unit assay
Recipient C57/Bl6 mice were sub-lethally irradiated (9.0 Gy) using a 137 Cs gamma-ray source and after 4 h these mice were tail-vein injected with 50,000 total BM cells from donor AHR FX or AHR Vav1 (prepared as described above) suspended in PBS. The recipient mice were euthanized after 8 or 12 days. Spleens were collected and fixed in Telleyesniczky's solution. The nodular colonies formed on the spleens were counted macroscopically [2].

Sorting and microarray analysis of Lin-CD48-CD150+ (SLAM+) cells
BM cells from femurs and tibias were harvested from each animal (6 to 8 mice) separately and depleted for lineage positive cells as previously described [6]. The Lin-cells were stained with fluorochrome-conjugated antibodies against Sca-1 (V450 Clone D7; BD Pharmingen), cKit (PeCy7 Clone 2B8; BD Pharmingen), CD34 (AF700 Clone Ram34; eBiosciences), CD48 (FITC Clone Hm48-1; BD Pharmingen), and CD150 (APC Clone 459911; R&D Systems). HSC were obtained by fluorescence-activated cell sorting lineage depleted BM cells [7]. Cells were sorted into RLT+ buffer (Qiagen) and placed at -80˚C. Total RNA was isolated from sorted LT-HSCs using an RNeasy Mini Kit (Qiagen). RNA was pre-amplified and cDNA was produced using a WT-Ovation PicoSL kit (Nugen). Each animal was ultimately prepared as a cDNA library for analysis using a distinct microarray chip. Microarray analysis was performed using Genechip Mouse Gene 2.0 ST Array (Affymetrix) by the UR Genomics Research Center. The IterPLIER algorithm was used to generate background-subtracted, quantile-normalized signals from the microarray data. Transcripts with a mean difference in expression of at least 1.5-fold and P<0.05 were examined for network/pathway associations by Ingenuity Pathway Analysis. Physiological functions were ascribed to the altered set of genes. Microarray data were also analyzed using Gene Set Enrichment Analysis (GSEA) as previously described [7]. The microarray data can be accessed through the Gene Expression Omnibus accession number GSE76276.

Reverse transcription (RT)-qPCR
LT-HSCs (SLAM+) were sorted as described above, and total RNA was extracted using an RNeasy Mini Kit (Qiagen). cDNA was prepared as described above and 10 ng of cDNA was used in qPCR reactions via a Bio-Rad CFX96 Real Time Thermal Cycler (Bio-Rad, Hercules, California), using primer/probe assays (Applied Biosystems). Expression of mRNA for each gene was normalized using the expression of Hprt and Gapdh. Gene expression was compared using the 2 -ΔΔT method [25]. Differences were considered significant when relative mRNA expression was 1.5-fold higher or lower than indicated controls with a p-value of less than 0.05.

Statistics
For analyses of two groups, the Student's t-test was used. For comparisons of two or more groups, a two-way ANOVA was used. For microarray data, The IterPLIER algorithm was used to generate background-subtracted, quantile-normalized signals from the microarray data and analyses were adjusted to account for multiple comparisons. Ingenuity Pathways Analysis (IPA) uses a proprietary platform knowledge base and Fischer's Exact Test to determine if pathways and subsets of genes are altered.

Validation of conditional deletion of Ahr gene in hematopoietic cells
To verify that targeted excision is occurring, we isolated genomic DNA from spleen, liver, kidney, thymus and BM from AHR FX and AHR Vav1 animals. PCR analyses revealed that hematopoietic tissues from AHR Vav1 mice display excision of Ahr ( Fig 1A). The tissues analyzed included the hematopoietic-derived cell enriched tissues BM, spleen, thymus. We determined that CD45 non-hematopoietic cells only display the unexcised allele. Western blotting confirmed that hematopoietic tissues from AHR Vav1 mice have greatly reduced or undetectable AHR protein ( Fig 1B). As an additional demonstration of functional loss of AHR, we treated isolated BM cells with the potent AHR agonist TCDD, and measured the expression level of the TCDD:AHR gene target Scinderin [3] by RT-qPCR. Relative to isolated Lin-BM cells from TCDD treated with control, Scinderin expression is 5-fold higher in Lin-BM cells from

Fig 1. Genotyping and gene induction in 8 week old AHR Vav1 mice. (A) Excised
Ahr is detectable in hematopoietic tissues of AHR Vav1 animals. Tissues from AHR Vav1 and AHR FX mice were isolated as described and DNA was extracted for PCR analysis using a three primer reaction that amplifies either 180 bp fragments (excised allele) or 140 bp (unexcised allele) fragments. (B) AHR Vav1 animals have reduced levels of AHR protein in hematopoietic cells. Various tissues (liver, spleen, thymus, and total bone marrow) were analyzed by western blot to detect AHR (~90kD). AHR FX mice (+) express the receptor in all tissues. AHR Vav1 (-) mice display reductions or lack of AHR in the tissues examined. Hepatoma cells were used as a positive control as they express large amounts of AHR.(C) Lineage negative cells from AHR Vav1 mice fail to upregulate Scinderin mRNA after 6h exposure to 10nM TCDD. Data expressed relative to DMSO vehicle, using HPRT as an endogenous control gene for normalization. N = 4 mice per treatment group. Relative fold change in expression was calculated using the 2 ΔΔCt method. AHR FX mice exposed to TCDD ex vivo (Fig 1C). In contrast, Lin-BM cells from AHR Vav1 mice failed to respond to TCDD, which further supports that functional deletion of AHR protein was achieved (Fig 1C). Some reports have suggested that Ahr excision is possible in nonhematopoietic tissues due to endothelial expression of Vav1-cre [26], but we did not observe this in our PCR screens. Combined with our confirmation that AHR protein levels are reduced in hematopoietic tissues in AHR Vav1 mice, and that they do not respond to TCDD, AHR Vav1 mice are a useful model for studying the hematopoietic role of AhR. Our data shows that we have deleted AhR within hematopoietic cells, and not other tissues.
Flanking Ahr with loxp sites and/or the expression of the Cre transgene could potentially have unintended off-target effects. AHR Vav1 animals were healthy and did not display any gross alterations to non-hematopoietic organs (S1 Fig). No difference was observed in total body mass, indicating that the mice grow at the same rate as controls. Heart, lung, thymus, kidney, liver and spleen mass were unaltered in AHR Vav1 mice relative to age-matched AHR FX controls. Together these data indicate that neither deletion of the Ahr gene in hematopoietic tissues nor expression of the Cre transgene is sufficient to induce observable gross changes in non-hematopoietic organs.
Alterations to peripheral blood cells can be reflective of defects in HSC function. AHR-KO mice display alterations to peripheral blood composition at 2-and 24-months [6, 7]. To determine if lack of AHR in hematopoietic cells is sufficient to produce similar alterations in peripheral blood, we examined complete blood counts and hematological parameters in 2and 18-month old AHR Vav1 mice compared to age-matched AHR FX mice. Young AHR Vav1 mice displayed no alterations to the total white blood cell count or in the percentage of lymphocytes, monocyte or granulocyte in peripheral blood (Fig 2A and 2C). However, as these mice age, there was significant decrease in total white blood cell (WBC) number. In addition, there was significant increase in monocytes and granulocytes, but a decrease in lymphocytes in older AHR Vav1 mice (Fig 2B and 2D).However, spleens from 8-week old AHR Vav1 mice did not display alterations in cellularity compared to AHR FX controls, nor were there significant differences in the percentage or number of T-cells, B-cells, myeloid cells and erythroid cells (S2 Fig). Given that it was previously reported that global AHR-KO mice display alterations in BM subpopulations [6], we examined the frequencies of hematopoietic subpopulations in the BM of AHR Vav1 mice. In contrast to global AHR-KO mice, young AHR Vav1 mice display no significant alterations in the percentage of LSK, MPP, CLP, CMP, ST-HSC or LT-HSC in BM cells (Fig 3A and 3B). Given that AhR-KO mice also displayed decreased expression of ROS detoxifying enzymes such as Stra13 [7], we examined ROS levels in AHR Vav1 mice. LSK cells from AHR Vav1 mice had a small, but statistically significant, enhanced oxidative capacity as indicated by DCFDA staining (Fig 3C).
In order to determine if there are functional alterations in the hematopoietic system of young AHR Vav1 mice, we utilized the spleen colony forming assay [27] (S3 Fig). We observed no significant alterations in colony forming units at either 8 or 12 days following irradiation and injection of AHR Vav1 or AHR FX cells. These data are consistent with the above data indicating no alterations in phenotype of BM from AHR Vav1 mice.
To determine whether conditional ablation of AHR from only the hematopoietic lineage affects LT-HSC function, we measured their ability to serially engraft and repopulate irradiated recipient mice. Serial transplantation forces LT-HSCs to exit quiescence and undergo regulated proliferation and differentiation, in order to repopulate blood lineages in the irradiated recipients. Thus, if conditionally ablating the AHR from only hematopoietic cells affects LT-HSC function, we would detect differences in cellular populations after serial transplantation. We observed a slight decrease in the total repopulation ability of cells from AHR Vav1 animals as indicated by the decreased number of CD45.2+ cells in primary recipients of AHR Vav1 cells (Fig 4A). However, AHR Vav1 cells appear to have enhanced proliferative potential at secondary transplant, indicated by the increase in the percentage of CD45.2+ cells in secondary recipients. This proliferative advantage did not persist to tertiary engraftment, as AHR Vav1 cells appear to exhaust prematurely when compared with AHR FX cells at the same time point.

AHR Vav1 mice have altered gene expression and signaling networks related to normal HSC function
To determine the possible differential expression of genes due to lack of AHR, we analyzed sorted LT-HSCs from 2-month old female mice. As hypothesized, lack of AHR expression in hematopoietic cells is sufficient to drive differences in gene expression relative to AHR FX controls ( Fig 5A). Array data were also analyzed by Gene Set Enrichment Analysis (GSEA). Enrichment was determined in a variety of genes sets related to hematological disease or proliferative capacity (Fig 5B, 5C, 5D and 5E). To examine the potential biological impact of the genes determined to be altered by microarray, we next utilized Ingenuity Pathway Analysis (IPA). IPA indicated that there are likely alterations in AHR Vav1 LT-HSCs in pathways such as Cell-to-Cell Signaling and Interaction, Cellular Development, and Cancer (Fig 6). Consistent with the hypothesized role of AHR as a regulator of the hematopoietic system, the genes changed in LT-HSCs of 2-month old AHR Vav1 participate in hematological system   development and function. Together, these proposed physiological functions generated by IPA are consistent with the hypothesis that lack of AHR is sufficient to induce alterations in gene expression in LT-HSCs, and that the induced alterations may play a role in the subsequent development of hematological disease or dysfunction. IPA was used to filter genes which change in LT-HSCs from both 8 week old AHR Vav1 and AHR-KO mice (AHR-KO microarray data from Gene Expression Omnibus database accession number: GSE 46976) and which also have putative AHR binding sites on their promoter (Supported by SABiosciences' proprietary database known as DECODE (DECipherment Of DNA Elements). These genes are presented in Table 2.
To examine the potential functional impact of lack of AHR in the hematopoietic system throughout aging, we repeated the array and GSEA/pathway analyses in 18-month old AHR Vav1 mice. These analyses indicated that LT-HSCs from old AHR Vav1 mice have differential gene expression compared to floxed controls (Fig 7A). Array data were also analyzed by GSEA. Lack of AhR alters hematopoietic stem cell gene expression Enrichment was determined in a variety of genes sets related to hematological disease or proliferative capacity and HSCs aging (Fig 7B-7G). IPA analysis indicated that there are alterations in AHR Vav1 LT-HSCs in genes functionally significant for hematopoietic stem cell function or activity (Fig 8). A different subset of altered genes was examined which possess putative AHR binding sites in aged AHR Vav1 and AHR-KO (AHR-KO microarray data from Gene Expression Omnibus database accession number: GSE 67378) are presented in Table 3. Ingenuity Pathway Analysiswas used to filter this list, and which also have putative AHR binding sites confirmed using SA Biosciences website. These data are presented Table 3.
To confirm that certain genes identified utilizing microarray and bioinformatics approaches are truly differentially expressed in AHR Vav1 mice, we chose to examine genes linked to hematopoietic development. We used qPCR to examine the expression of genes related to hematopoiesis at both ages, which included Birc6 and Stra13 (Fig 9A and 9B). Consistent with the hypothesis that AHR may regulate these genes, and hematopoietic aging in a

Discussion
The data presented here are consistent with the hypothesized role of AHR as a potential intrinsic regulator of genes in HSCs. In consideration of data previously reported for AHR-KO mice, where we observed altered functional capacity and abnormal proliferation [6], they also suggest that AHR signaling in non-hematopoietic "niche" tissue assists in the regulation of HSCs, and the absence of this signaling in AHR-null allele mice is partially responsible for producing the phenotypes observed.
Interestingly, AHR Vav1 mice do appear to phenocopy enhanced oxidative capacity of LSK cells, and this finding is consistent with a prior report in AHR-KO mice [7] and with the described crosstalk between AHR and the anti-oxidative transcription factor Nrf2. [28]. Functional assays of progenitor and HSC function indicate that AHR Vav1 mice are not altered at the level of progenitors as indicated by spleen colony forming units. This finding is consistent with the lack of alterations observed in BM progenitors assessed by flow cytometry. However, AHR Vav1 mice do display alterations in serial repopulation assays that resemble expansion and early exhaustion at the secondary and tertiary transplant analyses, respectively. While surprising that a defect only became apparent at the secondary stage of engraftment, these data are is consistent with AHR regulating at least some of the aspects of engraftment in BM, and with a previous report that activation of AHR in HSCs alters gene expression related to migration and trafficking [3]. The lack of phenocopy between AHR-KO mice and AHR Vav1 mice can potentially be due to the presence of AHR in non-hematopoietic cells in the AHR Vav1 mice. In addition, the young age of the mice examined could potentially play a role in the absence of the AHR-KO phenotype. Aging is a significant risk factor for hematological diseases [29], and dysfunction of hematopoietic system [30][31][32]. It is possible that the AHR Vav1 mice could develop a phenotype with aging that is more consistent with observed phenotypes in AHR-KO mice such as splenomegaly, white cell expansion, or LSK expansion [6,7]. Future studies will address this issue. However, notably there were significant deficits observed following the serial transplant of HSCs from young animals. Serial transplantation mimics some aspects of HSC aging. The observation that a defect becomes apparent in the secondary stage of engraftment with AHR Vav1 mice as donors is consistent with the hypothesis that AHR promotes long term fitness of hematopoietic stem cells. Serial repopulation mimics proliferative stress and aging, and it is intriguing that a defect became apparent at later iterations of engraftment. This is suggestive that expression of AHR is required for long term HSC maintenance and function.
In order to determine what gene expression changes could potentially contribute to HSC dysfunction, we examined gene expression profiles in LT-HSCs from AHR Vav1 mice. Gene expression profiles in LT-HSCs from young AHR Vav1 mice are substantially altered. Hierarchical clustering of the genes reveals alterations in genes such as FANCE, which is a critical component of DNA repair machinery in HSCs [33], and CFHR2 which may have regulatory roles for immune cells and complement activation [34]. Gene Set Enrichment Analysis indicates enrichment for genes in hematopoietic and cancer related gene sets from the Molecular Signatures Database. These genes include Jun and Fos, which are important regulators of HSC function [35,36]. The genes altered examined fall into gene sets and pathways that are likely to have functional impact on hematopoietic stem cell function or output.
Ingenuity Pathway Analysis indicated that the genes differentially expressed in AHR Vav1 mice are related to cell networks such as Cancer, Cell-to-Cell Signaling and Interaction, and Cellular Development. This finding is consistent with known and hypothesized roles for AHR in a variety of cell types [37][38][39]. The top biological functions predicted by IPA include Cellular Movement, Amino Acid Metabolism, Immune Cell Trafficking, Small Molecule Biochemistry and Hematological System Development and Function. These proposed networks are again consistent with known and hypothesized roles for AHR [3], and with the proposed activation of AHR by a variety of endogenous ligands [40][41][42][43]. qPCR confirmation of a subset of genes significant to the hematopoietic system revealed that at both young and old ages, AHR Vav1 mice have differential gene expression. Stra13 is involved in the detoxification of reactive oxygen species. We have previously identified Stra13 as differentially regulated in HSCs lacking AHR using a global deletion mouse (AHR-KO) model [7]. This finding suggests that AHR is an important regulator of genes which control ROS detoxification, as Stra13 has been decreased in both AhR-KO and AhR Vav1 mice. Birc6 is a regulator of apoptosis and has been described as being deregulated in acute myeloid leukemia [44]. Interestingly, knockdown of Birc6 in cell lines used to model promyelytic leukemia impairs neutrophil differentiation, and AhR regulation of Birc6 is suggested by our data. This finding is consistent with the hypothesis that AHR is a critical regulator of gene expression in LT-HSCs. Many of the genes we observed to be changed are differentially altered (directionality) between the two types of mouse model used to study AhR (globally deficient, vs HSC deficient). This lack of phenocopy is consistent with our hypothesis that non-hematopoietic effects are driving some of the changes in global AHR-KO animals, and further highlights the utility of using AHR Vav1 mice to identify genes directly regulated by AHR in hematopoietic cells.
Our lab has recently published work which suggests that AHR-KO mice have changes in the cellular composition of BM stromal cells [45]. We also observed higher BM cell counts in chimeric mice having WT hematopoietic cells transplanted into AHR-KO host. This study suggests that lack of AHR within BM stromal cells has a role in determining HSC phenotype and function in AHR-KO mice. HSCs from AHR-KO mice have reduced expression of Angiopoietin 1 (Angpt1). It has been reported that changes in Angpt1 gene expression in HSCs may induce secondary changes in BM niche [46]. Angpt1 is also expressed by niche osteoblast cells, which promotes quiescent HSCs in BM niche [47]. The observed phenotypic differences between AHR-KO and AHR Vav1 HSCs may be not only due to AHR cell-intrinsic regulation of HSCs, but an extrinsic effect of an AHR-regulated BM niche environment that may play a role in HSC homeostasis and function.  Lack of AhR alters hematopoietic stem cell gene expression

Down-regulated genes up-regulated genes in 18-month-old AHR-KO and AHR
Although we did not observe the same phenotype, function and gene profiles of AHR Vav1 HSCs as in AHR-KO mice, common changes in several genes were observed. The expression of pdp1 (up-regulated) and stra13 (down-regulated) were altered in both global and conditional AhR KO models. These genes are associated with aging and longevity, as well as oxidative stress [48,49]. When these mice age they have several common genes up-regulated (agbl4, birc6, anapc1) and down-regulated (runx1t1, perp); these play a role in megakaryopoiesis, apoptosis, cell cycle and aging related changes, apoptosis and leukemia [45,50,51].
Notably, Vav-1, and, in this model, Cre-recombinase, are expressed in hematopoietic cells at about mouse gestational day 11 [21,26]. As such, it is possible that despite the multiple gene changes in HSCs from AHR Vav1 mice, some compensatory gene and functional changes occurring (in HSCs and/or BM niche cells) during the lifetime of the AHR Vav1 animals could Lack of AhR alters hematopoietic stem cell gene expression contribute to the lack of complete phenocopy compared to AHR-KO mice. It is also possible that some AHR-dependent pre-programmed event taking place prior to gestational day 11 could "lock in" some aspects of HSC regulation. Other studies, such as the examination of an inducible KO model, may address some of these issues.
In conclusion, the data presented here suggest that lack of AHR in HSCs is sufficient to induce alterations in HSC gene expression, but these gene changes do not appear to induce significant alterations in HSC output/function at 2-months of age, other than in the context of serial transplantation, that were observed in AHR-KO mice. These findings indicate the need to examine the role of AHR signaling in non-hematopoietic stromal cells of the BM (or more distant tissues), and how such signaling can ultimately influence the function or activity of HSCs. The enhanced levels of oxidative species in both AHR Vav1 and AHR-KO mice are supportive of a role for AHR:Nrf2 crosstalk in maintaining HSC oxidative levels and may represent an important protective mechanism in HSC biology. These data also suggest the need to examine aging of AHR Vav1 mice to determine the impact that deficiency in AhR may have on the development of hematological disease.