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We aimed to characterize peritoneal macrophages from two novel mouse models that enable macrophage-specific overexpression of ABCA1 and ABCG1 via Cre recombinase. Since ABCA1/ABCG1 expression in macrophages is acknowledged to be anti-atherogenic, overexpression of these two transporters may result in a potent atheroprotective effect. However, there are no current animal models that permit overexpression of ABCA1/ABCG1 to precisely occur in macrophages. The purpose of this work was to use ex vivo strategies to characterize the peritoneal macrophages from two novel mouse models: ABCA1-LSL mice and ABCG1-LSL mice. Overexpression of ABCA1/ABCG1 in peritoneal macrophages is controlled by Cre recombinase, due to the LoxP-Stop-LoxP (LSL) system regulating ABC-transporter overexpression. In this study, we first characterized cells isolated from the peritoneal cavities of mice via immunofluorescent staining with macrophage markers CD11b and F4/80 to confirm that we were successful with peritoneal macrophage isolation. We next isolated peritoneal macrophages from ABCA1-LSL mice and ABCG1-LSL mice, and then we incubated these cells with gesicle particles containing Cre recombinase to measure ABC-transporter expression via immunoblotting and measure apoAI/HDL-mediated efflux using cholesterol efflux assays. In our experiments, we observed increases in ABCA1 protein expression and apoAI-mediated cholesterol efflux within ABCA1-LSL peritoneal macrophages, and increased ABCG1 protein expression and HDL-mediated cholesterol efflux in ABCG1-LSL peritoneal macrophages, when compared to corresponding control peritoneal macrophage groups. In conclusion, this report shows that ABCA1-LSL mice and ABCG1-LSL mice may possibly be utilized to cross with macrophage-specific Cre mice to study ABC-transporter overexpression precisely in macrophages, thereby providing valuable tools to dissect the impact of macrophage-specific ABC-transporter overexpression on atherogenesis.

Mammary epithelial progenitors are the normal cell-of-origin of breast cancer. We previously defined a population of p27+ quiescent hormone-responsive progenitor cells in the normal human breast whose frequency associates with breast cancer risk. Here, we describe that deletion of the Cdkn1b gene encoding the p27 cyclin-dependent kinase inhibitor in the estrogen-induced mammary tumor-susceptible ACI rat strain leads to a decrease in the relative frequencies of Cd49b+ mammary luminal epithelial progenitors and pregnancy-related differentiation. We show by comprehensive gene expression profiling of purified progenitor and differentiated mammary epithelial cell populations that p27 deletion has the most pronounced effects on luminal progenitors. Cdkn1b-/- females have decreased fertility, but rats that are able to get pregnant had normal litter size and were able to nurse their pups implying that loss of p27 in ACI rats does not completely abrogate ovarian function and lactation. Reciprocal mammary gland transplantation experiments indicate that the p27-loss-induced changes in mammary epithelial cells are not only caused by alterations in their intrinsic properties, but are likely due to altered hormonal signaling triggered by the perturbed systemic endocrine environment observed in Cdkn1b-/- females. We also observed a decrease in the frequency of mammary epithelial cells positive for progesterone receptor (Pr) and FoxA1, known direct transcriptional targets of the estrogen receptor (Erα), and an increase in phospho-Stat5 positive cells commonly induced by prolactin (Prl). Characterization of genome-wide Pr chromatin binding revealed distinct binding patterns in mammary epithelial cells of Cdkn1b+/+ and Cdkn1b-/- females and enrichment in genes with known roles in Notch, ErbB, leptin, and Erα signaling and regulation of G1-S transition. Our data support a role for p27 in regulating the pool size of hormone-responsive luminal progenitors that could impact breast cancer risk.

Mitral valve prolapse (MVP) is a common cardiac valve disease that often progresses to serious secondary complications requiring surgery. MVP manifests as extracellular matrix disorganization and biomechanically incompetent tissues in the adult setting. However, MVP has recently been shown to have a developmental basis, as multiple causal genes expressed during embryonic development have been identified. Disease phenotypes have been observed in mouse models with human MVP mutations as early as birth. This study focuses on the developmental function of DCHS1, one of the first genes to be shown as causal in multiple families with non-syndromic MVP. By using various biochemical techniques as well as mouse and cell culture models, we demonstrate a unique link between DCHS1-based cell adhesions and the septin-actin cytoskeleton through interactions with cytoplasmic protein Lix1-Like (LIX1L). This DCHS1-LIX1L-SEPT9 axis interacts with and promotes filamentous actin organization to direct cell-ECM alignment and valve tissue shape.

ObjectivesFamilies that contain multiple siblings affected with childhood-onset of systemic lupus erythematosus (SLE) likely have strong genetic predispositions. We performed whole-exome sequencing (WES) to identify familial rare risk variants and to assess their effects in lupus.MethodsSanger sequencing validated the two ultra-rare, predicted pathogenic risk variants discovered by WES and identified additional variants in 562 additional SLE patients. Effects of a splice site variant and a frameshift variant were assessed using a Minigene assay and CRISPR/Cas9-mediated knock-in (KI) mice, respectively.ResultsThe two familial ultra-rare, predicted loss-of-function (LOF) SAT1 variants exhibited X-linked recessive Mendelian inheritance in two unrelated African-American families. Each LOF variant was transmitted from the heterozygous unaffected mother to her two sons with childhood-onset SLE. The p.Asp40Tyr variant affected a splice donor site causing deleterious transcripts. The young hemizygous male and homozygous female Sat1p.Glu92Leufs*6 KI mice spontaneously developed splenomegaly, enlarged glomeruli with leukocyte infiltration, proteinuria and elevated expression of type I interferon inducible genes. SAT1 is highly expressed in neutrophils and encodes spermidine/spermine-N1-acetyltransferase 1 (SSAT1), a rate-limiting enzyme in polyamine catabolism. Young male KI mice exhibited neutrophil defects and decreased proportions of Foxp3+CD4+ T-cell subsets. Circulating neutrophil counts and proportions of Foxp3+CD4+ T cells correlated with decreased plasma levels of spermine in treatment naïve, incipient SLE patients.ConclusionsWe identified two novel SAT1 loss-of-function variants, showed the ability of the frameshift variant to confer murine lupus, highlighted the pathogenic role of dysregulated polyamine catabolism, and identified SAT1 LOF variants as new monogenic causes for SLE.

The distinct topographic Hox expression patterns observed in vascular smooth muscle cells (VSMCs) of the adult cardiovascular system suggest that these transcriptional regulators are critical for maintaining region-specific physiological properties of blood vessels. To test this proposition, we expanded the vascular Hoxc11 expression domain normally restricted to the lower limbs by utilizing an innovative integrated tetracycline regulatory system and Transgelin promoter elements to induce Hoxc11 expression universally in VSMCs of transgenic mice. Ectopic Hoxc11 expression in carotid arteries, aortic arch and descending aorta resulted in drastic vessel wall remodeling involving elastic laminae fragmentation, medial smooth muscle cell loss, and intimal lesion formation. None of these alterations were observed upon induction of Hoxc11 transgene expression in the femoral artery, i.e. the natural Hoxc11 activity domain, although this vessel was greatly enlarged, comparable to the topographically restricted vascular changes seen in Hoxc11−/− mice. To begin defining Hoxc11-controlled pathways of vascular remodeling, we performed immunolabeling studies in conjunction with co-transfection and chromatin immunoprecipitation (ChIP) assays using mouse vascular smooth muscle (MOVAS) cells. The results suggest direct transcriptional control of two members of the matrix metalloproteinase (Mmp) family, including Mmp2 and Mmp9 that are known as key players in the inception and progression of vascular remodeling events. In summary, the severe vascular abnormalities resulting from the induced dysregulated expression of a Hox gene with regional vascular patterning functions suggest that proper Hox function and regulation is critical for maintaining vascular functional integrity.

ObjectivesFamilies that contain multiple siblings affected with childhood onset of systemic lupus erythematosus (SLE) likely have strong genetic predispositions. We performed whole exome sequencing (WES) to identify familial rare risk variants and to assess their effects in lupus.MethodsSanger sequencing validated the two ultra-rare, predicted pathogenic risk variants discovered by WES and identified additional variants in 562 additional patients with SLE. Effects of a splice site variant and a frameshift variant were assessed using a Minigene assay and CRISPR/Cas9-mediated knock-in (KI) mice, respectively.ResultsThe two familial ultra-rare, predicted loss-of-function (LOF) SAT1 variants exhibited X-linked recessive Mendelian inheritance in two unrelated African–American families. Each LOF variant was transmitted from the heterozygous unaffected mother to her two sons with childhood-onset SLE. The p.Asp40Tyr variant affected a splice donor site causing deleterious transcripts. The young hemizygous male and homozygous female Sat1 p.Glu92Leufs*6 KI mice spontaneously developed splenomegaly, enlarged glomeruli with leucocyte infiltration, proteinuria and elevated expression of type I interferon-inducible genes. SAT1 is highly expressed in neutrophils and encodes spermidine/spermine-N1-acetyltransferase 1 (SSAT1), a rate-limiting enzyme in polyamine catabolism. Young male KI mice exhibited neutrophil defects and decreased proportions of Foxp3 +CD4+ T-cell subsets. Circulating neutrophil counts and proportions of Foxp3 +CD4+ T cells correlated with decreased plasma levels of spermine in treatment-naive, incipient SLE patients.ConclusionsWe identified two novel SAT1 LOF variants, showed the ability of the frameshift variant to confer murine lupus, highlighted the pathogenic role of dysregulated polyamine catabolism and identified SAT1 LOF variants as new monogenic causes for SLE.

Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13tm1Mrc) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1nu (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13tm1Mrc and Foxn1nu mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13tm1Mrc mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.

THE striking similarities in the structure, organization and anterior–posterior expression patterns between the marine Hox gene system and the Drosophila homeotic gene complexes 1,2 , called HOM-C (ref. 3), may point to highly conserved mechanisms for specifying positional identities (reviewed in ref. 4). Strong support for this concept lies in the observation of conserved colinearity between the genomic order of the Hox/HOM genes and their unique successive expression domains along the anterior-posterior axes of both mouse and fly embryos 1,2 . These unique and precise expression patterns appear to be facilitated by multiple cis -regulatory elements (reviewed in ref. 5). One of the few elements characterized in detail is the autoregulatory enhancer of the homeotic gene Deformed ( Dfd ) 6–9 , which supports expression in subregions of posterior head segments of Drosophila embryos 7 . Here we present evidence that this enhancer is capable of conferring reporter gene expression to a discrete subregion of the hindbrain in transgenic mouse embryos. Remarkably, this anterior-posterior subregion lies within the common anterior expression domain of the Dfd cognate Hox genes in the postotic hindbrain 10,11 . Our results indicate that the Dfd autoregulatory enhancer is part of a highly conserved mechanism for establishing region-specific gene expression along the anterior–posterior axis of the embryo.