Loss of EZH2 results in precocious mammary gland development and activation of STAT5-dependent genes.

Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activator EZH2 controls the differentiation clock of mammary epithelium. Ablation of Ezh2 from mammary stem cells resulted in precocious differentiation of alveolar epithelium during pregnancy and the activation of mammary-specific STAT5 target genes. This coincided with enhanced occupancy of these loci by STAT5, EZH1 and RNA Pol II. Limited activation of differentiation-specific genes was observed in mammary epithelium lacking both EZH2 and STAT5, suggesting a modulating but not mandatory role for STAT5. Loss of EZH2 did not result in overt changes in genome-wide and gene-specific H3K27me3 profiles, suggesting compensation through enhanced EZH1 recruitment. Differentiated mammary epithelia did not form in the combined absence of EZH1 and EZH2. Transplantation experiments failed to demonstrate a role for EZH2 in the activity of mammary stem and progenitor cells. In summary, while EZH1 and EZH2 serve redundant functions in the establishment of H3K27me3 marks and the formation of mammary alveoli, the presence of EZH2 is required to control progressive differentiation of milk secreting epithelium during pregnancy.

Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A.

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.

The STAT5-regulated miR-193b locus restrains mammary stem and progenitor cell activity and alveolar differentiation.

The transcription factor STAT5 mediates prolactin signaling and controls functional development of mammary tissue during pregnancy. This study has identified the miR-193b locus, also encoding miRNAs 365-1 and 6365, as a STAT5 target in mammary epithelium. While the locus was characterized by active histone marks in mammary tissue, STAT5 binding and expression during pregnancy, it was silent in most non-mammary cells. Inactivation of the miR-193b locus in mice resulted in elevated mammary stem/progenitor cell activity as judged by limiting dilution transplantation experiments of primary mammary epithelial cells. Colonies formed by mutant cells were larger and contained more Ki-67 positive cells. Differentiation of mammary epithelium lacking the miR-193b locus was accelerated during puberty and pregnancy, which coincided with the loss of Cav3 and elevated levels of Elf5. Normal colony development was partially obtained upon ectopically expressing Cav3 or upon siRNA-mediated reduction of Elf5 in miR-193b-null primary mammary epithelial cells. This study reveals a previously unknown link between the mammary-defining transcription factor STAT5 and a microRNA cluster in controlling mammary epithelial differentiation and the activity of mammary stem and progenitor cells.

Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration.

The transcription factors Signal Transducer and Activator of Transcription (STAT) 5A/B mediate prolactin-induced mammary development during pregnancy. However, it is not clear how the different processes, expansion and maturation of alveolar precursor cells and the differential induction of milk protein genes are regulated on a molecular level. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts mammary epithelial development during pregnancy and the regulation of target genes. The presence of only a single Stat5a or Stat5b allele was sufficient for the establishment of histologically undifferentiated alveolar units and two alleles permitted the execution of a differentiation program similar to that found with all four alleles. While one copy of Stat5 induced limited expression of target genes, two copies activated a lactation-like gene signature. Using ChIP-seq analyses on intact tissue under physiological conditions, we found that highly expressed and regulated genes were bound by STAT5 in their promoter proximal regions, whereas upstream binding had minor biological consequences. Remarkably, 80% of the genes bound by STAT5 in vivo were not under STAT5 control. RNA polymerase II intensity was directly proportional to STAT5 concentration only on STAT5 regulated genes providing mechanistic insight by which STAT5 activates mammary specific genes.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT) genomic binding patterns discerns cell-specific cis-regulatory modules.

BACKGROUND: Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription) family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. RESULTS: We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. CONCLUSIONS: Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

Cooperation of signalling pathways in embryonic mammary gland development.

Mammary glands become functional only in adult life but their development starts in the embryo. Initiation of the epithelial bud and ductal outgrowth are coordinated through short-range signals between epithelium and mesenchyme. Studies of natural and induced mouse mutants in which early mammary development is perturbed have identified genetic networks that regulate specific steps in these processes. Some of these signals contribute to aberrant mammary development in humans and are deregulated in cancer.

MiR-193b and miR-365-1 are not required for the development and function of brown fat in the mouse.

Generating heat and maintaining body temperature is the primary function of brown adipose tissue (BAT). Previous studies have implicated microRNAs, including miR-193b and miR-365-1, in BAT differentiation. We used mouse genetics to further understand the specific contributions of these two miRs. BAT function in mice with an inactivated miR-193b-365-1 locus, as determined by their response to the selective ß3 adrenergic receptor agonist CL316.243 and their tolerance to cold exposure, was normal and expression of genes associated with functional BAT, including Prdm16 and Ucp1, was unaffected. In addition, genome-wide expression profiles of miRNAs and mRNAs in BAT in the presence and absence of miR-193b-365-1 were determined. In summary, these data demonstrate, in contrast to earlier work, that the development, differentiation, and function of BAT do not require the presence of miR-193b and miR-365-1.

Thymic stromal lymphopoietin-mediated STAT5 phosphorylation via kinases JAK1 and JAK2 reveals a key difference from IL-7-induced signaling.

Thymic stromal lymphopoietin (TSLP) is a type I cytokine that plays essential roles in allergic/inflammatory skin and airway disorders, in helminth infections, and in regulating intestinal immunity. TSLP signals via IL-7Rα and a specific TSLPR subunit that is highly related to the common cytokine receptor γ chain, γ(c). Although TSLP has effects on a broad range of hematopoetic cells and can induce STAT5 phosphorylation, TSLP was reported to not signal via JAK kinases, and the mechanism by which TSLP regulates STAT5 phosphorylation has been unclear. We now demonstrate the role of JAK1 and JAK2 in TSLP-mediated STAT5 phosphorylation in mouse and human primary CD4(+) T cells, in contrast to the known activation of JAK1 and JAK3 by the related cytokine, IL-7. We also show that just as JAK1 interacts with IL-7Rα, JAK2 is associated with TSLPR protein. Moreover, we demonstrate the importance of STAT5 activation for TSLP-mediated survival and proliferation of CD4(+) T cells. These findings clarify the basis for TSLP-mediated signaling and provide an example wherein a cytokine uses JAK1 and JAK2 to mediate the activation of STAT5.

The miR-17/92 cluster is targeted by STAT5 but dispensable for mammary development.

Genome wide analysis revealed the miR-17/92 cluster as a STAT5 target. This cluster encodes six microRNAs, which predictably target genes that play a role in mammary gland development. In this study, we have deleted the miR-17/92 cluster in mammary stem cells and evaluated in the mouse its function during mammary gland development. Loss of the miR-17/92 cluster did not affect mammary development from prepuberty to lactation. Our studies demonstrated that, while expression of the miR-17/92 cluster is under control of the key mammary transcription factor STAT5, its presence is not required for normal mammary development or lactation.

The gene encoding the hematopoietic stem cell regulator CCN3/NOV is under direct cytokine control through the transcription factors STAT5A/B.

Cytokines control the biology of hematopoietic stem cells (HSCs) and progenitor cells in part through the transcription factors STAT5A/B. To investigate the target genes of STAT5A/B activated by cytokines in HSCs and progenitors, we performed microarray analyses using Lineage(-) Sca-1(+) c-Kit(+) (KSL) cells in the presence and absence of STAT5A/B. Stimulation with a mixture containing IL-3, IL-6, stem cell factor, thrombopoietin, and Flt3 ligand induced Ccn3/Nov mRNA over 100-fold in WT (control) but not Stat5a/b-null KSL cells. CCN3/NOV is a positive regulator of human HSC self-renewal and development of committed blood cells. Without stimulation, the Ccn3/Nov signal level was low in control KSL cells similar to Stat5a/b-null KSL cells. To determine which cytokine activates the Ccn3/Nov gene, we analyzed Lineage(-) c-Kit(+) (KL) and 32D cells using quantitative PCR and ChIP assays. Although stimulation with a mixture lacking IL-3 prevented the induction of Ccn3/Nov in control KL cells, IL-3 alone could induce Ccn3/Nov mRNA in control KL and 32D cells. ChIP assays using 32D cells revealed IL-3-induced binding of STAT5A/B to a γ-interferon-activated sequences site in the Ccn3/Nov gene promoter. This is the first report that Ccn3/Nov is directly induced by cytokines through STAT5A/B.

The transcription factors signal transducer and activator of transcription 5A (STAT5A) and STAT5B negatively regulate cell proliferation through the activation of cyclin-dependent kinase inhibitor 2b (Cdkn2b) and Cdkn1a expression.

UNLABELLED: Although the cytokine-inducible transcription factor signal transducer and activator of transcription 5 (STAT5) promotes proliferation of a wide range of cell types, there are cell-specific and context-specific cases in which loss of STAT5 results in enhanced cell proliferation. Here, we report that loss of STAT5 from mouse embryonic fibroblasts (MEFs) leads to enhanced proliferation, which was linked to reduced levels of the cell cycle inhibitors p15(INK4B) and p21(CIP1). We further demonstrate that growth hormone, through the transcription factor STAT5, enhances expression of the Cdkn2b (cyclin-dependent kinase inhibitor 2B) gene and that STAT5A binds to interferon-gamma-activated sequence sites within the promoter. We recently demonstrated that ablation of STAT5 from liver results in hepatocellular carcinoma upon CCl4 treatment. We now establish that STAT5, like in MEFs, activates expression of the Cdkn2b gene in liver tissue. Loss of STAT5 led to diminished p15(INK4B) and increased hepatocyte proliferation. CONCLUSION: This study for the first time demonstrates that cytokines, through STAT5, induce the expression of a key cell cycle inhibitor. These experiments therefore shed mechanistic light on the context-specific role of STAT5 as tumor suppressor.

MMTV-Cre transgenes can adversely affect lactation: considerations for conditional gene deletion in mammary tissue.

CRE-loxP-mediated inactivation and activation of genes in mouse mammary epithelium have been widely used to study genetic pathways in normal development and neoplastic transformation in vivo. In 1997, we generated three distinct mouse lines carrying an identical MMTV-Cre transgene (lines A, D, and F). Because the presence of CRE recombinase can adversely affect the physiology of nonmammary cells, we explored whether transgenic females display lactational defects. Whereas dams from line D nurse their pups and display overtly normal mammary development, line A shows some impairment during lactation and females from line F completely fail to nurse their litters. The ability to nurse a litter correlates with the extent of alveolar development and differentiation. This study demonstrates the importance of including appropriate "Cre-only" controls and provides guidelines to avoid problems in data interpretation.

Hematopoietic-specific Stat5-null mice display microcytic hypochromic anemia associated with reduced transferrin receptor gene expression.

Iron is essential for all cells but is toxic in excess, so iron absorption and distribution are tightly regulated. Serum iron is bound to transferrin and enters erythroid cells primarily via receptor-mediated endocytosis of the transferrin receptor (Tfr1). Tfr1 is essential for developing erythrocytes and reduced Tfr1 expression is associated with anemia. The transcription factors STAT5A/B are activated by many cytokines, including erythropoietin. Stat5a/b(-/-) mice are severely anemic and die perinatally, but no link has been made to iron homeostasis. To study the function of STAT5A/B in vivo, we deleted the floxed Stat5a/b locus in hematopoietic cells with a Tie2-Cre transgene. These mice exhibited microcytic, hypochromic anemia, as did lethally irradiated mice that received a transplant of Stat5a/b(-/-) fetal liver cells. Flow cytometry and RNA analyses of erythroid cells from mutant mice revealed a 50% reduction in Tfr1 mRNA and protein. We detected STAT5A/B binding sites in the first intron of the Tfr1 gene and found that expression of constitutively active STAT5A in an erythroid cell line increased Tfr1 levels. Chromatin immunoprecipitation experiments confirmed the binding of STAT5A/B to these sites. We conclude that STAT5A/B is an important regulator of iron update in erythroid progenitor cells via its control of Tfr1 transcription.

Acute myeloid leukemia-associated Mkl1 (Mrtf-a) is a key regulator of mammary gland function.

Transcription of immediate-early genes--as well as multiple genes affecting muscle function, cytoskeletal integrity, apoptosis control, and wound healing/angiogenesis--is regulated by serum response factor (Srf). Extracellular signals regulate Srf in part via a pathway involving megakaryoblastic leukemia 1 (Mkl1, also known as myocardin-related transcription factor A [Mrtf-a]), which coactivates Srf-responsive genes downstream of Rho GTPases. Here we investigate Mkl1 function using gene targeting and show the protein to be essential for the physiologic preparation of the mammary gland during pregnancy and the maintenance of lactation. Lack of Mkl1 causes premature involution and impairs expression of Srf-dependent genes in the mammary myoepithelial cells, which control milk ejection following oxytocin-induced contraction. Despite the importance of Srf in multiple transcriptional pathways and widespread Mkl1 expression, the spectrum of abnormalities associated with Mkl1 absence appears surprisingly restricted.

The transcription factors Stat5a/b are not required for islet development but modulate pancreatic beta-cell physiology upon aging.

In insulinoma cell lines proliferation and insulin gene transcription are stimulated by growth hormone and prolactin, which convey their signals through the transcription factors Stat5a and 5b (referred to as Stat5). However, the contribution of Stat5 to the physiology of beta-cells in vivo could not be assessed directly since Stat5-null mice die perinataly. To explore the physiological role of Stat5 in the mouse, the corresponding gene locus targeted with loxP sites was inactivated in beta-cells using two lines of Cre recombinase expressing transgenic mice. While the RIP-Cre transgene is active in pancreatic beta-cells and the hypothalamus, the Pdx1-Cre transgene is active in precursor cells of the endocrine and exocrine pancreas. Mice carrying two floxed Stat5alleles and a RIP-Cre transgene developed mild obesity, were hyperglycemic and exhibited impaired glucose tolerance. Since RIP-Cre transgenic mice by themselves display some glucose intolerance, the significance of these data is unclear. In contrast, mice, in which the Stat5 locus had been deleted with the Pdx1-Cre transgene, developed functional islets and were glucose tolerant. Mild glucose intolerance occurred with age. We conclude that Stat5 is not essential for islet development but may modulate beta-cell function.

Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation.

This study explored the functions of the signal transducers and activators of transcription 5a and 5b (referred to as Stat5 here) during different stages of mouse mammary gland development by using conditional gene inactivation. Mammary gland morphogenesis includes cell specification, proliferation and differentiation during pregnancy, cell survival and maintenance of differentiation throughout lactation, and cell death during involution. Stat5 is activated by prolactin, and its presence is mandatory for the proliferation and differentiation of mammary epithelium during pregnancy. To address the question of whether Stat5 is also necessary for the maintenance and survival of the differentiated epithelium, the two genes were deleted at different time points. The 110-kb Stat5 locus in the mouse was bracketed with loxP sites, and its deletion was accomplished by using two Cre-expressing transgenic lines. Loss of Stat5 prior to pregnancy prevented epithelial proliferation and differentiation. Deletion of Stat5 during pregnancy, after mammary epithelium had entered Stat5-mediated differentiation, resulted in premature cell death, indicating that at this stage epithelial cell proliferation, differentiation, and survival require Stat5.

Selective removal of the selenocysteine tRNA [Ser]Sec gene (Trsp) in mouse mammary epithelium.

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.

Subset- and tissue-defined STAT5 thresholds control homeostasis and function of innate lymphoid cells.

Innate lymphoid cells (ILCs) patrol environmental interfaces to defend against infection and protect barrier integrity. Using a genetic tuning model, we demonstrate that the signal-dependent transcription factor (TF) STAT5 is critical for accumulation of all known ILC subsets in mice and reveal a hierarchy of STAT5 dependency for populating lymphoid and nonlymphoid tissues. We apply transcriptome and genomic distribution analyses to define a STAT5 gene signature in natural killer (NK) cells, the prototypical ILC subset, and provide a systems-based molecular rationale for its key functions downstream of IL-15. We also uncover surprising features of STAT5 behavior, most notably the wholesale redistribution that occurs when NK cells shift from tonic signaling to acute cytokine-driven signaling, and genome-wide coordination with T-bet, another key TF in ILC biology. Collectively, our data position STAT5 as a central node in the TF network that instructs ILC development, homeostasis, and function and provide mechanistic insights on how it works at cellular and molecular levels.

Histone Demethylase KDM6A Controls the Mammary Luminal Lineage through Enzyme-Independent Mechanisms.

Establishment of the mammary luminal cell lineage is controlled primarily by hormones and through specific transcription factors (TFs). Previous studies have linked histone methyltransferases to the differentiation of mammary epithelium, thus opening the possibility of biological significance of counteracting demethylases. We have now demonstrated an essential role for the H3K27me3 demethylase KDM6A in generating a balanced alveolar compartment. Deletion of Kdm6a in the mammary luminal cell lineage led to a paucity of luminal cells and an excessive expansion of basal cells, both in vivo and in vitro The inability to form structurally normal ducts and alveoli during pregnancy resulted in lactation failure. Mutant luminal cells did not exhibit their distinctive transcription factor pattern and displayed basal characteristics. The genomic H3K27me3 landscape was unaltered in mutant tissue, and support for a demethylase-independent mechanism came from mice expressing a catalytically inactive KDM6A. Mammary tissue developed normally in these mice. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments demonstrated KDM6A binding to putative enhancers enriched for key mammary TFs and H3K27ac. This study demonstrated for the first time that the mammary luminal lineage relies on KDM6A to ensure a transcription program leading to differentiated alveoli. Failure to fully implement this program results in structurally and functionally impaired mammary tissue.

Signal transducer and activator of transcription 5 (STAT5) paralog dose governs T cell effector and regulatory functions.

The transcription factor STAT5 is fundamental to the mammalian immune system. However, the relationship between its two paralogs, STAT5A and STAT5B, and the extent to which they are functionally distinct, remain uncertain. Using mouse models of paralog deficiency, we demonstrate that they are not equivalent for CD4(+) 'helper' T cells, the principal orchestrators of adaptive immunity. Instead, we find that STAT5B is dominant for both effector and regulatory (Treg) responses and, therefore, uniquely necessary for immunological tolerance. Comparative analysis of genomic distribution and transcriptomic output confirm that STAT5B has fargreater impact but, surprisingly, the data point towards asymmetric expression (i.e. paralog dose), rather than distinct functional properties, as the key distinguishing feature. Thus, we propose a quantitative model of STAT5 paralog activity whereby relative abundance imposes functional specificity (or dominance) in the face of widespread structural homology.