Summary Maintenance of single layered endothelium, squamous endothelial cell shape, and formation of a patent vascular lumen all require defined endothelial cell polarity. Loss of β1 integrin (Itgb1) in nascent endothelium leads to disruption of arterial endothelial cell polarity and lumen formation. The loss of polarity is manifested as cuboidal shaped endothelial cells, dysregulated levels and mis-localization of normally polarized cell-cell adhesion molecules, as well as decreased expression of the polarity gene Par3 (pard3). β1 integrin and Par3 are both localized to the endothelial layer, with preferential expression of Par3 in arterial endothelium. Luminal occlusion is also exclusively noted in arteries, and is partially rescued by replacement of Par3 protein in β1 deficient vessels. Combined, our findings demonstrate that β1 integrin functions upstream of Par3 as part of a molecular cascade required for endothelial cell polarity and lumen formation.

CD4 + Foxp3 + regulatory T (T reg ) cells are important for maintaining immune tolerance. Understanding the molecular mechanism that regulates T reg differentiation will facilitate the development of effective therapeutic strategies against autoimmune diseases. We report here that the SUMO E3 ligase PIAS1 restricts the differentiation of natural T reg cells by maintaining a repressive chromatin state of the Foxp3 promoter. PIAS1 acts by binding to the Foxp3 promoter to recruit DNA methyltransferases and heterochromatin protein 1 for epigenetic modifications. Pias1 deletion caused promoter demethylation, reduced histone H3 methylation at Lys 9 , and enhanced promoter accessibility. Consistently, Pias1 −/− mice displayed an increased natural T reg cell population and were resistant to the development of experimental autoimmune encephalomyelitis. Our studies have identified an epigenetic mechanism that negatively regulates the differentiation of natural T reg cells.Naturally occurring thymus-derived regulatory T (nT reg ) cells play a critical role in the maintenance of self-tolerance and homeostasis within the immune system (1-4). The transcription factor Foxp3 controls the development and function of T reg cells (5-7). Several regulatory DNA elements within the Foxp3 locus have been suggested to modulate Foxp3 expression, including the promoter region and conserved noncoding sequences (6-9). Although the transcription factors that positively regulate Foxp3 expression are well characterized (10-13), little is known about the negative regulation of Foxp3.PIAS1 (protein inhibitor of the activated signal transducer and activator of transcription STAT1) is a SUMO E3 ligase that binds to chromatin to repress transcription (14,15). The recruitment of PIAS1 to chromatin requires it to be phosphorylated on Ser 90 . This is induced by a variety of immune regulatory stimuli, including TCR (T cell receptor) activation (16). PIAS1 was phosphorylated on Ser 90 in freshly isolated thymocytes and splenocytes (17) (Fig. 1A). There was a small increase in the percentage of thymic single-positive CD4 + or CD8 + T cells in Pias1 −/− mice, although the total T cell number was not significantly altered ( fig. S1, A and B).The frequency of both thymic and splenic CD4 + Foxp3 + nT reg cells was † To whom correspondence should be addressed. bliu@ucla.edu (B.L.); kshuai@mednet.ucla.edu (K.S.). * These authors contributed equally to this manuscript. (Fig. 1B). In addition, the number of thymic CD4 + Foxp3 + nT reg cells was also significantly increased in Pias1 −/− mice (about 135%) (Fig. 1B). However, the mean intensity of Foxp3 expression in Foxp3 + cells was not altered in Pias1 −/− cells ( fig. S1C). Moreover, Pias1 disruption had no significant effect on the in vivo proliferation or survival of T reg cells ( fig. S2).To test whether PIAS1 directly regulates the intrinsic differentiation potential of nT reg cells, bone marrow from wild-type and Pias1 −/− mice depleted of CD4 + and CD8 + T cells was transplanted into sublethall...

Loss of the CDK inhibitor p27KIP1 is widely linked with poor prognosis in human cancer. In Wnt10b-expressing mammary tumors, levels of p27 KIP1 were extremely low; conversely, Wnt10b-null mammary cells expressed high levels of this protein, suggesting Wnt-dependent regulation of p27 KIP1 . Interestingly we found that Wnt-induced turnover of p27 KIP1 was independent from classical SCF SKP2 -mediated degradation in both mouse and human cells. Instead, turnover required Cullin 4A and Cullin 4B, components of an alternative E3 ubiquitin ligase induced in response to active Wnt signaling. We found that CUL4A was a novel Wnt target gene in both mouse and human cells and that CUL4A physically interacted with p27 KIP1 in Wnt-responding cells. We further demonstrated that both Cul4A and Cul4B were required for Wnt-induced p27 KIP1 degradation and S-phase progression. CUL4A and CUL4B are therefore components of a conserved Wnt-induced proteasome targeting (WIPT) complex that regulates p27 KIP1 levels and cell cycle progression in mammalian cells.[Keywords: Basal-like breast cancer; reprogramming; Wnt-induced proteasome targeting (WIPT); mammary progenitor cells; mammary stem cell (MSC); Cdkn1b] Supplemental material is available at http://www.genesdev.org.

Epigenetic gene silencing by histone modifications and DNA methylation is essential for cancer development. The molecular mechanism that promotes selective epigenetic changes during tumorigenesis is not understood. We report here that the PIAS1 SUMO ligase is involved in the progression of breast tumorigenesis. Elevated PIAS1 expression was observed in breast tumor samples. PIAS1 knockdown in breast cancer cells reduced the subpopulation of tumor-initiating cells, and inhibited breast tumor growth in vivo. PIAS1 acts by delineating histone modifications and DNA methylation to silence the expression of a subset of clinically relevant genes, including breast cancer DNA methylation signature genes such as cyclin D2 and estrogen receptor, and breast tumor suppressor WNT5A. Our studies identify a novel epigenetic mechanism that regulates breast tumorigenesis through selective gene silencing.

The selective and temporal DNA methylation plays an important role in the self-renewal and differentiation of hematopoietic stem cells (HSCs), but the molecular mechanism that controls the dynamics of DNA methylation is not understood. Here, we report that the PIAS1 epigenetic pathway plays an important role in regulating HSC self-renewal and differentiation. PIAS1 is required for maintaining the quiescence of dormant HSCs and the long-term repopulating capacity of HSC. Pias1 disruption caused the abnormal expression of lineage-associated genes. Bisulfite sequencing analysis revealed the premature promoter demethylation of Gata1, a key myeloerythroid transcription factor and a PIAS1-target gene, in Pias1 À/À HSCs. As a result, Pias1 disruption caused the inappropriate induction of Gata1 in HSCs and common lymphoid progenitors (CLPs). The expression of other myeloerythroid genes was also enhanced in CLPs and lineage-negative progenitors, with a concurrent repression of B cell-specific genes. Consistently, Pias1 disruption caused enhanced myeloerythroid, but reduced B lymphoid lineage differentiation. These results identify a novel role of PIAS1 in maintaining the quiescence of dormant HSCs and in the epigenetic repression of the myeloerythroid program.