Pluripotency factors functionally premark cell-type-restricted enhancers in ES cells.

Enhancers for embryonic stem (ES) cell-expressed genes and lineage-determining factors are characterized by conventional marks of enhancer activation in ES cells1-3, but it remains unclear whether enhancers destined to regulate cell-type-restricted transcription units might also have distinct signatures in ES cells. Here we show that cell-type-restricted enhancers are 'premarked' and activated as transcription units by the binding of one or two ES cell transcription factors, although they do not exhibit traditional enhancer epigenetic marks in ES cells, thus uncovering the initial temporal origins of cell-type-restricted enhancers. This premarking is required for future cell-type-restricted enhancer activity in the differentiated cells, with the strength of the ES cell signature being functionally important for the subsequent robustness of cell-type-restricted enhancer activation. We have experimentally validated this model in macrophage-restricted enhancers and neural precursor cell (NPC)-restricted enhancers using ES cell-derived macrophages or NPCs, edited to contain specific ES cell transcription factor motif deletions. DNA hydroxyl-methylation of enhancers in ES cells, determined by ES cell transcription factors, may serve as a potential molecular memory for subsequent enhancer activation in mature macrophages. These findings suggest that the massive repertoire of cell-type-restricted enhancers are essentially hierarchically and obligatorily premarked by binding of a defining ES cell transcription factor in ES cells, dictating the robustness of enhancer activation in mature cells

lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs.

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours

Epithelial Cell Integrin Β1 is Required for Developmental Angiogenesis in the Pituitary Gland

As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non–cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development

ATR inhibition facilitates targeting of leukemia dependence on convergent nucleotide biosynthetic pathways.

Leukemia cells rely on two nucleotide biosynthetic pathways, de novo and salvage, to produce dNTPs for DNA replication. Here, using metabolomic, proteomic, and phosphoproteomic approaches, we show that inhibition of the replication stress sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) reduces the output of both de novo and salvage pathways by regulating the activity of their respective rate-limiting enzymes, ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK), via distinct molecular mechanisms. Quantification of nucleotide biosynthesis in ATR-inhibited acute lymphoblastic leukemia (ALL) cells reveals substantial remaining de novo and salvage activities, and could not eliminate the disease in vivo. However, targeting these remaining activities with RNR and dCK inhibitors triggers lethal replication stress in vitro and long-term disease-free survival in mice with B-ALL, without detectable toxicity. Thus the functional interplay between alternative nucleotide biosynthetic routes and ATR provides therapeutic opportunities in leukemia and potentially other cancers.Leukemic cells depend on the nucleotide synthesis pathway to proliferate. Here the authors use metabolomics and proteomics to show that inhibition of ATR reduced the activity of these pathways thus providing a valuable therapeutic target in leukemia

Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells.

In the mammalian genome, the clustered protocadherin (cPCDH) locus provides a paradigm for stochastic gene expression with the potential to generate a unique cPCDH combination in every neuron. Here we report a chromatin-based mechanism that emerges during the transition from the naive to the primed states of cell pluripotency and reduces, by orders of magnitude, the combinatorial potential in the human cPCDH locus. This mechanism selectively increases the frequency of stochastic selection of a small subset of cPCDH genes after neuronal differentiation in monolayers, 10-month-old cortical organoids and engrafted cells in the spinal cords of rats. Signs of these frequent selections can be observed in the brain throughout fetal development and disappear after birth, except in conditions of delayed maturation such as Down's syndrome. We therefore propose that a pattern of limited cPCDH-gene expression diversity is maintained while human neurons still retain fetal-like levels of maturation

Understanding Enhancer Role in Transcriptional Response

In genetics, an enhancer is a short (50-1500 bp) region of DNA that can be bound with proteins (activators) to activate transcription of a gene or transcription. Despite their discovery more than 35 years ago, the fundamental principles by which enhancers are activated and regulate their coding gene transcriptional targets in metazoans have remained poorly understood. The molecular mechanisms responsible for orchestrating and integrating genome-wide transcriptional responses to diverse signaling pathways critical for developmental, physiological, and pathological regulation are still widely unknown. The Rosenfeld lab has made an effort to study these molecular mechanisms, focused on previously unsuspected aspects of enhancer function, chromosomal structure, and subnuclear architectural interactions. These strategies, which underlie genome-wide transcriptional responses in the endocrine and central nervous systems and are critical for physiological and behavioral processes in all vertebrates, are orchestrated by the network of genomic enhancers. Our recent findings have substantially altered concepts regarding the roles of noncoding RNAs (ncRNAs), mechanisms of enhancer activation and function, and nuclear architecture as critical aspects of regulated gene expression programs. This work has uncovered unexpected aspects of enhancer function, highlighting their functioning as regulated transcription units in dynamic alterations in nuclear architecture, by specific epigenomic strategies with therapeutic implications for many common diseases. We have studied the role of enhancers in a variety of biological systems: pituitary corticotrope, prostate cancer, breast cancer, and neuronal cells

Understanding Enhancer Role in Transcriptional Response

In genetics, an enhancer is a short (50-1500 bp) region of DNA that can be bound with proteins (activators) to activate transcription of a gene or transcription. Despite their discovery more than 35 years ago, the fundamental principles by which enhancers are activated and regulate their coding gene transcriptional targets in metazoans have remained poorly understood. The molecular mechanisms responsible for orchestrating and integrating genome-wide transcriptional responses to diverse signaling pathways critical for developmental, physiological, and pathological regulation are still widely unknown. The Rosenfeld lab has made an effort to study these molecular mechanisms, focused on previously unsuspected aspects of enhancer function, chromosomal structure, and subnuclear architectural interactions. These strategies, which underlie genome-wide transcriptional responses in the endocrine and central nervous systems and are critical for physiological and behavioral processes in all vertebrates, are orchestrated by the network of genomic enhancers. Our recent findings have substantially altered concepts regarding the roles of noncoding RNAs (ncRNAs), mechanisms of enhancer activation and function, and nuclear architecture as critical aspects of regulated gene expression programs. This work has uncovered unexpected aspects of enhancer function, highlighting their functioning as regulated transcription units in dynamic alterations in nuclear architecture, by specific epigenomic strategies with therapeutic implications for many common diseases. We have studied the role of enhancers in a variety of biological systems: pituitary corticotrope, prostate cancer, breast cancer, and neuronal cells

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