FoxP3 associates with enhancer-promoter loops to regulate Treg-specific gene expression

Gene expression programs are specified by higher-order chromatin structure and enhancer-promoter loops (EPL). T regulatory cells (Treg) identity is dominantly specified by the transcription factor FoxP3, whose mechanism of action is unclear. We applied proximity-ligation with chromatin immunoprecipitation (HiChIP) in Treg and closely related conventional CD4+ T cells (Tconv). EPL identified by H3K27Ac HiChIP showed a range of connection intensity, with some super-connected genes. TF-specific HiChIP showed that FoxP3 interacts with EPLs at a large number of genes, including some not differentially expressed in Treg vs Tconv, but enriched at the core Treg signature loci that it upregulates. FoxP3 association correlates with heightened H3H27Ac looping, as ascertained by analysis of FoxP3-deficient Treg-like cells. There was marked asymmetry in the loci where FoxP3 associated at the enhancer- or the promoter-side of EPLs, with enrichment for different transcriptional cofactors. FoxP3 EPL intensity distinguished gene clusters identified by single-cell ATAC-seq as co-varying between individual Tregs, supporting a direct transactivation model for FoxP3 in determining Treg identity.

Adipose MDM2 regulates systemic insulin sensitivity

The intimate association between obesity and type II diabetes urges for a deeper understanding of adipocyte function. We and others have previously delineated a role for the tumor suppressor p53 in adipocyte biology. Here, we show that mice haploinsufficient for MDM2, a key regulator of p53, in their adipose stores suffer from overt obesity, glucose intolerance, and hepatic steatosis. These mice had decreased levels of circulating palmitoleic acid [non-esterified fatty acid (NEFA) 16:1] concomitant with impaired visceral adipose tissue expression of Scd1 and Ffar4. A similar decrease in Scd and Ffar4 expression was found in in vitro differentiated adipocytes with perturbed MDM2 expression. Lowered MDM2 levels led to nuclear exclusion of the transcriptional cofactors, MORC2 and LIPIN1, and thereby possibly hampered adipocyte function by antagonizing LIPIN1-mediated PPARγ coactivation. Collectively, these data argue for a hitherto unknown interplay between MDM2 and MORC2/LIPIN1 involved in balancing adipocyte function.

Krüppel-homolog 1 exerts anti-metamorphic and vitellogenic functions in insects via phosphorylation-mediated recruitment of specific cofactors

Background The zinc-finger transcription factor Krüppel-homolog 1 (Kr-h1) exerts a dual regulatory role during insect development by preventing precocious larval/nymphal metamorphosis and in stimulating aspects of adult reproduction such as vitellogenesis. However, how Kr-h1 functions both as a transcriptional repressor in juvenile metamorphosis and an activator in adult reproduction remains elusive. Here, we use the insect Locusta migratoria to dissect the molecular mechanism by which Kr-h1 functions as activator and repressor at these distinct developmental stages. Results We report that the kinase PKCα triggers Kr-h1 phosphorylation at the amino acid residue Ser154, a step essential for its dual functions. During juvenile stage, phosphorylated Kr-h1 recruits a corepressor, C-terminal binding protein (CtBP). The complex of phosphorylated Kr-h1 and CtBP represses the transcription of Ecdysone induced protein 93F (E93) and consequently prevents the juvenile-to-adult transition. In adult insects, phosphorylated Kr-h1 recruits a coactivator, CREB-binding protein (CBP), and promotes vitellogenesis by inducing the expression of Ribosomal protein L36. Furthermore, Kr-h1 phosphorylation with the concomitant inhibition of E93 transcription is evolutionarily conserved across insect orders. Conclusion Our results suggest that Kr-h1 phosphorylation is indispensable for the recruitment of transcriptional cofactors, and for its anti-metamorphic and vitellogenic actions in insects. Our data shed new light on the understanding of Kr-h1 regulation and function in JH-regulated insect metamorphosis and reproduction.

Emerging Principles in the Transcriptional Control by YAP and TAZ

Yes-associated protein (YAP) and TAZ are transcriptional cofactors that sit at the crossroad of several signaling pathways involved in cell growth and differentiation. As such, they play essential functions during embryonic development, regeneration, and, once deregulated, in cancer progression. In this review, we will revise the current literature and provide an overview of how YAP/TAZ control transcription. We will focus on data concerning the modulation of the basal transcriptional machinery, their ability to epigenetically remodel the enhancer–promoter landscape, and the mechanisms used to integrate transcriptional cues from multiple pathways. This reveals how YAP/TAZ activation in cancer cells leads to extensive transcriptional control that spans several hallmarks of cancer. The definition of the molecular mechanism of transcriptional control and the identification of the pathways regulated by YAP/TAZ may provide therapeutic opportunities for the effective treatment of YAP/TAZ-driven tumors.

Epigenetic Regulation of Gonadotropin Hormone Beta Subunit Gene Expression

Gonadotropin releasing hormone (GnRH) from the hypothalamus regulates the synthesis and secretion of gonadotropin hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH regulates steroidogenesis in both sexes and ovulation in females, while FSH stimulates folliculogenesis in females and spermatogenesis in males. LH and FSH are heterodimers of a common alpha subunit and a unique beta subunit, which provides biological specificity and is the rate limiting factor in hormone synthesis. Immediate early genes, early growth response 1 (Egr1) and fos proto-oncogene (cFos) are critical in the induction of LH-beta and FSH-beta subunits by GnRH, respectfully. However, gaps exist in our understanding of developmental initiation and hormonal regulation of gonadotropin gene expression. Specifically, epigenetic mechanisms that may play a role in beta subunit transcriptional regulation are unknown. The aim of this work was to identify transcriptional cofactors that are recruited to gonadotropin beta-subunit promoters with or without GnRH. Transcription factors interact with cofactors that recruit chromatin remodeling enzymes in order to regulate transcription. Identification of cofactors may explain tight regulation of gonadotropin hormone levels in reproductive physiology. Previous studies identified regions on the beta-subunit promoters that are necessary for GnRH responsiveness. These regions were used to pull down interacting proteins that bind to these response elements using nuclear extracts from the immortalized mature gonadotrope cell line, LβT2. Using a discovery proteomics approach, we identified different transcriptional cofactors that are recruited to beta subunit promoters with or without GnRH. Approximately 1500-2000 proteins were identified per pulldown. As expected, proteins known to interact with beta subunit promoters, such as Egr1, cFos and cJun were identified in the DNA pulldown experiments as positive controls. We identified 63 proteins unique for LH-beta promoter under control conditions and 60 unique for FSH-beta promoter, of which 7 proteins for LH-beta and 8 proteins for FSH-beta may play a role as corepressors. We further identified 97 proteins that were pulled down with the LH-beta promoter following GnRH treatment, of which 9 proteins were also pulled down with Egr1 as potential coactivator candidates. We also identified 72 proteins that were pulled down with the FSH-beta promoter following GnRH treatment, of which 6 proteins were pulled down with cFos as potential coactivator candidates. Functional studies to identify roles of these cofactors in gonadotropin hormone expression are in progress. The identification of epigenetic regulators will allow for better understanding of the transcriptional regulation of gonadotropin beta-subunit gene expression, which is critical for reproductive function.

Folliculin: A Regulator of Transcription Through AMPK and mTOR Signaling Pathways

Folliculin (FLCN) is a tumor suppressor gene responsible for the inherited Birt-Hogg-Dubé (BHD) syndrome, which affects kidneys, skin and lungs. FLCN is a highly conserved protein that forms a complex with folliculin interacting proteins 1 and 2 (FNIP1/2). Although its sequence does not show homology to known functional domains, structural studies have determined a role of FLCN as a GTPase activating protein (GAP) for small GTPases such as Rag GTPases. FLCN GAP activity on the Rags is required for the recruitment of mTORC1 and the transcriptional factors TFEB and TFE3 on the lysosome, where mTORC1 phosphorylates and inactivates these factors. TFEB/TFE3 are master regulators of lysosomal biogenesis and function, and autophagy. By this mechanism, FLCN/FNIP complex participates in the control of metabolic processes. AMPK, a key regulator of catabolism, interacts with FLCN/FNIP complex. FLCN loss results in constitutive activation of AMPK, which suggests an additional mechanism by which FLCN/FNIP may control metabolism. AMPK regulates the expression and activity of the transcriptional cofactors PGC1α/β, implicated in the control of mitochondrial biogenesis and oxidative metabolism. In this review, we summarize our current knowledge of the interplay between mTORC1, FLCN/FNIP, and AMPK and their implications in the control of cellular homeostasis through the transcriptional activity of TFEB/TFE3 and PGC1α/β. Other pathways and cellular processes regulated by FLCN will be briefly discussed.

Temporal mechanisms of myogenic specification in human induced pluripotent stem cells

Understanding the mechanisms of myogenesis in human induced pluripotent stem cells (hiPSCs) is a prerequisite to achieving patient-specific therapy for diseases of skeletal muscle. hiPSCs of different origin show distinctive kinetics and ability to differentiate into myocytes. To address the unique cellular and temporal context of hiPSC differentiation, we perform a longitudinal comparison of the transcriptomic profiles of three hiPSC lines that display differential myogenic specification, one robust and two blunted. We detail temporal differences in mechanisms that lead to robust myogenic specification. We show gene expression signatures of putative cell subpopulations and extracellular matrix components that may support myogenesis. Furthermore, we show that targeted knockdown of ZIC3 at the outset of differentiation leads to improved myogenic specification in blunted hiPSC lines. Our study suggests that β-catenin transcriptional cofactors mediate cross-talk between multiple cellular processes and exogenous cues to facilitate specification of hiPSCs to mesoderm lineage, leading to robust myogenesis.

A new perspective on the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

A new perspective on the evolution of the interaction between the Vg/VGLL1-3 proteins and the TEAD transcription factors

The most downstream elements of the Hippo pathway, the TEAD transcription factors, are regulated by several cofactors, such as Vg/VGLL1-3. Earlier findings on human VGLL1 and here on human VGLL3 show that these proteins interact with TEAD via a conserved amino acid motif called the TONDU domain. Surprisingly, our studies reveal that the TEAD-binding domain of Drosophila Vg and of human VGLL2 is more complex and contains an additional structural element, an Ω-loop, that contributes to TEAD binding and in vivo function. To explain this unexpected structural difference between proteins from the same family, we propose that, after the genome-wide duplications at the origin of vertebrates, the Ω-loop present in an ancestral VGLL gene has been lost in some VGLL variants. These findings illustrate how structural and functional constraints can guide the evolution of transcriptional cofactors to preserve their ability to compete with other cofactors for binding to transcription factors.

Division of labor between YAP and TAZ in non-small cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide. The paralogous transcriptional cofactors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ, also called WWTR1), the main downstream effectors of the Hippo signal transduction pathway, are emerging as pivotal determinants of malignancy in lung cancer. Traditionally, studies have tended to consider YAP and TAZ as functionally redundant transcriptional cofactors, with similar biological impact. However, there is growing evidence that each of them also possesses distinct attributes. Here, we sought to systematically characterize the division of labor between YAP and TAZ in non-small cell lung cancer (NSCLC), the most common histological subtype of lung cancer. Employing representative NSCLC cell lines, as well as patient-derived data, we show that the two paralogs orchestrate non-overlapping transcription programs in this cancer type: whereas YAP preferentially regulates gene sets associated with cell division and cell cycle progression, TAZ preferentially regulates genes associated with extracellular matrix organization. Concordantly, depletion of YAP, but not TAZ, leads to growth arrest, while YAP overexpression promotes cell proliferation. Likewise, depletion of TAZ, but not YAP, compromises cell migration, whereas TAZ overexpression enhances migration. Importantly, the differential effects of YAP vs TAZ on key cellular processes are also associated with differential response to anti-cancer therapies. Uncovering the different activities and downstream effects of YAP and TAZ may thus facilitate better stratification of lung cancer patients for anti-cancer therapies.