Paracrine FGF1 signaling directs pituitary architecture and size.

Organ architecture is established during development through intricate cell-cell communication mechanisms, yet the specific signals mediating these communications often remain elusive. Here, we used the anterior pituitary gland that harbors different interdigitated hormone-secreting homotypic cell networks to dissect cell-cell communication mechanisms operating during late development. We show that blocking differentiation of corticotrope cells leads to pituitary hypoplasia with a major effect on somatotrope cells that directly contact corticotropes. Gene knockout of the corticotrope-restricted transcription factor Tpit results in fewer somatotropes, with less secretory granules and a loss of cell polarity, resulting in systemic growth retardation. Single-cell transcriptomic analyses identified FGF1 as a corticotrope-specific Tpit dosage-dependent target gene responsible for these phenotypes. Consistently, genetic ablation of FGF1 in mice phenocopies pituitary hypoplasia and growth impairment observed in Tpit-deficient mice. These findings reveal FGF1 produced by the corticotrope cell network as an essential paracrine signaling molecule participating in pituitary architecture and size.

The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling.

The anterior and intermediate lobes of the pituitary gland derive from the surface ectoderm. They provide a simple system to assess mechanisms of developmental identity established by tissue determinants. Each lobe contains a lineage expressing the hormone precursor pro-opiomelanocortin (POMC): the corticotropes and melanotropes. The T-box transcription factor Tpit controls terminal differentiation of both lineages. We now report on the unique role of Pax7 as a selector of intermediate lobe and melanotrope identity. Inactivation of the Pax7 gene results in loss of melanotrope gene expression and derepression of corticotrope genes. Pax7 acts by remodeling chromatin and allowing Tpit binding to a new subset of enhancers for activation of melanotrope-specific genes. Thus, the selector function of Pax7 is exerted through pioneer transcription factor activity. Genome-wide, the Pax7 pioneer activity is preferentially associated with composite binding sites that include paired and homeodomain motifs. Pax7 expression is conserved in human and dog melanotropes and defines two subtypes of pituitary adenomas causing Cushing's disease. In summary, expression of Pax7 provides a unique tissue identity to the pituitary intermediate lobe that alters Tpit-driven differentiation through pioneer and classical transcription factor activities.

Mutations in NFKB2 and potential genetic heterogeneity in patients with DAVID syndrome, having variable endocrine and immune deficiencies.

BACKGROUND: DAVID syndrome is a rare condition combining anterior pituitary hormone deficiency with common variable immunodeficiency. NFKB2 mutations have recently been identified in patients with ACTH and variable immunodeficiency. A similar mutation was previously found in Nfkb2 in the immunodeficient Lym1 mouse strain, but the effect of the mutation on endocrine function was not evaluated. METHODS: We ascertained six unrelated DAVID syndrome families. We performed whole exome and traditional Sanger sequencing to search for causal genes. Lym1 mice were examined for endocrine developmental anomalies. RESULTS: Mutations in the NFKB2 gene were identified in three of our families through whole exome sequencing, and in a fourth by direct Sanger sequencing. De novo origin of the mutations could be demonstrated in three of the families. All mutations lie near the C-terminus of the protein-coding region, near signals required for processing of NFΚB2 protein by the alternative pathway. Two of the probands had anatomical pituitary anomalies, and one had growth and thyroid hormone as well as ACTH deficiency; these findings have not been previously reported. Two children of one of the probands carried the mutation and have to date exhibited only an immune phenotype. No mutations were found near the C-terminus of NFKB2 in the remaining two probands; whole exome sequencing has been performed for one of these. Lym1 mice, carrying a similar Nfkb2 C-terminal mutation, showed normal pituitary anatomy and expression of proopiomelanocortin (POMC). CONCLUSIONS: We confirm previous findings that mutations near the C-terminus of NFKB2 cause combined endocrine and immunodeficiencies. De novo status of the mutations was confirmed in all cases for which both parents were available. The mutations are consistent with a dominant gain-of-function effect, generating an unprocessed NFKB2 super-repressor protein. We expand the potential phenotype of such NFKB2 mutations to include additional pituitary hormone deficiencies as well as anatomical pituitary anomalies. The lack of an observable endocrine phenotype in Lym1 mice suggests that the endocrine component of DAVID syndrome is either not due to a direct role of NFKB pathways on pituitary development, or else that human and mouse pituitary development differ in its requirements for NFKB pathway function.

Transcription cofactors TRIM24, TRIM28, and TRIM33 associate to form regulatory complexes that suppress murine hepatocellular carcinoma.

TRIM24 (TIF1α), TRIM28 (TIF1ß), and TRIM33 (TIF1γ) are three related cofactors belonging to the tripartite motif superfamily that interact with distinct transcription factors. TRIM24 interacts with the liganded retinoic acid (RA) receptor to repress its transcriptional activity. Germ line inactivation of TRIM24 in mice deregulates RA-signaling in hepatocytes leading to the development of hepatocellular carcinoma (HCC). Here we show that TRIM24 can be purified as at least two macromolecular complexes comprising either TRIM33 or TRIM33 and TRIM28. Somatic hepatocyte-specific inactivation of TRIM24, TRIM28, or TRIM33 all promote HCC in a cell-autonomous manner in mice. Moreover, HCC formation upon TRIM24 inactivation is strongly potentiated by further loss of TRIM33. These results demonstrate that the TIF1-related subfamily of TRIM proteins interact both physically and functionally to modulate HCC formation in mice.

Negative control of Smad activity by ectodermin/Tif1gamma patterns the mammalian embryo.

The definition of embryonic potency and induction of specific cell fates are intimately linked to the tight control over TGFbeta signaling. Although extracellular regulation of ligand availability has received considerable attention in recent years, surprisingly little is known about the intracellular factors that negatively control Smad activity in mammalian tissues. By means of genetic ablation, we show that the Smad4 inhibitor ectodermin (Ecto, also known as Trim33 or Tif1gamma) is required to limit Nodal responsiveness in vivo. New phenotypes, which are linked to excessive Nodal activity, emerge from such a modified landscape of Smad responsiveness in both embryonic and extra-embryonic territories. In extra-embryonic endoderm, Ecto is required to confine expression of Nodal antagonists to the anterior visceral endoderm. In trophoblast cells, Ecto precisely doses Nodal activity, balancing stem cell self-renewal and differentiation. Epiblast-specific Ecto deficiency shifts mesoderm fates towards node/organizer fates, revealing the requirement of Smad inhibition for the precise allocation of cells along the primitive streak. This study unveils that intracellular negative control of Smad function by ectodermin/Tif1gamma is a crucial element in the cellular response to TGFbeta signals in mammalian tissues.

Tripartite motif 24 (Trim24/Tif1α) tumor suppressor protein is a novel negative regulator of interferon (IFN)/signal transducers and activators of transcription (STAT) signaling pathway acting through retinoic acid receptor α (Rarα) inhibition.

Recent genetic studies in mice have established that the nuclear receptor coregulator Trim24/Tif1α suppresses hepatocarcinogenesis by inhibiting retinoic acid receptor α (Rara)-dependent transcription and cell proliferation. However, Rara targets regulated by Trim24 remain unknown. We report that the loss of Trim24 resulted in interferon (IFN)/STAT pathway overactivation soon after birth (week 5). Despite a transient attenuation of this pathway by the induction of several IFN/STAT pathway repressors later in the disease, this phenomenon became more pronounced in tumors. Remarkably, Rara haplodeficiency, which suppresses tumorigenesis in Trim24(-/-) mice, prevented IFN/STAT overactivation. Moreover, together with Rara, Trim24 bound to the retinoic acid-responsive element of the Stat1 promoter and repressed its retinoic acid-induced transcription. Altogether, these results identify Trim24 as a novel negative regulator of the IFN/STAT pathway and suggest that this repression through Rara inhibition may prevent liver cancer.

TRIM involvement in transcriptional regulation.

Members of the tripartite motif (TRIM) protein family are found in all multicellular eukaryotes and function in a wide range of cellular processes such as cell cycle regulation, differentiation, development, oncogenesis and viral response. Over the past few years, several TRIM proteins have been reported to control gene expression through regulation of the transcriptional activity of numerous sequence-specific transcription factors. These proteins include the transcriptional intermediary factor 1 (TIF1) regulators, the promyelocytic leukemia tumor suppressor PML and the RET finger protein (RFP). In this chapter, we will consider the molecular interactions made by these TRIM proteins and will attempt to clarify some of the molecular mechanisms underlying their regulatory effect on transcription.

Arterial calcifications and increased expression of vitamin D receptor targets in mice lacking TIF1alpha.

Calcification of arteries is a major risk factor for cardiovascular mortality in humans. Using genetic approaches, we demonstrate here that the transcriptional intermediary factor 1alpha (TIF1alpha), recently shown to function as a tumor suppressor in murine hepatocytes, also participates in a molecular cascade that prevents calcifications in arterioles and medium-sized arteries. We further provide genetic evidence that this function of TIF1alpha is not exerted in hepatocytes. The sites of ectopic calcifications in mutant mice lacking TIF1alpha resemble those seen in mice carrying an activating mutation of the calcium sensor receptor (Casr) gene and, in TIF1alpha-deficient kidneys, Casr expression is increased together with that of many other vitamin D receptor (VDR) direct target genes, namely Car2, Cyp24a1, Trpv5, Trpv6, Calb1, S100g, Pthlh, and Spp1. Thus, our data indicate that TIF1alpha represses the VDR pathway in kidney and suggest that an up-regulation of Casr expression in this organ could account for ectopic calcifications generated upon TIF1alpha deficiency. Interestingly, the calcifying arteriopathy of TIF1alpha-null mutant mice shares features with the human age-related Mönckeberg's disease and, overall, the TIF1alpha-null mutant pathological phenotype supports the hypothesis that aging is promoted by increased activity of the vitamin D signaling pathway.

Pituitary cell translation and secretory capacities are enhanced cell autonomously by the transcription factor Creb3l2.

Translation is a basic cellular process and its capacity is adapted to cell function. In particular, secretory cells achieve high protein synthesis levels without triggering the protein stress response. It is unknown how and when translation capacity is increased during differentiation. Here, we show that the transcription factor Creb3l2 is a scaling factor for translation capacity in pituitary secretory cells and that it directly binds ~75% of regulatory and effector genes for translation. In parallel with this cell-autonomous mechanism, implementation of the physiological UPR pathway prevents triggering the protein stress response. Knockout mice for Tpit, a pituitary differentiation factor, show that Creb3l2 expression and its downstream regulatory network are dependent on Tpit. Further, Creb3l2 acts by direct targeting of translation effector genes in parallel with signaling pathways that otherwise regulate protein synthesis. Expression of Creb3l2 may be a useful means to enhance production of therapeutic proteins.

Pioneer and nonpioneer factor cooperation drives lineage specific chromatin opening.

Pioneer transcription factors are characterized by having the unique property of enabling the opening of closed chromatin sites, for implementation of cell fates. We previously found that the pioneer Pax7 specifies melanotrope cells through deployment of an enhancer repertoire, which allows binding of Tpit, a nonpioneer factor that determines the related lineages of melanotropes and corticotropes. Here, we investigate the relation between these two factors in the pioneer mechanism. Cell-specific gene expression and chromatin landscapes are defined by scRNAseq and chromatin accessibility profiling. We find that in vivo deployment of the melanotrope enhancer repertoire and chromatin opening requires both Pax7 and Tpit. In cells, binding of heterochromatin targets by Pax7 is independent of Tpit but Pax7-dependent chromatin opening requires Tpit. The present work shows that pioneer core properties are limited to the ability to recognize heterochromatin targets and facilitate nonpioneer binding. Chromatin opening per se may be provided through cooperation with nonpioneer factors.