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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.

Pioneer transcription factors establish new cell-fate competence by triggering chromatin remodeling. However, many features of pioneer action, such as their kinetics and stability, remain poorly defined. Here, we show that Pax7, by opening a unique repertoire of enhancers, is necessary and sufficient for specification of one pituitary lineage. Pax7 binds its targeted enhancers rapidly, but chromatin remodeling and gene activation are slower. Enhancers opened by Pax7 show a loss of DNA methylation and acquire stable epigenetic memory, as evidenced by binding of nonpioneer factors after Pax7 withdrawal. This work shows that transient Pax7 expression is sufficient for stable specification of cell identity.

The specification of cell fate is critical for proper cell differentiation and organogenesis. In endocrine tissues, this process leads to the differentiation, often a multistep process, of hormone-producing cells. This process is driven by a combination of transcription factors (TFs) that includes general factor, tissue-restricted, and/or cell-restricted factors. The last 2 decades have seen the discovery of many TFs of restricted expression and function in endocrine tissues. These factors are typically critical for expression of hormone-coding genes as well as for differentiation and proper function of hormone-producing cells. Further, genes encoding these tissue-restricted TFs are themselves subject to mutations that cause hormone deficiencies. Although the model that emerged from these 2 decades is one in which a specific combination of TFs drives a unique cell specification and genetic program, recent findings have led to the discovery of TFs that have the unique property of being able to remodel chromatin and thus modify the epigenome. Most importantly, such factors, known as pioneer TFs, appear to play critical roles in programming the epigenome during the successive steps involved in cell specification. This review summarizes our current understanding of the mechanisms for pioneer TF remodeling of chromatin. Currently, very few TFs that have proven pioneer activity are known, but it will be critical to identify these factors and understand their mechanisms of action if we are to harness the potential of regenerative therapies in endocrinology.

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.

As model of organogenesis, the pituitary gland is a relatively simple tissue; yet, we understand little of the mechanisms that determine organ size, cell number and allocation of cells to different lineages. While the discovery of cell-restricted transcription factors has led to significant insight into the mechanisms controlling differentiation and cell-specific gene expression, we still need to integrate these processes with control of organ development. The identification of pituitary stem cells has suggested mechanisms for maintenance of adult pituitary but these findings again highlight the crucial role of cell cycle control for determination of progenitor and differentiated cell numbers. We recently described the mechanisms for progenitor cell cycle exit in early pituitary development that critically depend on the cell cycle inhibitor p57Kip2. It appears that cell cycle control is independent of differentiation, indicating that separate regulatory mechanisms must be involved in each process. The role of p57Kip2 appears to be restricted to progenitor cell cycle exit and it is rather the related p27Kip1 that prevents re-entry into the cycle of differentiated cells. While these data revealed a new transient intermediate between progenitors and differentiated cells, they also raised new questions and suggested that separate signals may control differentiation and cell cycle., (Copyright (c) 2010 S. Karger AG, Basel.)

Patterning and differentiation signals are often believed to drive the developmental program, including cell cycle exit of proliferating progenitors. Taking advantage of the spatial and temporal separation of proliferating and differentiated cells within the developing anterior pituitary gland, we investigated the control of cell proliferation during organogenesis. Thus, we identified a population of noncycling precursors that are uniquely marked by expression of the cell cycle inhibitor p57(Kip2) and by cyclin E. In p57(Kip2-/-) mice, the developing pituitary is hyperplastic due to accumulation of proliferating progenitors, whereas overexpression of p57(Kip2) leads to hypoplasia. p57(Kip2)-dependent cell cycle exit is not required for differentiation, and conversely, blockade of cell differentiation, as achieved in Tpit(-/-) pituitaries, does not prevent cell cycle exit but rather leads to accumulation of p57(Kip2)-positive precursors. Upon differentiation, p57(Kip2) is replaced by p27(Kip1). Accordingly, proliferating differentiated cells are readily detected in p27(Kip1-/-) pituitaries but not in wild-type or p57(Kip2-/-) pituitaries. Strikingly, all cells of p57(Kip2-/-);p27(Kip1-/-) pituitaries are proliferative. Thus, during normal development, progenitor cell cycle exit is controlled by p57(Kip2) followed by p27(Kip1) in differentiated cells; these sequential actions, taken together with different pituitary outcomes of their loss of function, suggest hierarchical controls of the cell cycle that are independent of differentiation.

NeuroD1(BETA2) and Tpit are cell-specific activators of pituitary proopiomelanocortin (POMC) gene transcription. Expression of both factors slightly precedes that of POMC at embryonic d 12.5 of mouse pituitary development. We now report that NeuroD1(BETA2) is required for early corticotroph differentiation. In agreement with the transcriptional synergism observed between Tpit and basic helix-loop-helix dimers containing NeuroD1(BETA2), POMC expression is delayed in NeuroD1-deficient mice. However, this differentiation defect does not reflect a change of corticotroph commitment as revealed by Tpit expression. The delay of corticotroph terminal differentiation is transient and coincides with the developmental window of NeuroD1 expression in corticotrophs. In contrast to their requirement in other NeuroD1-expressing cells, the neurogenin genes do not appear to be necessary for corticotroph differentiation. Taken together with a similar requirement of Tpit for corticotroph differentiation but not for commitment, the present data indicate that the POMC promoter is a point of convergence for independent corticotroph differentiating signals.

Somatic adrenal stemcells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01-0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified. In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3β-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished in Pbx1 haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficient Tpit-/- mice was significantly wider compared with wild-type adrenals (Tpit-/- 259±10.7 vs Tpit+/- 100±12.3%; P!0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit-/- 0.45±0.16 vs Tpit+/- 0.13±0.04%; P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%; P=0.33 and 0.09%), providing indirect evidence for a stem cell 'arrest' in Tpit-/- mice and the role of ACTH in adrenocortical stem cell modulation and differentiation. [ABSTRACT FROM AUTHOR]

Adult tissue homeostasis and regeneration rely on tissue stem cell populations that generate committed precursors and differentiated cells while maintaining a pool of stem cells. In adult skeletal muscle, such cells, called satellite cells, remain quiescent at the periphery of muscle fibers. Upon injury they undergo activation, proliferation and differentiation to replace damaged fibers and also self-renew to reconstitute the muscle stem cell pool. During regeneration, the transition from a quiescent muscle stem cell to a differentiated fiber is accompanied by major metabolic changes. Such changes, and notably the switch from a glycolytic proliferative progenitor state to an oxidative post-mitotic differentiated state, require extensive mitochondrial biogenesis that takes place at the onset of differentiation and leads to increased ROS production. However, it is unclear whether this enhanced ROS production/mitochondrial content reflects an adaptation to the rising energy demand or whether it constitutes an essential regulation element of the differentiation program.To investigate the potential role of this metabolic switch and more specifically of reactive oxygen species during muscle regeneration, we took advantage of mouse mutants for Pitx2 and Pitx3 genes. Both genes are involved in foetal myogenesis where they have been identified as key regulators of the redox state preventing excessive ROS levels and DNA damage as cells undergo differentiation. We have now analyzed adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem cell compartment. Double mutant satellite cells undergo senescence with impaired regeneration after injury, whereas in single Pitx3 mutants, premature differentiation occurs. We show that these effects are directly linked to dose-dependent changes in ROS levels and can be reversed by lowering ROS with the N-acetylcystein, supporting the notion that a controlled increase in ROS is required for differentiation of muscle stem cells. [ABSTRACT FROM AUTHOR]

Summary: During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation. [Copyright &y& Elsevier]

Pro-opiomelanocortin (POMC) is expressed in two lineages of the pituitary, the anterior lobe corticotrophs and the intermediate lobe melanotrophs. POMC expression in these two lineages is highly dependent on the cell-restricted transcription factor Tpit. As Tpit intervenes relatively late in differentiation of those lineages, we have been searching for other transcription factors that may participate in their gene expression program. On the basis of similarity with the Tpit expression profile, we identified Ets variant gene 1 (Etvl/Er81) as a putative POMC transcription factor. Using Etvl -lacZ knockin mice, we describe preferential Etvl expression in pituitary POMC cells and also in posterior lobe pituicytes. We further show that Etv1 enhances POMC transcription on its own and in synergy with Tpit. The Ets-binding site located within the Tpit/Pitx regulatory element is necessary for Etv1 activity in POMC-expressing AtT-20 cells but dispensable for synergy with Tpit. Etv1 and Tpit interact together in coimmunoprecipitation experiments. Furthermore, Etv1 is present at the POMC promoter, and siRNA-mediated knockdown of Etv1 in AtT-20 cells produces a significant decrease in POMC expression. Etv1 knockout pituitaries show normal POMC cell distribution and normal POMC mRNA abundance, suggesting compensation by other factors. The coordinate expression of Etv1 with POMC cell differentiation and its interaction with the highly cell-restricted Tpit factor indicate that Etv1 participates in a combinatorial code for pituitary cell-specific gene expression. [ABSTRACT FROM AUTHOR]